Thermophile

Biosci Rep, 1997 Dec, 17(6), 529 - 35
Mitochondrial dehydrogenases in different taxa of tetrahymena: effect of insulin; Kohidai L et al.; Mitochondrial dehydrogenase activity was measured in seven taxa of Tetrahymena (T . pyriformis G1 . T . hegewishi, T . malaccensis, T . pigmentosa, T . shapiro, T . thermophila CU-399, T . thermophila MS-1) . Enzyme activity was different in the taxa investigated . Insulin reduced enzyme activity in six of the seven taxa studied . The duration of activity reduction was relatively long (5-10 min.) in most of the cases, and in T . hegewishi this lasted up to the end of the measurements (30 min.) . There was no interrelation between the basic dehydrogenase activity of the taxon and the effect of insulin . There was also no correlation between the degree of relationship (of the taxa) and the dehydrogenase profile after insulin treatment.

Lipids, 1998 Mar, 33(3), 319 - 26
Lipids of Thermococcus hydrothermalis, an archaea isolated from a deep-sea hydrothermal vent; Lattuati A et al.; The membrane lipids of a deep-sea hydrothermal vent archaea, Thermococcus hydrothermalis, were isolated, purified, and structurally characterized . On the basis of acid methanolysis and spectroscopic studies, the polar lipids, amounting to 4.5% (w/w) of the dry cells, comprised diphytanyl glycerol diethers and dibiphytanyldiglycerol tetraethers, in a 45:55 ratio . No cyclopentane ring was present in the tetraethers . From the neutral lipids, accounting for 0.4% (w/w) of the dry cells, besides low amounts of di- and tetraethers occurring in a free form, four acyclic tetraterpenoid hydrocarbons, di- and tri-unsaturated were identified . All were structurally related to lycopane . The presence of these hydrocarbons provides some evidence that lycopane, widely distributed in oceans, could be derived, at least partially, from the hydrocarbons synthesized by some thermophilic Archaea . Finally, analysis of the uninoculated culture medium indicates that fatty acid derivatives and some steroid and triterpenoid compounds identified in the lipidic extract of the archaea originated from the culture medium.

FEBS Lett, 1998 Mar 27, 425(2), 204 - 8
Cloning, sequencing and expression of the genes encoding the sodium translocating N5-methyltetrahydromethanopterin : coenzyme M methyltransferase of the methylotrophic archaeon Methanosarcina mazei Gö1; Lienard T et al.; The N5-methyltetrahydromethanopterin:coenzyme M methyltransferase of Methanosarcina mazei Go1 is a membrane-associated, corrinoid-containing protein that uses a transmethylation reaction to drive an energy-conserving sodium ion pump . The eight open reading frames encoding the eight different subunits of the methyltransferase were identified and sequenced . All of these subunits are shown to be heterologously expressed in minicells of the Escherichia coli mutant DK6 . Sequence comparisons with the methyltransferases of thermophilic and hypothermophilic methanogenic archaea are presented . The participation of the gene product of mtrD in sodium ion translocation as well as a consensus sequence of a corrinoid binding motif in MtrA are discussed.

J Biol Chem, 1998 Mar 27, 273(13), 7193 - 6
Protein components contribute to active site architecture for eukaryotic ribonuclease P; True HL et al.; In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity . The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components . We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes . Protein components from the Tetrahymena thermophila and human RNase P holoenzymes form specific products in photoreactions containing {4-thio}-uridine-labeled pre-tRNAGln . The HeLa RNase P RNA in neither the presence nor the absence of holoenzyme protein components formed cross-link products to the pre-tRNAGln probe . Parallel photo-cross-linking experiments with the Escherichia coli RNase P holoenzyme revealed that only the bacterial RNase P RNA forms specific substrate photoadducts . A protein-rich active site for the eukaryotic RNase P represents one unique feature that distinguishes holoenzyme organization between bacteria and eukaryotes.

Biospectroscopy, 1998, 4(1), 1 - 15
Cyanide binding and active site structure in heme-copper oxidases: normal coordinate analysis of iron-cyanide vibrations of a3(2+)CN- complexes of cytochromes ba3 and aa3; Kim Y et al.; The cyanide isotope-sensitive low-frequency vibrations of ferrous cyano complexes of cytochrome a3 are studied for cytochrome ba3 from Thermus thermophilus and cytochrome aa3 from bovine heart . Cyanide complexes of ba3 display three isotope sensitive frequencies at 512, 485, and 473 cm-1 . The first is primarily an Fe-C stretching motion, whereas the lower wavenumber modes are bending motions . These iron-cyanide vibrations are independent of the redox levels of the other metal centers in the protein . On the other hand, the fully reduced bovine derivative complexed with cyanide gives rise to a bending vibration at 503 cm-1 and a stretching vibration at 469 cm-1 . That is, the ordering of the stretching and bending frequencies is reversed from that of the bacterial protein . These results are analyzed by normal coordinate calculations to obtain comparative models for the binuclear O2 reducing site of the two proteins . We find that the observed frequencies are consistent with a linear Fe-C-N group and larger Fe-C stretching force constant (2.558 mdyn/A) for ba3 and a slightly bent Fe-C-N group (angle approximately 170 degrees) and a smaller Fe-C stretching force constant (2.335 mdyn/A) for aa3 . Thus, there are significant differences in the interaction of cyanide with ferrous a3 in the two proteins that are most likely caused by a weaker proximal histidine interaction and stronger peripheral heme electron withdrawing effects in ba3 . Possible sources of these protein-induced effects are discussed . Using the analysis developed here, comparison of the FeCN stretching and bending frequencies of the ferrous bovine a3-CN complex to those obtained from the ferric a3-CN complex suggests that upon conversion of the resting to the fully reduced protein, a conformational change occurs that constrains the ligand binding site.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2801 - 6
The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures; Villeret V et al.; The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 degrees C . To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 A and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria . Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties . A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme . We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry . The enzyme displays Michaelis-Menten kinetics . A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif . Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme . The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.

Nature, 1998 Mar 26, 392(6674), 353 - 8
The complete genome of the hyperthermophilic bacterium Aquifex aeolicus; Deckert G et al.; Aquifex aeolicus was one of the earliest diverging, and is one of the most thermophilic, bacteria known . It can grow on hydrogen, oxygen, carbon dioxide, and mineral salts . The complex metabolic machinery needed for A . aeolicus to function as a chemolithoautotroph (an organism which uses an inorganic carbon source for biosynthesis and an inorganic chemical energy source) is encoded within a genome that is only one-third the size of the E . coli genome . Metabolic flexibility seems to be reduced as a result of the limited genome size . The use of oxygen (albeit at very low concentrations) as an electron acceptor is allowed by the presence of a complex respiratory apparatus . Although this organism grows at 95 degrees C, the extreme thermal limit of the Bacteria, only a few specific indications of thermophily are apparent from the genome . Here we describe the complete genome sequence of 1,551,335 base pairs of this evolutionarily and physiologically interesting organism.

Protein Sci, 1998 Mar, 7(3), 698 - 705
Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution; Akanuma S et al.; We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell . The leuB gene encoding B . subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T . thermophilus . The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above . Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants . Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained . DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine . The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme . Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.

Biochim Biophys Acta, 1998 Mar 9, 1396(2), 215 - 27
A thermophilic nitrate reductase is responsible for the strain specific anaerobic growth of Thermus thermophilus HB8; Ramirez-Arcos S et al.; T . thermophilus HB8 contains a nitrate reductase gene cluster which is absent from closely related strains . This cluster encodes 4 ORFs (a-d) similar in organization and protein sequence to those encoded by respiratory nitrate reductase operons (narGHJI) of Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Thiosphaera pantothropha . The highest similarity is shown between the proteins encoded by the ORFa, ORFb and ORFd, and the structural components of the mesophilic nitrate reductases NarG (alpha), NarH (beta), and NarI (gamma) proteins, whilst ORFc encodes a protein which showed lower similarity to NarJ, a protein of unknown function encoded between narH and narI genes in all the nar cluster so far sequenced . This T . thermophilus HB8 narGHJI cluster is strongly induced by the combined effect of nitrate and low oxygen concentration, giving rise to the synthesis of an enzyme whose optimal temperature and pH was determined to be 80 degrees C, and pH 10, respectively . We also demonstrate that insertional inactivation of the narG and narH genes of this cluster results in strictly aerobic mutants, showing its sole responsibility in the strain specific ability of T . thermophilus HB8 to grow anaerobically.

Biochim Biophys Acta, 1998 Feb 17, 1382(2), 324 - 32
A stable, molten-globule-like cytochrome c; Wittung-Stafshede P; Expression of cytochrome c from Thermus thermophilus in Escherichia coli (E . coli) leads to a protein with characteristics of a molten globule . Unfolding induced by guanidine hydrochloride (GdHCl) shows that E . coli-expressed cytochrome c has lower stability (and less cooperativity of unfolding) compared to the protein extracted from Thermus thermophilus, even though the two proteins have identical amino-acid sequences . Moreover, Soret and far-UV circular dichroism signals differ for the two proteins, suggesting a distorted heme environment and more side-chain dynamics of E . coli-expressed cytochrome c . Still, tryptophan fluorescence in E . coli-expressed cytochrome c is quenched as in native protein, and the iron coordinates in a low-spin form . Amino-acid sequencing indicates the presence of only one covalent cysteine-linkage to the heme in E . coli-expressed cytochrome c (normally, there are two linkages), a possible explanation for the trapped, molten-globule-like structure . The features of this non-native protein may be of interest for interpretation of cytochrome c folding kinetics in vitro, since a molten globule may be an intermediate on the folding pathway.

Genetics, 1998 Mar, 148(3), 1109 - 15
High frequency intragenic recombination during macronuclear development in Tetrahymena thermophila restores the wild-type SerH1 gene; Deak JC et al.; Macronuclear development in ciliates is characterized by extensive rearrangement of genetic material, including sequence elimination, chromosome fragmentation and telomere addition . Intragenic recombination is a relatively rare, but evolutionarily important phenomenon occurring in mitosis and meiosis in a wide variety of organisms . Here, we show that high frequency intragenic recombination, on the order of 30%, occurs in the developing amitotic macronucleus of the ciliate Tetrahymena thermophila . Such recombination, occurring between two nonsense transition mutations separated by 726 nucleotides, reproducibly restores wild-type expression of the SerH1 surface protein gene, thus mimicking complementation in trans heterozygotes . Recombination must be considered a potentially important aspect of macronuclear development, producing gene combinations not present in the germinal micronucleus.

Curr Microbiol, 1998 Apr, 36(4), 202 - 6
Permeabilization of Streptococcus thermophilus and Lactobacillus delbrueckii subsp . bulgaricus with ethanol; Somkuti GA et al.; Streptococcus thermophilus and Lactobacillus delbrueckii subsp . bulgaricus cultures were treated with ethanol and tested for viability and beta-galactosidase activity . Exposure of the biomass of test cultures to 30%-55% ethanol (vol/vol) caused a 100% loss of viability and up to 15-fold increase in measurable beta-galactosidase activity in both streptococci and lactobacilli . Ethanol-treated cell suspensions could be stored for up to 6 months without loss of enzyme activity . The nonviable permeabilized biomass of the more active S . thermophilus was used to achieve up to 80% hydrolysis of lactose in aqueous solutions and non-fat milk.

Genes Dev, 1998 Mar 1, 12(5), 721 - 33
Interaction of recombinant Tetrahymena telomerase proteins p80 and p95 with telomerase RNA and telomeric DNA substrates; Gandhi L et al.; Telomerase is a specialized reverse transcriptase that catalyzes telomeric repeat addition at the ends of existing telomeres or fragmented chromosomes . Two telomerase proteins from Tetrahymena thermophila, p80 and p95, were identified on the basis of their association with telomerase activity and telomerase RNA . Here we have produced recombinant versions of these proteins to characterize their functions in the ribonucleoprotein . Our findings indicate that the two proteins can form a complex whose association is independent of RNA . Each protein interacts directly with telomerase RNA, but the p80/p95 complex binds RNA with an affinity substantially greater than either protein alone . We have also characterized the DNA binding properties of p95 and show that it interacts with telomeric substrate DNAs with a specificity characteristic of the functionally defined Tetrahymena telomerase substrate anchor site . Together, these findings suggest a model in which protein-nucleic acid interactions separable from the active site contribute to positioning the template and primer, rather than exclusively the direct nucleic acid-active site interaction typical of other polymerases.

Biochemistry, 1998 Mar 3, 37(9), 3172 - 7
Effect of redox state on the folding free energy of a thermostable electron-transfer metalloprotein: the CuA domain of cytochrome oxidase from Thermus thermophilus; Wittung-Stafshede P et al.; The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein) . The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium . This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant . The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C . For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol . The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form . The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site . The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 565 - 71
Characterization of a cellobiose dehydrogenase from Humicola insolens; Schou C et al.; The major cellobiose dehydrogenase (oxidase) (CBDH) secreted by the soft-rot thermophilic fungus Humicola insolens during growth on cellulose has been isolated and purified . It was shown to be a haemoflavoprotein with a molecular weight of 92 kDa and a pI of 4.0, capable of oxidizing the anomeric carbon of cellobiose, soluble cellooligosaccharides, lactose, xylobiose and maltose . Possible electron acceptors are 2,6-dichlorophenol-indophenol (DCPIP), Methylene Blue, 3,5-di-t-butyl-1,2-benzoquinone, potassium ferricyanide, cytochrome c and molecular oxygen . The oxidation of the prosthetic groups by oxygen was monitored at 449 nm for the flavin group and at 562 nm for the haem group . The curves were very similar to those of the cellobiose dehydrogenase from Phanerochaete chrysosporium, suggesting a similar mechanism . The pH-optima for the oxidation varied remarkably depending on the electron acceptor . For the organic electron acceptors, the pH-optima ranged from pH 4 for Methylene Blue to pH 7 for DCPIP and the benzoquinone . In the case of the FeIII-containing electron acceptors, the enzyme displayed alkaline pH-optima, in contrast to the properties of cellobiose dehydrogenases from Phanerochaete chrysosporium and Myceliophthora (Sporotrichum) thermophila . The enzyme has optimal activity at 65 degrees C.

Biosci Biotechnol Biochem, 1998 Feb, 62(2), 197 - 200
Effects of lactic acid bacteria on binding and absorption of mutagenic heterocyclic amines; Terahara M et al.; Effects of binding heterocyclic amines to cells of lactic acid bacteria on theirs absorption were investigated . Cells of Lactobacillus delbrueckii subsp . bulgaricus 2038 and Streptococcus thermophilus 1131 bind both 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) . The binding of strain 1131 cells to Trp-P-1 was maximum in the pHs from 4 to 8, but strain 2038 cells bound Trp-P-1 and MeIQx only slightly at pH 7 . We investigated the absorption of heterocyclic amines by the small intestine of F344 rats in the presence of these bacterial cells, using an in situ loop technique . The absorption of Trp-P-1 by the small intestine was significantly lower in the presence of strain 1131 cells than in the absence of the cells, but the presence of strain 2038 cells had no effect on Trp-P-1 absorption . Perhaps strain 1131 cells bind to Trp-P-1 at the same pH as that of the small intestine (pH 6-7) and thus decrease its absorption.

FEMS Microbiol Lett, 1998 Mar 15, 160(2), 205 - 8
BstB7SI (R decreases CCGGY), a thermostable isoschizomer of Cfr10I, from a strain of Bacillus stearothermophilus isolated from oil-contaminated soil in Kuwait; al-Awadhi S et al.; Isolates of thermophilic bacteria from desert soil in Kuwait, heavily contaminated with crude oil, have been screened for the presence of restriction endonuclease activity . One of the isolates (B7S), identified as Bacillus stearothermophilus, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda bacteriophage DNA at 65 degrees C . A type II restriction endonuclease (BstB7SI) has been partially purified from this isolate . BstB7SI recognises the six-base sequence RCCGGY (R = A or G; Y = T or C) and hydrolyses the phosphodiester bond in both strands of the DNA substrate between the first and second bases of the recognition sequence 5'-R decreases CCGGY-3'producing four-base 5' overhangs . BstB7SI is therefore an isoschizomer of the mesophilic prototype restriction endonuclease Cfr10I . BstB7SI has a pH optimum of 9.7, requires 10 mM MgCl2 and 75 mM NaCl for maximum activity, and retains full enzyme activity when incubated for 5 min at temperatures up to 70 degrees C.

Biochem Mol Biol Int, 1998 Feb, 44(2), 391 - 7
Studies on the heterologous expression of BstVI restriction endonuclease in Escherichia coli; Saavedra C et al.; Bacterial restriction and modification systems must be regulated to avoid self-restriction . It is generally accepted that cognate DNA methyltransferases normally protects both, the host's chromosome and extrachromosomal elements from the activity of their endonuclease counterparts . When the bstVIRM genes from Bacillus stearothermophilus V were subcloned into Escherichia coli, several clones exhibiting a r+m- phenotype were originated . The present work was undertaken to analyze the possibility that mechanisms other than DNA methylation could account for the viability of these cells . No evidence was found for an inhibitory agent or endonuclease compartmentation . In vivo experiments showed that lambda phage multiplication was poorly restricted by the heterologous enzyme . The restricting activity against the incoming phase increased however when phage adsortion was performed at higher temperatures . Analogous experiments in which a DNA-repair deficient strain was used as a host for the thermophilic R-M system suggested, to some extent, the participation of the repair machinery in the viability of r+m- clones.

Biochem Mol Biol Int, 1998 Feb, 44(2), 269 - 82
An archaeal DNA binding protein from thermophilic Sulfolobus acidocaldarius forms different types of complexes with DNA; Sreenivas K et al.; We have characterized a DNA binding protein (DBNP-B) from the thermoacidophilic archaeon Sulfolobus acidocaldarius with respect to its interaction with single and double stranded DNA . The protein in solution exists predominantly as dimer as indicated by cross linking studies . Binding of DBNP-B to etheno DNA and poly (dA) resulted in fluorescence enhancement and hyperchromicity respectively . Ethidium bromide intercalated into DNA was completely displaced by DBNP-B . DNase I digestion of dsDNA was increased at subsaturating concentration of the protein and was inhibited at higher concentrations . These results and electron microscopy indicate that the protein forms different types of novel complexes with DNA at different protein to DNA ratios.

Biochim Biophys Acta, 1998 Feb 2, 1379(2), 297 - 301
Small angle neutron scattering analysis of thermal stability of 23S rRNA and the intact 50S subunits of Sulfolobus solfataricus; Briganti G et al.; The ribosomes of the extremely thermophilic archaebacterium, Sulfolobus solfataricus, are very resistant to thermal denaturation (optimal growth temperature 87 degrees C), remaining essentially intact up to above 90 degrees C . However, the separate ribosomal components (rRNA and r-proteins) are less thermally stable than the ribosome as a whole, indicating that the mode of interaction of all of the components within the ribonucleoprotein particle play an essential role in determining thermal stability . To get some insight into the structural features of the thermophilic ribosome, we performed small angle neutron scattering (SANS) measurements at various temperatures on Sulfolobus solfataricus intact large ribosomal subunits (50S) and deproteinated large ribosomal subunit RNA (23S) . Even if the scattering profiles suggest the presence of supramolecular aggregates in all of the samples and at all of the investigated temperatures, the measured form factors indicated for both samples that, at temperatures above 70 degrees C, the suspended particles underwent a structural rearrangement . This finding is likely to reflect single particles' properties, since S . solfataricus ribosomes are known to be biologically activated only above 60 degrees C, and there are indications that such activation requires a conformational rearrangement of the particle . A remarkable superimposition of the percentage variation of the volume from neutron scattering and of the absorbency increment with respect to temperature supports this view.

Mol Biol Cell, 1998 Feb, 9(2), 497 - 511
Proteolytic processing and Ca2+-binding activity of dense-core vesicle polypeptides in Tetrahymena; Verbsky JW et al.; Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal . The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied . Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors . These share little overall amino acid identity but are nonetheless predicted to have structural similarity . In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation . In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo . Core assembly and postexocytic dispersal are compartment-specific events . Two likely regulatory factors are proteolytic processing and exposure to calcium . We asked whether these might directly influence the conformations of core proteins . Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements . Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release . The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles.

Microbios, 1997, 91(368-369), 181 - 90
Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival, and proliferation in Tetrahymena thermophila; Straarup EM et al.; Autocrine factors prevent cell death in the ciliate Tetrahymena thermophila, a unicellular eukaryote, in a chemically defined medium . At certain growth conditions these factors are released at a sufficient concentration by > 500 cells ml-1 to support cell survival and proliferation . The protein kinase C activators phorbol 12-myristate 13-acetate (PMA) or 1-oleyl 2-acetate glycerol (OAG) when added to 250 cells ml-1 supported cell survival and proliferation . In the presence of the serine and threonine kinase inhibitor staurosporine the cells died both at 250 cells ml-1 in cultures supplemented with either PMA or OAG, or at 2,500 cells ml-1 . At 500 cells ml-1 PMA induced the in vivo phosphorylation of at least six proteins . The myelin basic protein fragment 4-14 was phosphorylated in vitro in crude extracts of a culture of 250,000 cells ml-1 . Both the in vivo and the in vitro phosphorylation were inhibited by staurosporine.

Biochemistry, 1998 Mar 10, 37(10), 3369 - 76
Kinetic role of electrostatic interactions in the unfolding of hyperthermophilic and mesophilic rubredoxins; Cavagnero S et al.; The temperature dependence of the unfolding kinetics of rubredoxins from the hyperthermophile Pyrococcus furiosus (RdPf) and the mesophile Clostridium pasteurianum (RdCp) has been studied . Results show that RdPf unfolds much more slowly, under all experimentally accessible temperature regimes, than RdCp and other typical mesophilic proteins . Rates of RdCp and RdPf unfolding decrease upon increasing the pH above 2 and diverge dramatically at pH 7 . As shown by detailed electrostatic energy calculations, this is the result of a differential degree of protonation of the negatively charged amino acids, which causes distinct electrostatic configurations as a function of pH . We propose that ion pairs, particularly those that are placed in key surface positions, may play a kinetic role by mildly clamping the protein and thereby influencing the nature and the number of the vibrational normal modes that are thermally accessible upon unfolding . More generally, these modes are also likely to be affected by the favorable electrostatic configurations, which we have shown to be directly linked to the extremely slow unfolding rates of RdPf at neutral pH . Even at pH 2, in the absence of any salt bridges, the unfolding rates of RdPf are much smaller than those of RdCp . This is ascribed to presently unidentified structural elements of nonelectrostatic nature . Since electrostatic effects influence the unfolding kinetics of both mesophilic and thermophilic rubredoxins, these findings may be of general significance for proteins.

J Biotechnol, 1997 Jan 3, 59(3), 203 - 11
Purification and characterisation of an intracellular X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus ACA-DC 4; Tsakalidou E et al.; An intracellular X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus ACA-DC 4, isolated from traditional Greek yoghurt, was purified by anion exchange and gel filtration chromatography . A single band of molecular weight of about 80,000 appeared in SDS-PAGE; by gel filtration it was shown that the native enzyme was dimeric . The peptidase showed optimum activity on glycyl-prolyl 4-nitroanilide at pH 7.0 and at 50 degrees C, with K(m) = 3.1 mM and Vmax = 3500 U mg-1; over 50 degrees C the enzyme activity declined rapidly . It was inactivated by PMSF; sulfhydryl group reagents and metal chelators had little effect on enzyme activity.

Biochemistry, 1998 Feb 24, 37(8), 2639 - 47
Activation of methyl-SCoM reductase to high specific activity after treatment of whole cells with sodium sulfide; Becker DF et al.; Here, we report a method to generate the active form of methyl-SCoM reductase (MCR) from Methanosarcina thermophila . The protocol involves adding sodium sulfide to a growing cell culture prior to harvest to yield a "ready" (MCRox1) state of the enzyme . This method can also generate a ready state of the Methanobacterium thermoautotrophicum (strain Marburg) MCR . Experiments using sodium 35S-labeled sulfide indicate the ready state that is generated involves a Ni-S adduct . As was shown earlier for the Mb . thermoautotrophicum MCRox1 {Goubeaud, M., Schreiner, G . and Thauer, R . K . (1997) Eur . J . Biochem . 17, 2374-2377}, this ready state is converted to the highly active MCRred1 form by reductive activation with Ti(III) citrate . The reduction of MCRox1 to MCRred1 with concomitant increase in activity demonstrated that MCRred1 is the active form of MCR from Ms . thermophila . We also observed the loss of the 35S-sulfide label from the enzyme when MCRox1 was converted to MCRred1 . Other states of MCR could be generated in the whole cells by adding different potential ligands to the cell medium; for example, the MCRox2 state was generated by treating cells with sodium sulfite or sodium dithionite.

J Membr Biol, 1998 Mar 1, 162(1), 51 - 7
Purification and characterization of a novel chemorepellent receptor from Tetrahymena thermophila; Kuruvilla HG et al.; Chemosensory transduction and adaptation are important aspects of signal transduction mechanisms in many cell types, ranging from prokaryotes to differentiated tissues such as neurons . The eukaryotic ciliated protozoan, Tetrahymena thermophila, is capable of responding to both chemoattractants (O'Neill et al., 1985; Leick, 1992; Kohidai, Karsa & Csaba, 1994, 1995) and chemorepellents (Francis & Hennessey, 1995; Kuruvilla, Kim & Hennessey, 1997) . An example of a nontoxic, depolarizing chemorepellent in Tetrahymena is extracellular lysozyme (Francis & Hennessey, 1995; Hennessey, Kim & Satir, 1995) . Lysozyme is an effective chemorepellent at micromolar concentrations, binds to a single class of externally facing membrane receptors and prolonged exposure (10 min) produces specific chemosensory adaptation (Kuruvilla et al., 1997) . We now show that this lysozyme response is initiated by a depolarizing chemoreceptor potential in Tetrahymena and we have purified the membrane lysozyme receptor by affinity chromatography of solubilized Tetrahymena membrane proteins . The solubilized, purified protein is 42 kD and it exhibits saturable, high affinity lysozyme binding . Polyclonal antibodies raised against this 42 kD receptor block the in vivo lysozyme chemoresponse . This is not only the first time that a chemoreceptor potential has been recorded from Tetrahymena but also the first time that a chemorepellent receptor has been purified from any unicellular eukaryote.

J Bacteriol, 1998 Mar, 180(6), 1480 - 7
Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila; Maupin-Furlow JA et al.; The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized . The biochemical properties revealed novel features of the archaeal 20S proteasome . A fully active 20S proteasome could be assembled in vitro with purified native alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the beta prosubunits or assembly of the 20S proteasome . A protein complex with a molecular mass intermediate to those of the alpha7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes . The heterologously produced M . thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides . Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity) . Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity . The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates . The 20S proteasome also hydrolyzed large proteins . Replacement of the nucleophilic Thr1 residue with an Ala in the beta subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity . Replacement of beta subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Ogamma nucleophile . Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.

RNA, 1998 Mar, 4(3), 268 - 75
Experimental evolution of complexity: in vitro emergence of intermolecular ribozyme interactions; Hanczyc MM et al.; In the course of evolving variants of the Tetrahymena thermophila Group I ribozyme for improved DNA cleavage in vitro, we witnessed the unexpected emergence of a derived molecular species, capable of acting as a partner for the ribozyme, but no longer autocatalytic . This new RNA species exhibits a deletion in the catalytic core and participates in a productive intermolecular interaction with an active ribozyme, thus insuring its survival in the population . These novel RNA molecules have evolved a precise catalytic interaction with the Group I ribozyme and depend for their survival on the continued presence of active catalysts . This interaction hints at the complexity that may inevitably arise even in simple evolving systems.

Dig Dis Sci, 1998 Jan, 43(1), 133 - 7
Management of lactose maldigestion by consuming milk containing lactobacilli; Lin MY et al.; The influence of nonfermented milk containing L . acidophilus or L . bulgaricus on lactose utilization by lactose maldigesters was investigated . Nonfermented milks containing L . acidophilus or L . bulgaricus at 10(8) and 10(9) CFU/ml were prepared using 2% low-fat milk . Lactose maldigestion was monitored by measuring breath hydrogen at hourly intervals for 8 hr following consumption of 400 ml of each diet . Nonfermented milk containing L . acidophilus B at 10(8) CFU/ml were not effective in reducing breath hydrogen and symptoms . Nonfermented milk containing L . acidophilus B at 10(9) CFU/ml only slightly decreased breath hydrogen production; however, the symptoms were significantly improved . Nonfermented milks containing L . bulgaricus 449 at 10(8) and 10(9) CFU/ml were effective in reducing breath hydrogen and symptoms . The results for bulgaricus milk were all significant . In this study, L . acidophilus B and L . bulgaricus 449 were chosen because of their similar beta-galactosidase activity and bile sensitivity . L . acidophilus and L . bulgaricus are both thermophilic lactobacilli and an active transport (permease) system is found in both species for lactose transport . The major factor affecting in vivo lactose digestion in this study appears to be the bacterial cell wall/membrane structures . That the cell wall/membrane structures of L . acidophilus are different from those of L . bulgaricus can be indirectly proven by the results of sonication time for maximum beta-galactosidase activity measurement . The results of this study indicate that L . bulgaricus is usually a better choice than L . acidophilus for manufacturing nonfermented milks for lactose maldigesters.

Nat Struct Biol, 1998 Mar, 5(3), 229 - 35
Conservation of rapid two-state folding in mesophilic, thermophilic and hyperthermophilic cold shock proteins; Perl D et al.; The cold shock protein CspB from Bacillus subtilis is only marginally stable, but it folds extremely fast in a simple N reversible U two-state reaction . The corresponding cold shock proteins from the thermophile Bacillus caldolyticus and the hyperthermophile Thermotoga maritima show strongly increased conformational stabilities, but unchanged very fast two-state refolding kinetics . The absence of intermediates in the folding of B . subtilis CspB is thus not a corollary of its low stability . Rather, two-state folding and an unusually native-like activated state of folding seem to be inherent properties of these small all-beta proteins . There is no link between stability and folding rate, and numerous sequence positions exist which can be varied to modulate the stability without affecting the rate and mechanism of folding.

Virology, 1998 Feb 15, 241(2), 345 - 56
Evolution of Streptococcus thermophilus bacteriophage genomes by modular exchanges followed by point mutations and small deletions and insertions; Desiere F et al.; Comparative sequence analysis of 40% of the genomes from two prototype Streptococcus thermophilus bacteriophages (lytic group I phage phi Sfi19 and the cos site containing temperate phage phi Sfi21) suggested two processes in the evolution of their genomes . In a first evolutionarily distant phase the basic genome structure was apparently constituted by modular exchanges . Over the 17-kb-long DNA segment analyzed in the present report, we observed clusters of genes with similarity to genes from Leuconostoc oenos phage L10, Lactococcus lactis phage BK5-T, and Streptococcus pneumoniae phage Dp-1 . A chimeric protein was predicted for orf 1291 which showed similarity to both phage BK5-T and phage Dp-1 proteins . The very large orf 1626 gene product showed similarity to two adjacent genes from the Lactobacillus delbrueckii phage LL-H and further phage proteins (Lactococcus lactis, Bacillus subtills) . The similarities were localized to distinct parts of this apparently multifunctional protein . The putative phi Sfi19 lysin showed similarity to both lysins of phages and cellular enzymes . In a second, evolutionarily more recent, phase the S, thermophilus phage genomes apparently diversified by point mutations and small deletions/insertions . Over the investigated 17-kb DNA region phi Sfi19 differed from phi Sfi21 by 10% base pair changes, the majority of which were point mutations (mainly at the third codon position), while a third of the base pair differences were contributed by small deletions/insertions . The base pair changes were unevenly distributed . Over the Leuconostoc phage-related DNA the change rate was high, while over the Lactococcus and S . pneumoniae phage-related DNA the change rate was low . We speculate that the degree of base pair change could provide relative time scales for the modular exchange reactions observed in S . thermophilus phages.

Genome Res, 1998 Feb, 8(2), 91 - 9
Mapping the germ-line and somatic genomes of a ciliated protozoan, Tetrahymena thermophila; Orias E; Ciliates are among the very few eukaryotes in which the powers of molecular biology, conventional genetics, and microbial methodology can be synergistically combined . Because ciliates also are distant relatives of vertebrates, fungi, and plants, the sequencing of a ciliate genome will be of import to our understanding of eukaryotic biology . Tetrahymena thermophila is the only ciliate in which a systematic genetic mapping of DNA polymorphisms has begun . Tetrahymena has many biological features that make it a specially or uniquely useful experimental system for fundamental research in cell and molecular biology and for biotechnological applications . A key factor in the usefulness of Tetrahymena is the speed, facility, and versatility with which it can be cultivated under a wide range of nutrient conditions, temperature, and scale . This article describes the progress made in genetically and physically mapping the genomes of T . thermophila: the micronuclear (germ-line) genome, which is not transcriptionally expressed, and the macronuclear (somatic) fragmented genome, which is actively expressed and determines the cell's phenotype.

Nucleic Acids Res, 1998 Feb 15, 26(4), 903 - 10
Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8; Mikawa T et al.; The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8 . Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa . Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein . The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased . The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T . thermophilus MutM protein was active in E.coli . The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity . Size-exclusion chromatography indicated that T . thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution . Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30% . Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C . Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively . Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.

Int J Food Microbiol, 1997 Sep 16, 38(2-3), 235 - 8
Incidence and biochemical characteristics of Bacillus flora in Sardinian dairy products; Cosentino S et al.; This study was planned to assess the frequency and level of Bacillus spp . contamination in Sardinian dairy products and to evaluate some food-spoilage-related characteristics of the strains isolated . Of the 378 dairy products tested, 265 (70%) were found to contain Bacillus spp . The overall level of contamination ranged from less than 10 cfu per ml or gram up to a maximum of 1200 cfu . A total of 483 strains, belonging to 14 species, have been isolated from the 265 positive samples . The most frequently isolated psychotropic species were B . cereus (18.6% of total isolates), B . coagulans and B . mycoides . B . subtilis, B . licheniformis and B . pumilis were the most common mesophilic strains and B . stearotermophilus was the dominant thermophilic species . Most strains showed strong enzymatic activity, as indicated by the high percentage of isolates capable of hydrolysing casein, gelatin, starch and liquids . As regards possible health hazards . 72% of the B . cereus strains tested showed evidence of toxin production using a reversed passive latex agglutination assay.

J Biochem (Tokyo), 1997 Dec, 122(6), 1092 - 104
Crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile, Bacillus coagulans: two strategies for thermostabilization of protein structures; Tsuchiya D et al.; The crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans (BcIPMDH) has been determined by the X-ray method . BcIPMDH is a dimeric enzyme composed of two identical subunits, each of which takes an open alpha/beta structure with 11 alpha-helices and 14 beta-strands . The polypeptide is folded into two domains . The first domain is composed of residues 1-101 and 257-356, and the second domain, of residues 102-256 . The latter domains of the two subunits are associated with one another by a dyad axis to make the dimer, locally forming a beta-sheet and a four-helix bundle . As compared with the structure of the enzyme from the extreme thermophile Thermus thermophilus (TtIPMDH), a new short beta-sheet (residues 329-330 and 340-341) absent in TtIPMDH is formed by the insertion of 5 residues in BcIPMDH . In terms of determinants for thermostabilization, both consistent and inconsistent changes were found between the two enzymes . The regions including inconsistent changes are formed by different usages of the determinants for stabilizing the loops at different levels . Those in BcIPMDH contain some structural redundancies in length of amino acid sequence and flexibility of residues, which seem to be unnecessary for the enzymatic reaction . Such redundancies are also found in the primary structure of the enzyme of the mesophile Bacillus subtilis, but these parts are more stabilized in BcIPMDH by hydrogen bonds and salt bridges . On the other hand, TtIPMDH is stabilized by reducing such redundant parts . This contrast suggests that different strategies may be preferred for thermostabilization, depending on temperature.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 999 - 1003
In vitro assembly of a ribonucleoprotein particle corresponding to the platform domain of the 30S ribosomal subunit; Agalarov SC et al.; A fragment of the 16S RNA of Thermus thermophilus corresponding to the central domain (nucleotides 547-895) has been prepared by transcription in vitro . Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle . This particle contained five T . thermophilus proteins corresponding to Escherichia coli ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit . When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit . We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.

J Bacteriol, 1998 Mar, 180(5), 1129 - 34
Identification of essential glutamates in the acetate kinase from Methanosarcina thermophila; Singh-Wissmann K et al.; Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO- + ATP<-->CH3CO2PO3(2-) + ADP) . A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J . A . Todhunter and D . L . Purich, Biochem . Biophys . Res . Commun . 60:273-280, 1974) . Alignment of the amino acid sequences for the acetate kinases from E . coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates . These glutamates were replaced in the M . thermophila enzyme to determine if any are essential for catalysis . The histidine-tagged altered enzymes were produced in E . coli and purified to electrophoretic homogeneity by metal affinity chromatography . Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism . Replacement of E385 influenced the Km values for acetate and ATP with only moderate decreases in k(cat), which suggests that this residue is involved in substrate binding but not catalysis . The unaltered acetate kinase was not inactivated by N-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site . Replacement of E97 decreased the Km value for acetate but not ATP, suggesting this residue is involved in binding acetate . Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.

J Bacteriol, 1998 Mar, 180(5), 1103 - 9
Cloning and characterization of transcription of the xylAB operon in Thermoanaerobacter ethanolicus; Erbeznik M et al.; The genes encoding xylose isomerase (xylA) and xylulose kinase (xylB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus were found to constitute an operon with the transcription initiation site 169 nucleotides upstream from the previously assigned (K . Dekker, H . Yamagata, K . Sakaguchi, and S . Udaka, Agric . Biol . Chem . 55:221-227, 1991) promoter region . The bicistronic xylAB mRNA was processed by cleavage within the 5'-terminal portion of the XylB-coding sequence . Transcription of xylAB was induced in the presence of xylose, and, unlike in all other xylose-utilizing bacteria studied, was not repressed by glucose . The existence of putative xyl operator sequences suggested that xylose utilization is controlled by a repressor-operator mechanism . The T . ethanolicus xylB gene coded for a 500-amino-acid-residue protein with a deduced amino acid sequence highly homologous to those of other XylBs . This is the first report of an xylB nucleotide sequence and an xyLAB operon from a thermophilic anaerobic bacterium.

FEMS Microbiol Lett, 1998 Mar 1, 160(1), 17 - 23
alpha-Amylase gene of thermophilic Streptomyces sp . TO1: nucleotide sequence, transcriptional and amino acid sequence analysis; Mellouli L et al.; The nucleotide sequence of a 1860-bp region encoding a thermostable alpha-amylase of Streptomyces sp . TO1 was determined . Frame analysis revealed the presence of a 1359-bp long open reading frame (amy TO1) encoding a 453 amino acid protein with a deduced M(r) of 49 kDa . Northern blot analysis revealed that amy TO1 gene was expressed as approximately 1.5-kbp monocistronic transcript in both SL1326/pLM1 and Streptomyces sp . TO1 strains . Primer extension experiments indicated that the transcriptional start site lies 30 bp upstream of the ATG start codon, and allowed the identification of -35 (TTGCTG) and -10 (TACGCG) eubacterial-like promoter sequences . Amy TO1 exhibits strong amino acid identities with those from other Streptomyces species with a maximum of 78% with S . thermoviolaceus alpha-amylase . Nevertheless, subtle amino acid changes such as the substitution of four conserved residues found at similar positions in other Streptomyces alpha-amylases by proline residues, and the substitution of three conserved hydrophilic amino acids by hydrophobic ones in Amy TO1 might account for the thermostable properties of Amy TO1.

Microbiology, 1998 Feb, 144 ( Pt 2), 479 - 92
Genes and enzymes of the acetyl cycle of arginine biosynthesis in the extreme thermophilic bacterium Thermus thermophilus HB27; Baetens M et al.; An arginine biosynthetic gene cluster, argC-argJ, of the extreme thermophilic bacterium Thermus thermophilus HB27 was isolated by heterologous complementation of an Escherichia coli acetylornithinase mutant . The recombinant plasmid (pTHM1) conferred ornithine acetyltransferase activity to the E . coli host, implying that T . thermophilus uses the energetically more economic pathway for the deacetylation of acetylornithine . pTHM1 was, however, unable to complement an E . coli argA mutant and no acetylglutamate synthase activity could be detected in E . coli argA cells containing pTHM1 . The T . thermophilus argJ-encoded enzyme is thus monofunctional and is unable to use acetyl-CoA to acetylate glutamate (contrary to the Bacillus stearothermophilus homologue) . Alignment of several ornithine acetyltransferase amino acid sequences showed no obvious pattern that could account for this difference; however, the monofunctional enzymes proved to have shorter N-termini . Sequence analysis of the pTHM1 3.2 kb insert revealed the presence of the argC gene (encoding N-acetylglutamate-5-semialdehyde dehydrogenase) upstream of the argJ gene . Alignment of several N-acetylglutamate-5-semialdehyde dehydrogenase amino acid sequences allowed identification of two strongly conserved putative motifs for cofactor binding: a putative FAD-binding site and a motif reminiscent of the NADPH-binding fingerprint . The relationship between the amino acid content of both enzymes and thermostability is discussed and an effect of the GC content bias is indicated . Transcription of both the argC and argJ genes appeared to be vector-dependent . The argJ-encoded enzyme activity was twofold repressed by arginine in the native host and was inhibited by ornithine . Both upstream of the argC gene and downstream of the argJ gene an ORF with unknown function was found, indicating that the organization of the arginine biosynthetic genes in T . thermophilus is new.

Microbiology, 1998 Feb, 144 ( Pt 2), 457 - 65
Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile 'Anaerocellum thermophilum' with separate glycosyl hydrolase family 9 and 48 catalytic domains; Zverlov V et al.; A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum' . Full-length CelA and a truncated enzyme species designated CelA' were purified to homogeneity from culture supernatants . CelA has an apparent molecular mass of 230 kDa . The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and beta-glucan . Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 degrees C (CM-cellulase) and 85 degrees C (Avicelase) . Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation . The CelA-encoding gene was isolated from genomic DNA of 'A . thermophilum' by PCR and the nucleotide sequence was determined . The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the N-terminus of CelA purified from 'A . thermophilum' . Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family III cellulose-binding domain linked by Pro-Thr-Ser-rich regions . The enzyme is most closely related to CelA of Caldicellulosiruptor saccharolyticus (sequence identities of 96 and 97% were found for the N- and C-terminal catalytic domains, respectively) . Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain . Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound to microcrystalline cellulose . Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

Structure, 1998 Jan 15, 6(1), 101 - 8
tRNA(Pro) anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase; Cusack S et al.; BACKGROUND: Most aminoacyl-tRNA synthetases (aaRSs) specifically recognize all or part of the anticodon triplet of nucleotides of their cognate tRNAs . Class IIa and class IIb aaRSs possess structurally distinct tRNA anticodon-binding domains . The class IIb enzymes (LysRS, AspRS and AsnRS) have an N-terminal beta-barrel domain (OB-fold); the interactions of this domain with the anticodon stem-loop are structurally well characterised for AspRS and LysRS . Four out of five class IIa enzymes (ProRS, ThrRS, HisRS and GlyRS, but not SerRS) have a C-terminal anticodon-binding domain with an alpha/beta fold, not yet found in any other protein . The mode of RNA binding by this domain is hitherto unknown as is the rationale, if any, behind classification of anticodon-binding domains for different aaRSs . RESULTS: The crystal structure of Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) in complex with tRNA(Pro) has been determined at 3.5 A resolution by molecular replacement using the native enzyme structure . One tRNA molecule, of which only the lower two-thirds is well ordered, is found bound to the synthetase dimer . The C-terminal anticodon-binding domain binds to the anticodon stem-loop from the major groove side . Binding to tRNA by ProRSTT is reminiscent of the interaction of class IIb enzymes with cognate tRNAs, but only three of the anticodon-loop bases become splayed out (bases 35-37) rather than five (bases 33-37) in the case of class IIb enzymes . The two anticodon bases conserved in all tRNA(Pro), G35 and G36, are specifically recognised by ProRSTT . CONCLUSIONS: For the synthetases possessing the class IIa anticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception of HisRS), the two anticodon bases 35 and 36 are sufficient to uniquely identify the cognate tRNA (GG for proline, GU for threonine, CC for glycine), because these amino acids occupy full codon groups . The structure of ProRSTT in complex with its cognate tRNA shows that these two bases specifically interact with the enzyme, whereas base 34, which can be any base, is stacked under base 33 and makes no interactions with the synthetase . This is in agreement with biochemical experiments which identify bases 35 and 36 as major tRNA identity elements . In contrast, class IIb synthetases (AspRS, AsnRS and LysRS) have a distinct anticodon-binding domain that specifically recognises all three anticodon bases . This again correlates with the requirements of the genetic code for cognate tRNA identification, as the class IIb amino acids occupy half codon groups.

Eur J Biochem, 1998 Feb 1, 251(3), 744 - 57
Glycyl-tRNA synthetase from Thermus thermophilus--wide structural divergence with other prokaryotic glycyl-tRNA synthetases and functional inter-relation with prokaryotic and eukaryotic glycylation systems; Mazauric MH et al.; The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae . To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter-relations with those of other origins analyzed . Alignments of the protein sequence of the dimeric thermophilic glycyl-tRNA synthetase (Gly-tRNA synthetase) derived from its gene with sequences of other dimeric Gly-tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases . Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly-tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes . Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems . Efficient glycylations involving partners from T . thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases . However, Gly-tRNA synthetase from T . thermophilus acylates eukaryotic tRNA(Gly) while the charging ability of the E . coli enzyme is restricted to prokaryotic tRNA(Gly) . A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly-tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme . The consensus sequence of the tRNAs charged by the various Gly-tRNA synthetases reveals conservation of only G1-C72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10-Y25)-G45 triplet involved in tRNA folding . Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set . These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T . thermophilus in this phylogeny.

J Mol Evol, 1998 Jan, 46(1), 1 - 17
The path from the RNA world; Poole AM et al.; We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes . The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis . By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides . With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA --> RNP --> protein, the first proteins in the "breakthrough organism" (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic . Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the "introns-first" theory . Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a "eukaryotic-like" genome organization (a fragmented linear genome with multiple centers of replication) . The steps from the proposed ribo-organism RNA genome --> eukaryotic-like DNA genome --> prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome --> eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA --> RNP --> protein . A likely molecular mechanism, "plasmid transfer," is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture . Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry . A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history.

Structure, 1997 Nov 15, 5(11), 1475 - 83
Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability; Auerbach G et al.; BACKGROUND: Phosphoglycerate kinase (PGK) is essential in most living cells both for ATP generation in the glycolytic pathway of aerobes and for fermentation in anaerobes . In addition, in many plants the enzyme is involved in carbon fixation . Like other kinases, PGK folds into two distinct domains, which undergo a large hinge-bending motion upon catalysis . The monomeric 45 kDa enzyme catalyzes the transfer of the C1-phosphoryl group from 1, 3-bisphosphoglycerate to ADP to form 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP . For decades, the conformation of the enzyme during catalysis has been enigmatic . The crystal structure of PGK from the hyperthermophilic organism Thermotoga maritima (TmPGK) represents the first structure of an extremely thermostable PGK . It adds to a series of four known crystal structures of PGKs from mesophilic via moderately thermophilic to a hyperthermophilic organism, allowing a detailed analysis of possible structural determinants of thermostability . RESULTS: The crystal structure of TmPGK was determined to 2.0 A resolution, as a ternary complex with the product 3-phosphoglycerate and the product analogue AMP-PNP (adenylyl-imido diphosphate) . The complex crystallizes in a closed conformation with a drastically reduced inter-domain angle and a distance between the two bound ligands of 4.4 A, presumably representing the active conformation of the enzyme . The structure provides new details of the catalytic mechanism . An inter-domain salt bridge between residues Arg62 and Asp200 forms a strap to hold the two domains in the closed state . We identify Lys197 as a residue involved in stabilization of the transition state phosphoryl group, and so term it the 'phosphoryl gripper' . CONCLUSIONS: The hinge-bending motion of the two domains upon closure of the structure, as seen in the Trypanosoma PGK structure, is confirmed . This closed conformation obviously occurs after binding of both substrates and is locked by the Arg62-Asp200 salt bridge . Re-orientations in the conserved active-site loop region around Thr374 also bring both domains into direct contact in the core region of the former inter-domain cleft, to form the complete catalytic site . Comparison of extremely thermostable TmPGK with less thermostable homologues reveals that its increased rigidity is achieved by a raised number of intramolecular interactions, such as an increased number of ion pairs and additional stabilization of alpha helix and loop regions . The covalent fusion with triosephosphate isomerase might represent an additional stabilization strategy.

Comp Immunol Microbiol Infect Dis, 1997 Sep, 20(4), 335 - 44
{Thermophilic bacteria resistant to antibiotics in traditional public baths}; Filali FR et al.; Three thermophilic bacteria strains, designated strain BS1, BS2 and BS3, resistant to beta-lactam antibiotics, and leaving at an optimal temperature for growth of about 50 degrees C, were isolated from traditional baths in Meknes-city in Morocco . Physiological and biochemical studies showed that these organisms belong to Gram positive Bacilli . They could not be identified with the Bergey's Manuel of Systematic Bacteriology (1986) . The dosage of beta-lactamase during the exponential growth phase has revealed that the strain BS3 produces a maximal amount of this enzyme . Studies aimed at determining the optimal conditions for incubation and growth have been performed in order to optimize the excretion of beta-lactamase by BS3 cells and thus facilitate the purification and and characterization of this enzyme.

Virology, 1998 Feb 1, 241(1), 61 - 72
Comparison of the lysogeny modules from the temperate Streptococcus thermophilus bacteriophages TP-J34 and Sfi21: implications for the modular theory of phage evolution; Neve H et al.; A 7.6-kb DNA segment covering the putative lysogeny module of the pac-site-containing temperate Streptococcus thermophilus bacteriophage TP-J34 was sequenced . Sequence alignment with the lysogeny module from the cos-site-containing S . thermophilus bacteriophage phiSfi21 revealed areas of high sequence conservation (e.g., over the int gene), interspersed with regions of low or no sequence similarity (e.g., over the cro gene) . Four of the six sharp transition zones from high to low sequence conservation were found within open reading frames coding for the CI repressor, the Anti-repressor, the Immunity protein, and a protein of unknown function . The transition points in the cI and ant genes appear to separate gene segments coding for distinct functional domains of these proteins . In addition, these two transition points were located at or near the deletion sites observed in spontaneous phage phiSfi21 deletion mutants, thus suggesting these transition points as recombinational hotspots . Furthermore, the sequence at the transition point in the cI gene resembles the attachment site of the phage, suggesting the involvement of the phage integrase in at least some of the exchange reactions . Contrary to the initial formulation of the modular theory of phage evolution the unit of the evolutionary exchange in streptococcal phages is not a group of functional genes, but can be as small as a single gene . Exchange reactions can also occur within genes, possibly between gene segments encoding distinct protein domains .

EMBO J, 1998 Jan 2, 17(1), 27 - 36
Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus; Beissinger M et al.; Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis . We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints . The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands . The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants . This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex . The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI . This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S . elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f . As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.

Biochem J, 1998 Jan 15, 329 ( Pt 2), 303 - 12
Multiple forms of DNA polymerase from the thermo-acidophilic eubacterium Bacillus acidocaldarius: purification, biochemical characterization and possible biological role; De Falco M et al.; Two DNA polymerase isoenzymes, called DpA and DpB on the basis of their elution order from DEAE cellulose, were purified to homogeneity from the thermo-acidophilic eubacterium Bacillus acidocaldarius . The enzymes are weakly acidophilic proteins constituted by a single subunit of 117 and 103 kDa respectively . DpA and DpB differ in thermostability, in thermophilicity, in sensitivity to assay conditions and in resistance to sulphydryl-group blocking agents such as N-ethylmaleimide and p-hydroxymercuriobenzoate . They differ also in synthetic template-primer utilization, in the apparent Km for dNTPs and in processivity . In particular, DpA utilizes more effic iently synthetic templates-primers such as poly(dA).poly(dT), poly(dT) . (rA)12-18 and poly(rA).(dT)12-18 and presents a greater tendency to accept dNTP analogues modified in the sugar or in the base ring, such as cytosine beta-d-arabinofuranoside 5'-triphosphate, 2',3'-dideoxyribonucleosides 5'-triphosphate, butylphenyl-dGTP and digoxigenin-conjugated dUTP . In addition, DpA presents an exonuclease activity that preferentially hydrolyses DNA in the 5'-3' direction, whereas DpB lacks this activity . The possible biological role of the enzymes is discussed.

Nucleic Acids Res, 1998 Jan 15, 26(2), 391 - 406
Similarities and differences among 105 members of the Int family of site-specific recombinases; Nunes-Duby SE et al.; Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences . In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold . With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre . Two structured regions also harbor some pronounced differences . The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions . The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures . The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins . The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed . In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution . These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).

J Cell Sci, 1998 Jan, 111 ( Pt 1), 131 - 40
Mutational analysis of regulated exocytosis in Tetrahymena; Melia SM et al.; Genetic analysis of regulated exocytosis can be accomplished in ciliates, since mutants defective in stimulus-dependent secretion of dense-core vesicles can be identified . In Tetrahymena thermophila, secretion in wild-type cells can result in their encapsulation by the proteins released from vesicle cores . Cells with defects in secretion were isolated from mutagenized homozygous cells that were generated using a highly efficient method . Screening was based both on a visual assay for encapsulation, and on a novel panning step using differential centrifugation to take advantage of the selective mobility of mutants that fail to encapsulate upon stimulation . 18 mutants with defects in several ordered steps have been identified . Defects in a set of these could be localized to three stages: granule formation, transport to cell surface docking sites, and exocytosis itself . Mutants with defects in this last stage can be ordered into successive steps based on several criteria, including their responsiveness to multiple secretagogues and Ca2+ ionophores . The results of both somatic and genetic complementation on selected pairs also help to characterize the defective factors.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 96 - 100
C-terminal regions of D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure; Kim GJ et al.; Most microbial D-hydantoinases have been reported to have catalytic properties similar to those of mammalian dihydropyrimidinases . Comparison of the primary structures of microbial D-hydantoinases with mammalian dihydropyrimidinases revealed that the amino acid homology is about 37% and functionally important residues are rigidly conserved at identical positions . Interestingly, however, the C-terminal regions were found to be completely mismatched with each other . In order to investigate the possible role of the C-terminal regions, we deleted the C-terminal regions of the D-hydantoinases from two thermophilic Bacilli and compared the catalytic and structural properties of the mutant enzymes with those of wild-type enzymes . As a result, the C-terminal region was found not to be essential for catalysis, but it does affect the oligomeric structure of the enzyme.

J Bacteriol, 1998 Feb, 180(4), 921 - 31
Molecular characterization of a phage-inducible middle promoter and its transcriptional activator from the lactococcal bacteriophage phi31; Walker SA et al.; An inducible middle promoter from the lactococcal bacteriophage phi31 was isolated previously by shotgun cloning an 888-bp fragment (P15A10) upstream of the beta-galactosidase (beta-Gal) gene (lacZ.st) from Streptococcus thermophilus (D . J . O'Sullivan, S . A . Walker, S . G . West, and T . R . Klaenhammer, Bio/Technology 14:82-87, 1996) . The promoter showed low levels of constitutive beta-Gal activity which could be induced two- to threefold over baseline levels after phage infection . During this study, the fragment was subcloned and characterized to identify a smaller, tightly regulated promoter fragment which allowed no beta-Gal activity until after phage infection . This fragment, defined within nucleotides 566 to 888 (P(566-888); also called fragment 566-888), contained tandem, phage-inducible transcription start sites at nucleotides 703 and 744 (703/744 start sites) . Consensus -10 regions were present upstream of both start sites, but no consensus -35 regions were identified for either start site . A transcriptional activator, encoded by an open reading frame (ORF2) upstream of the 703/744 start sites, was identified for P(566-888) . ORF2 activated P(566-888) when provided in trans in Escherichia coli . In addition, when combined with pTRK391 (P15A10::lacZ.st) in Lactococcus lactis NCK203, an antisense ORF2 construct was able to retard induction of the phage-inducible promoter as measured by beta-Gal activity levels . Finally, gel shift assays showed that ORF2 was able to bind to promoter fragment 566-888 . Deletion analysis of the region upstream from the tandem promoters identified a possible binding site for transcriptional activation of the phage promoters . The DNA-binding ability of ORF2 was eliminated upon deletion of part of this region, which lies centered approximately 35 bp upstream of start site 703 . Deletion analysis and mutagenesis studies also elucidated a critical region downstream of the 703/744 start sites, where mutagenesis resulted in a two- to threefold increase in beta-Gal activity . With these improvements, the level of expression achieved by an explosive-expression strategy was elevated from 3,000 to 11,000 beta-Gal units within 120 min after induction.

Ann N Y Acad Sci, 1997 Nov 21, 829, 326 - 40
First production-level bioremediation of explosives-contaminated soil in the United States; Emery DD et al.; Umatilla Army Depot Activity (UMDA) near Hermiston, Oregon was the location of the first production-level bioremediation of explosives-contaminated soil in the U.S . Soil from munitions washout lagoons contained high concentrations of TNT (2,4,6-Trinitrotoluene) and RDX (Hexahydro-1,3,5-trinitro-1,3,5- triazine) as well as HMX (Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) . In addition to these primary contaminants, laboratory tests were performed for Tetryl (Methyl-2,4,6-trinitrophenylnitramine), 4-Am-DNT (4-Amino-2,6-dinitrotoluene), 2-Am-DNT (2-Amino-4,6-dinitrotoluene), 2,4 DNT (2,4-Dinitrotoluene), 2,6 DNT (2,6-Dinitrotoluene), 1,3,5-TNB (1,3,5-Trinitrobenzene), 1,3,-DNB (1,3-Dinitrobenzene) and NB (Nitrobenzene) during the pilot-scale treatability tests . The clean-up goal established by the Record of Decision (ROD) was 30 mg/kg each for TNT and RDX . Degradation progress was monitored using immunoassay field screening Methods SW 846,4050 and 4051 . Confirmational analysis consisted of EPA Method 8330 . Treatment time on a 2,700 cubic yard batch (810 cubic yards of soil) was 10-12 days . A composting technique developed by the Army Environmental Center and implemented by Bioremediation Service, Inc., Portland, Oregon was used at the site . Agricultural waste products (or amendments including cow manure, chicken manure, potato waste, sawdust and alfalfa) were blended with the contaminated soil during treatment . Specialized soil turning equipment mixed the compost for optimum biological action and homogeneity . Homogeneity of the compost mix ensured rapid degradation of all contaminants . Physical and chemical properties were closely monitored to ensure that thermophilic bacteria played a dominant role in the degradation process . Nearly 5,000 cubic yards of soil have been successfully treated, and more than 70% of all analyses indicate non-detectable levels of both TNT and RDX . The U.S . Army Corps of Engineers estimates that over $2.6 million is being saved using bioremediation at Umatilla.

Arch Microbiol, 1997 Dec, 168(6), 536 - 9
Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus; Aalen N et al.; NADP+-specific glutamate dehydrogenase (EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic, strictly anaerobic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 . The native enzyme (263 kDa) is composed of subunits of mol . mass 46 kDa, suggesting a hexameric structure . The temperature optimum for enzyme activity was > 95 degrees C . The enzyme was highly thermostable, having a half-life of 140 min at 100 degrees C . Potassium phosphate, KCl, and NaCl enhanced the thermal stability and increased the rate of activity three- to fourfold . The N-terminal 26-amino-acid sequence showed a high degree of similarity to glutamate dehydrogenases from Pyrococcus spp . and Thermococcus spp.

Arch Microbiol, 1997 Dec, 168(6), 493 - 503
Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen . nov; Bodrossy L et al.; Two methanotrophic bacteria with optimum growth temperatures above 40 degrees C were isolated . Thermotolerant strain LK6 was isolated from agricultural soil, and the moderately thermophilic strain OR2 was isolated from the effluent of an underground hot spring . When compared to the described thermophilic methanotrophs Methylococcus capsulatus and Methylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus . However, their 16S rRNA gene sequences are markedly different from the sequence of Methylococcus thermophilus ( approximately 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the gamma-subgroup of the Proteobacteria related to extant Type I methanotrophs . Further phenotypic characterisation showed that the isolates possess particulate methane monooxygenase (pMMO) but do not contain soluble methane monooxygenase . The nucleotide sequence of a gene encoding pMMO (pmoA) was determined for both isolates and for Methylomonas gracilis . PmoA sequence comparisons confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously described Type I methanotrophs . We propose that strains OR2 and LK6, together with the misclassified thermophilic strains Methylomonas gracilis VKM-14LT and Methylococcus thermophilus IMV-B3122, comprise a new genus of thermophilic methanotrophs, Methylocaldum gen . nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile.

Arch Microbiol, 1997 Dec, 168(6), 473 - 9
Megaplasmids in Thermus oshimai isolates from two widely separated geographical areas: restriction fragment profiling and DNA homology; Moreira LM et al.; Megaplasmid DNA was detected in ten isolates belonging to the recently described thermophilic eubacterial species Thermus oshimai and isolated from hot springs in Portugal (eight isolates) and Iceland (two isolates) . The estimated size of the large plasmids purified from T . oshimai SPS-18 from S . Pedro do Sul, Portugal, and from isolate JK-91 from Hveragerdhi-Hengill, Iceland, was 214 and 275 kb, respectively . No sequence homologous to isolate SPS-18 megaplasmid is present in chromosomal DNA as indicated by Southern hybridization analysis . Overall examination of the HindIII fragment profiles of megaplasmid DNAs purified from isolates from the same geographical area gave similar but not always identical restriction profiles on agarose gels . Restriction fragment length polymorphism (RFLP) was higher for megaplasmids present in isolates purified from the Portuguese and Icelandic isolates than for megaplasmids from the same hot spring . Megaplasmid RFLP correlated with previous results obtained on the polymorphism of macrorestriction patterns of whole genomic DNA and with the RFLP of co-resident small plasmid DNA that was found in one half of the isolates examined . The 16-kb HindIII-HindIII fragment from isolate SPS-18 megaplasmid showed DNA-DNA homology with restriction fragments of similar size generated by the large plasmids present in all the other isolates, even in those from hot springs of widely separated geographical areas . This suggests a high degree of sequence conservation in T . oshimai megaplasmids.

Biochim Biophys Acta, 1998 Jan 8, 1379(1), 53 - 60
Tepidopterin, 1-O-(L-threo-biopterin-2'-yl)-beta-N-acetylglucosamine from Chlorobium tepidum; Cho SH et al.; A novel pterin compound, designated as tepidopterin, was detected from a thermophilic photosynthetic green sulphur bacterium, Chlorobium tepidum . The amount of tepidopterin inside the cell was estimated to be 2-5 micromol g(-1) dry weight of cell . This compound was purified through a high performance liquid chromatography using preparative DeltaPak C18 column . This compound was characterized by chromatographic behavior and by absorption and fluorescence properties . Its structure was determined to be 1-O-(L-threo-biopterin-2'-yl)-beta-N-acetylglucosamine by 1D- and 2D-NMR spectroscopy, mass spectrometry and CD . The relative amount of tetrahydrotepidopterin was estimated to be 96.7% inside the cell, that of dihydrotepidopterin 2.9%, and that of fully oxidized tepidopterin 0.4% . The amount of tepidopterin within the cell increased continuously until the beginning of the stationary phase of the cell growth.

Microbiology, 1998 Jan, 144 ( Pt 1), 167 - 75
Isoschizomers of the restriction endonuclease TaqI (T/CGA) requiring different metal ion concentrations and having a range of thermal stabilities from Thermus species from different continents; Welch SG et al.; One-hundred-and-fifty-two isolates of the genus Thermus, collected from hot springs on four continents, were screened for evidence of the presence of the thermophilic Type II restriction endonuclease TaqI (T/CGA) . The presence of isoschizomers of TaqI in 27 of the isolates, originating from hot springs in New Zealand, Iceland, USA, Japan, mainland Portugal and the island of Sao Miguel in the Azores, is reported . Six of the TaqI-containing isolates from diverse geographical locations, identified by means of DNA/DNA homology and 16S rRNA sequence alignment as belonging to the Thermus species T . aquaticus, T . filiformis, T . thermophilus, T . scotoductus and T . brockianus, were selected for comparative studies . The TaqI isoschizomers from each of the six isolates were partially purified . They differed in their magnesium ion requirements, isoelectric points, subunit molecular masses and thermal stability.

Biochemistry (Mosc), 1997 Nov, 62(11), 1196 - 201
The telomere and telomerase: nucleic acid-protein complexes acting in a telomere homeostasis system . A review; Blackburn EH; The tandemly repeated DNA sequence of telomeres is typically specified by the ribonucleoprotein enzyme telomerase . Telomerase copies part of its intrinsic RNA moiety to synthesize one strand of the telomeric repeat DNA Recent work, taken together with many observations over the past years, has led to the concept of a telomere homeostasis system . We have analyzed the interplay between two key physical components of this system: structural components of the telomere itself and of telomerase . Here we review some of these recent studies . The experimental method used in common in these studies was to make mutations in the template sequence of telomerase RNA, which caused various phenotypes . First, mutating specific residues in the ciliate Tetrahymena thermophila and yeast showed that these residues are required for critical aspects of the enzymatic action of telomerase . Second, certain mutated telomeric sequences caused a strong anaphase block in Tetrahymena micronuclei . Third, specific template mutations in the telomerase RNA gene led to varying degrees of telomere elongation in Tetrahymena and the yeast Kluyveromyces lactis . For some of the K . lactis mutations, the loss of length unregulated elongation was directly related to loss of binding to K . lactis Rap 1p protein . Using K . lactis carrying alterations in the telomerase RNA template, and in the gene encoding the Rap 1p protein, we found that a crucial determinant of telomere length homeostasis is the nature of the duplex DNA-Rap 1p protein complex on the very end repeat of the telomere . We propose that this complex plays a key role in regulating access of telomerase to the telomere.

J Biol Chem, 1998 Jan 9, 273(2), 865 - 70
ATP synthesis by F0F1-ATP synthase independent of noncatalytic nucleotide binding sites and insensitive to azide inhibition; Bald D et al.; ATP hydrolyzing activity of a mutant alpha3beta3gamma subcomplex of F0F1-ATP synthase (DeltaNC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E . , Honda, M., Allison, W . S., Dou, C., and Yoshida, M . (1997) J . Biol . Chem . 272, 8215-8221) . This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites . We reconstituted F0F1-ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis . Remarkably, DeltaNC F0F1-ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation . ATP synthesis by DeltaNC F0F1-ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form . The time course of ATP synthesis by DeltaNC F0F1-ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen . Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal . These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.

Biotechniques, 1997 Nov, 23(5), 904 - 6, 908, 910
Isolation of full-length RNA from a thermophilic cyanobacterium; Luo XZ et al.; Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR . It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation . Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium . The assay of M . laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods . The mRNA isolated by several conventional methods was completely degraded . A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press . This method produced high-quality, full-length mRNA in high yield . Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR . This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate.

FEBS Lett, 1997 Dec 29, 420(2-3), 139 - 42
Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation; Lechler A et al.; The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA(Phe) was studied . Removal of domain 8 of the beta subunit resulted in drastic reduction of the dissociation constant of the FRS x tRNA(Phe) complex . Neither the removal of arginine 2 of the beta subunit, which makes the only major contact between domains beta1-5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS . Thus, the body comprising domains 1-5 of the beta subunit may not be essential for efficient aminoacylation of tRNA(Phe) by the FRS and rather be involved in other functions.

Rocz Panstw Zakl Hig, 1997, 48(3), 263 - 8
{Action of physical agents on microorganisms}; Strus M; Among numerous physical agents exerting their deleterious effect on microorganisms only a few have been applied to sterilisation or disinfection used for medical purposes . Temperature is the most important agent, which from one side in a very wide range enables supporting of metabolic processes of psycho-, mezo- and thermophilic microorganisms, but beyond these limits causes their death . High temperature induces at first damage of cytoplasmic membrane and then denaturation of RNA leading to death . On the other hand, a low temperature slowly decreasing below 0 degree C induces crystallisation of water in cells and destruction of cytoplasmic structures . Ultraviolet radiation causes mutations resulting in stopping of DNA replication in all forms of the microorganisms . The same way of the lethal activity is exerted by ionising radiation . Its kinetic energy induces mutations affecting not single bases but also whole operons making gene expression impossible . Gaseous plasma is a new physical agent applied recently to sterilisation . High frequency energy initiates generation of the plasma from hydrogen peroxide vapours in a high vacuum and creates reactive species particles from the vapours that collide and kill microorganisms . On the other hand, application of ultrasound radiation to killing of microorganisms needs for further studies because of a high variability depending upon used frequency and energy . It is not known, for example, if destruction of microorganisms by ultrasounds is related to a phenomenon of cavitation or thermal energy . Nevertheless, even a range of frequency and energy used in commercial microwave ovens kills vegetative cells of coliform rods in about 15 minutes.

J Clin Microbiol, 1998 Jan, 36(1), 41 - 7
Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase; Poyart C et al.; We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S . acidominimus, S . agalactiae, S . alactolyticus, S . anginosus, S . bovis, S . constellatus, S . canis, S . cricetus, S . downei, S . dysgalactiae, S . equi subsp . equi, S . equi subsp . zooepidemicus, S . equinus, S . gordonii, S . iniae, S . intermedius, S . mitis, S . mutans, S . oralis, S . parasanguis, S . pneumoniae, S . porcinus, S . pyogenes, S . salivarius, S . sanguis, S . sobrinus, S . suis, S . thermophilus, and S . vestibularis) . Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences . We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species . We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

Protein Eng, 1997 Aug, 10(8), 905 - 14
Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri; Voorhorst WG et al.; The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J . and de Vos,W.M . (1996) J . Biol . Chem., 271, 20426-20431) . Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri . Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures . Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e . subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization . Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions . These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues . The latter are often present in putative N-glycosylation sites . Results from modelling of known substrates in the substrate-binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.

Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13487 - 92
B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli; Jordan A et al.; The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus . Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT . dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C . The TM and DR genes were cloned by PCR from peptide sequence information . The TM gene was sequenced completely and expressed in Escherichia coli . The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria . They can also be aligned to the sequence of the large protein of the aerobic E . coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12 . Structure determinations of the E . coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site . From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

Arch Latinoam Nutr, 1995 Sep, 45(3), 207 - 12
{Comparison of methods for the detection and enumeration of lactic acid bacteria in yogurt}; Briceno AG et al.; It is generally agreed that the population of lactic acid bacteria in yogurt must be not less than 10(6) ufc/g . Viability of the lactic flora until the end of shelf-life is affected for many factors, which has incidence in the recuperability of this microflora . MRS and LEE agar were selected for the total count of lactic bacteria, the M17 was used for the S salivarius ssp thermophilus and the RCA for L . delbrueckii ssp bulgaricus . Different methodologies were used for detection and enumeration of this bacteria: direct plate count; thermal treatment and recuperation of the injured cells in Soy Tripticase broth . The enumeration was done at time zero and 3 hours after the start of the fermentative process and during storage at 4, 12 and 21 days . The results shown an excellent recuperability of the lactic flora in the selected media and methods that were used: however the enumeration was significatively lower in the RCA agar . The counts in the LEE agar shown a better recuperability . The thermal treatment affected negatively the counts of lactic flora and the repair method shown better results in the yogurt sample during storage . pH and acidity were determined at the beginning and during the storage period . It was observed a pH decrease because of the lactic acid production at the end of shelf-life.

Mol Cell Probes, 1997 Oct, 11(5), 337 - 47
A rapid and sensitive method for non-isotopic quantitation of HIV-1 RNA using thermophilic SDA and flow cytometry; Mehrpouyan M et al.; Thermophilic strand displacement amplification (tSDA) is an isothermal DNA amplification technique that proceeds at 55-60 degrees C using both a thermostable restriction enzyme and a DNA polymerase . A modification of this system has been developed that allows the simultaneous amplification and detection of a DNA target by the addition of a detector probe to the reaction . This tSDA system has been further modified into a flow cytometry-based, bead capture assay for quantitation of HIV-1 RNA . A biotinylated capture probe and digoxygenin-dUTP have been incorporated into the tSDA reaction . The resulting double labelled amplicons are captured on strepavidin beads, and a fluorescent signal is generated on the beads by staining with fluorescent anti-digoxygenin antibody . The assay has a linear dynamic range of three orders of magnitude with a lower detection limit at 250 HIV-1 RNA molecules.

Biol Chem, 1997 Oct, 378(10), 1199 - 203
Thermal denaturation of human cystatin C and two of its variants; comparison to chicken cystatin; Zerovnik E et al.; Thermal denaturation of the recombinant human cystatin C, an 8-residue shorter variant (Leu-9 cystatin C), and the W106S mutant were measured using differential scanning calorimetry (DSC) . The finding that Leu-9 cystatin C is of similar stability to the full length protein is in accordance with its nearly normal inhibitory activity . The variant W106S cystatin C exhibits a higher melting temperature by 4 degrees than the wild-type protein . This contrasts with its reduced inhibitory activity and represents an example where activity changes are due to local effects and are not correlated to stability . From the ratio between Van't Hoff and calorimetric enthalpies it is judged that recombinant human cystatin C and Leu-9 cystatin C are dimeric prior to thermal unfolding whereas W106S cystatin C is monomeric . Melting temperatures and estimated stabilities for some other members of the cystatin superfamily of the cysteine proteinase inhibitors are presented which have been recorded previously or were collected for this study (chicken cystatin) . It is concluded that thermal stability of human cystatin C (Tm = 82 degrees C) is placed in between the more stable human stefin A (Tm = 95 degrees C) and the less stable human stefin B (Tm = 66 degrees C) whereas chicken cystatin behaves as a thermophilic protein, melting above 115 degrees C . To illustrate secondary structure changes, thermal denaturations of the recombinant human cystatin C and of W106S cystatin C were followed by circular dichroism in the far UV . It was found that the change in tertiary structure (revealed by DSC) precedes the major change in secondary structure.

Biochim Biophys Acta, 1997 Sep 12, 1353(3), 253 - 65
Cloning, sequencing, and expression of dnaK-operon proteins from the thermophilic bacterium Thermus thermophilus; Osipiuk J et al.; We propose that the dnaK operon of Thermus thermophilus HB8 is composed of three functionally linked genes: dnaK, grpE, and dnaJ . The dnaK and dnaJ gene products are most closely related to their cyanobacterial homologs . The DnaK protein sequence places T . thermophilus in the plastid Hsp70 subfamily . In contrast, the grpE translated sequence is most similar to GrpE from Clostridium acetobutylicum, a Gram-positive anaerobic bacterium . A single promoter region, with homology to the Escherichia coli consensus promoter sequences recognized by the sigma70 and sigma32 transcription factors, precedes the postulated operon . This promoter is heat-shock inducible . The dnaK mRNA level increased more than 30 times upon 10 min of heat shock (from 70 degrees C to 85 degrees C) . A strong transcription terminating sequence was found between the dnaK and grpE genes . The individual genes were cloned into pET expression vectors and the thermophilic proteins were overproduced at high levels in E . coli and purified to homogeneity . The recombinant T . thermophilus DnaK protein was shown to have a weak ATP-hydrolytic activity, with an optimum at 90 degrees C . The ATPase was stimulated by the presence of GrpE and DnaJ . Another open reading frame, coding for ClpB heat-shock protein, was found downstream of the dnaK operon.

Biochim Biophys Acta, 1997 Sep 12, 1353(3), 217 - 23
Cloning of the phytases from Emericella nidulans and the thermophilic fungus Talaromyces thermophilus; Pasamontes L et al.; Phytases (EC 3.1.3.8) belong to the family of histidine acid phosphatases . We have cloned the phytases of the fungi Emericella nidulans and Talaromyces thermophilus . The putative enzyme encoded by the E . nidulans sequence consists of 463 amino acids and has a Mr of 51785 . The protein deduced from the T . thermophilus sequence consists of 466 amino acids corresponding to a Mr of 51450 . Both predicted amino acid sequences exhibited high identity (48% to 67%) to known phytases . This high level of identity allowed the modelling of all available fungal phytases based on the three-dimensional structure coordinates of the Aspergillus niger phytase . By this approach we identified 21 amino acids which are conserved in fungal phyA phytases and are part of the residues forming the substrate pocket . Furthermore, potential glycosylation sites were identified and compared between the aforementioned phytases and the A . niger phytase.

Plant Mol Biol, 1997 Nov, 35(4), 407 - 16
Lumenal proteins involved in respiratory electron transport in the cyanobacterium Synechocystis sp . PCC6803; Manna P et al.; Cyanobacterial thylakoids catalyze both photosynthetic and respiratory activities . In a photosystem I-less Synechocystis sp . PCC 6803 strain, electrons generated by photosystem II appear to be utilized by cytochrome oxidase . To identify the lumenal electron carriers (plastocyanin and/or cytochromes c553, c550, and possibly cM) that are involved in transfer of photosystem II-generated electrons to the terminal oxidase, deletion constructs for genes coding for these components were introduced into a photosystem I-less Synechocystis sp . PCC 6803 strain, and electron flow out of photosystem II was monitored in resulting strains through chlorophyll fluorescence yields . Loss of cytochrome c553 or plastocyanin, but not of cytochrome c550, decreased the rate of electron flow out of photosystem II . Surprisingly, cytochrome cM could not be deleted in a photosystem I-less background strain, and also a double-deletion mutant lacking both plastocyanin and cytochrome c553 could not be obtained . Cytochrome cM has some homology with the cytochrome c-binding regions of the cytochrome Caa3-type cytochrome oxidase from Bacillus spp . and Thermus thermophilus . We suggest that cytochrome cM is a component of cytochrome oxidase in cyanobacteria that serves as redox intermediate between soluble electron carriers and the cytochrome aa3 complex, and that either plastocyanin or cytochrome c553 can shuttle electrons from the cytochrome b6f complex to cytochrome cM.

Appl Microbiol Biotechnol, 1997 Dec, 48(6), 709 - 13
Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass; Kadam KL et al.; A Saccharomyces-cerevisiae-based simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass is limited to an operating temperature of about 37 degrees C, and even a small increase in temperature can have a deleterious effect . This points to a need for a more thermotolerant yeast . To this end, S . cerevisiae D5A and a thermotolerant yeast, Candida acidothermophilum, were tested at 37 degrees C, 40 degrees C, and 42 degrees C using dilute-acid-pretreated poplar as substrate . At 40 degrees C, C . acidothermophilum produced 80% of the theoretical ethanol yield, which was higher than the yield from S . cerevisiae D5A at either 37 degrees C or 40 degrees C . At 42 degrees C, C . acidothermophilum showed a slight drop in performance . On the basis of preliminary estimates, SSF with C . acidothermophilum at 40 degrees C can reduce cellulase costs by about 16% . Proportionately greater savings can be realized at higher temperatures if such a high-temperature SSF is feasible . This demonstrates the advantage of using thermophilic or thermotolerant yeasts.

Biochemistry, 1998 Jan 27, 37(4), 1007 - 14
Mutations in the nucleotide binding domain of the alpha subunits of the F1-ATPase from thermophilic Bacillus PS3 that affect cross-talk between nucleotide binding sites; Grodsky NB et al.; Inactivation of MF1 (bovine mitochondrial F1-ATPase) with 5'-p-fluorosulfonylbenzoylethenoadenosine is caused by labeling alpha Y244 {Verburg, J . G., and Allison, W . S . (1990) J . Biol . Chem . 265, 8065-8074} . In the crystal structure {Abrahams, J.P., Leslie, A . G . W., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628}, alpha Y244 is hydrogen bonded to alpha R304 which is also hydrogen bonded to alpha Y300 . The catalytic properties of mutant alpha 3 beta 3 gamma subcomplexes of the TF1-ATPase from the thermophilic Bacillus PS3 containing the alpha F244C, alpha R304C, and alpha Y300C substitutions have been examined . Each has unique features for hydrolyzing ATP and forming inhibitory ADP-fluoroaluminate complexes in catalytic sites . Unlike wild-type, the (alpha R304C)3 beta 3 gamma and (alpha Y300C)3 beta 3 gamma subcomplexes entrap inhibitory MgADP in a catalytic site during turnover which fails to dissociate when ATP binds to noncatalytic sites . Although the hydrolytic properties of the (alpha F244C)3 beta 3 gamma subcomplex and wild-type are similar, the mutant forms ADP-fluoroaluminate complexes 7 times faster than wild-type when Al3+ and F- are added to it in the presence of excess ADP and Mg2+ . It also resists inhibition by high Mg2+ concentrations in the assay medium . At least one noncatalytic site of the (alpha F244C)3 beta 3 gamma subcomplex has increased affinity for ADP, indicating that the enhanced rate of formation of the ADP-fluoroaluminate complex reflects augmented cooperativity between noncatalytic and catalytic sites . The rate of formation of the ADP-fluoroaluminate complex in (alpha Y300C)3 beta 3 gamma increases only 40% when MgADP in bound to two catalytic sites rather than one, compared to a 9-fold increase exhibited by wild type . When Al3+ and F- are added to the (alpha Y300C)3 beta 3 gamma subcomplex after incubation with excess ADP and Mg2+, ADP-fluoroaluminate complexes are formed in three catalytic sites rather than two observed with the other subcomplexes . Reconciliation of the catalytic properties of the mutant subcomplexes in terms of the crystal structure suggests that alpha F244, alpha R304, and alpha Y300 of TF1 are part of a pathway that propagates conformational signals from one catalytic site to another.

Biochimie, 1997 Sep, 79(8), 523 - 6
Preparation of a 'beheaded' derivative of the 30S ribosomal subunit; Ulitin AB et al.; Oligodesoxyribonucleotide-directed cleavage of protein-deficient Thermus thermophilus derivatives of the 30S ribosomal subunits with RNase H is described . A homogeneous RNP fragment has been isolated as a result of the cleavage and subsequent purification in the sucrose gradient . It corresponds to the central and 5' domains of the 30S ribosomal subunit . The high compactness of the fragment in solution suggests that it can be considered as a 'beheaded' derivative of the 30S ribosomal subunit . The absence of a reconstitution stage in isolation of the 22S RNP fragment provides for its preparation in large amounts.

J Med Microbiol, 1998 Jan, 47(1), 5 - 16
Isolation, identification and increasing importance of 'free-living' amoebae causing human disease; Szenasi Z et al.; Amphizoic small amoebic protozoa are capable of existing both in 'free-living' and in 'parasitic' form depending on the actual conditions . Two genera (Naegleria and Acanthamoeba) have become recognised as opportunist human parasites . Since the first description in 1965 of a lethal case of primary amoebic meningoencephalitis (PAM) caused by Naegleria, many more (mostly lethal) cases have been reported, while granulomatous amoebic encephalitis (GAE), as well as eye (keratinitis, conjunctivitis, etc.), ear, nose, skin and internal organ infections caused by Acanthamoeba have also occurred in rapidly increasing numbers . Both pathogenic and non-pathogenic species of Naegleria and Acanthamoeba are found worldwide in water, soil and dust, where they provide a potential source of infection . Successful differential diagnosis and appropriate (specific) therapy depends on precise laboratory identification of the 'free-living' amoebae . In most cases, isolation from the environment can be achieved, but identification and differentiation of the pathogenic and non-pathogenic strains is not easy . The methods presently available do not fulfil completely the requirements for specificity, sensitivity and reliability . Morphological criteria are inadequate, while thermophilic character, pH dependency and even virulence in infected mice, are not unambiguous features of pathogenicity of the different strains . More promising are molecular methods, such as restriction endonuclease digestion of whole-cell DNA or mitochondrial DNA, as well as iso-enzyme profile analysis after iso-electric focusing and staining for acid phosphatase and propionyl esterase activity . Use of appropriate monoclonal antibodies has also yielded promising results in the differentiation of human pathogenic and non-pathogenic strains . However, quicker, simpler, more specific and reliable methods are still highly desirable . The significance of endosymbiosis (especially with Legionella strains) is not well understood . The results of a systematic survey in Hungary for the isolation and identification of 'free-living' amoebae, including an investigation of the Hungarian amoebic fauna, the isolation of possibly pathogenic Naegleria strains and of some Acanthamoeba strains from eye diseases, as well as the finding of a case of endosymbiosis, are also reported here.

J Appl Microbiol, 1997 Dec, 83(6), 685 - 92
Isolation and growth physiology of novel thermoactinomycetes; Yallop CA et al.; A total of 34 thermophilic isolates identified as members of the genus Thermoactinomyces by a range of chemotaxonomic, microscopic and determinative biochemical tests, were isolated from two acid soils . Growth studies in shake flask and fermenter identified the isolates to be moderately acidophilic with growth occurring between pH 4.5 and 6.0 with optima at pH 5.0 . The isolates differed considerably from known Thermoactinomyces cultures in their pH profile, colony morphology and in several biochemical tests . Extracellular enzyme activities are identified and partially characterized in terms of their thermostability, pH and temperature profiles from crude supernatant fluid samples . Optimal protease, amylase and pullulanase activity was observed at pH 5.0-5.5 and 75-80 degrees C with each showing T(50) values of 10, 30 and 30 min, respectively . A highly thermotolerant extracellular esterase was also identified which retained 50% activity after 8 h at 90 degrees C . This is the first report of an acidophilic member of the genus Thermoactinomyces.

J Biochem (Tokyo), 1997 Nov, 122(5), 1004 - 9
Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the CuA-cytochrome c domain from the thermophilic Bacillus caa3-type cytochrome c oxidase; Uchida A et al.; The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques . The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E . coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid . The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase . The chimeric subunit II was confirmed to bind to heme C . These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.

J Bacteriol, 1998 Jan, 180(2), 388 - 94
Biochemical and genetic characterization of an FK506-sensitive peptidyl prolyl cis-trans isomerase from a thermophilic archaeon, Methanococcus thermolithotrophicus; Furutani M et al.; A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus . The PPIase activity was inhibited by FK506 but not by cyclosporine . The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration . The enzyme was thermostable, with the half-lives of its activity at 90 and 100 degrees C being 90 and 30 min, respectively . The catalytic efficiencies (k(cat)/Km) measured at 15 degrees C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 microM(-1) s(-1), respectively, in chymotrypsin-coupled assays . The purified enzyme was sensitive to FK506 and therefore was called MTFK (M . thermolithotrophicus FK506-binding protein) . The MTFK gene (462 bp) was cloned from an M . thermolithotrophicus genomic library . The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea . The relationship between the thermostable nature of MTFK and its structure is discussed.

J Bacteriol, 1998 Jan, 180(2), 366 - 76
Novel division level bacterial diversity in a Yellowstone hot spring; Hugenholtz P et al.; A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S . M . Barns, R . E . Fundyga, M . W . Jeffries, and N . R . Page, Proc . Natl . Acad . Sci . USA 91:1609-1613, 1994) . Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned . Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined . These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences . A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions . The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions . Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria . The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction . Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers . Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial . These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments . Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

J Biol Chem, 1997 Dec 19, 272(51), 32294 - 300
Photoinactivation of the F1-ATPase from spinach chloroplasts by dequalinium is accompanied by derivatization of methionine beta183; Ren HM et al.; In contrast to the F1-ATPases from bovine mitochondria and the thermophilic Bacillus PS3, which are reversibly inhibited by dequalinium in the absence of irradiation, the Mg2+-ATPase activity of heat- or dithiothreitol-activated chloroplast F1 (CF1) from spinach chloroplasts is slightly stimulated by dequalinium . Conversely, dequalinium is a partial inhibitor (maximal inhibition is 85-90%) of the Ca2+-ATPase of CF1 activated by heat, dithiothreitol, or octylglucoside . The Mg2+- and Ca2+-ATPase activities of CF1 respond differently in the presence of lauryl dimethylamine oxide (LDAO) in the assay medium . Whereas the Mg2+-ATPase activity of heat- or dithiothreitol-activated CF1 is stimulated up to 14-fold by increasing concentrations of LDAO, the Ca2+-ATPase is inhibited in a biphasic manner by increasing concentrations of LDAO . In the presence of LDAO, dequalinium does not stimulate the heat-activated Mg2+-ATPase over that promoted by LDAO alone . That dequalinium slightly stimulates Mg2+-ATPase activity although it inhibits Ca2+-ATPase activity can be reconciled by assuming that dequalinium binds to two sites in CF1, a stimulatory site that also binds LDAO and an inhibitory site . By acting as a partial inhibitor of the Mg2+-ATPase activity that it activates, the combined effect of dequalinium is modest stimulation . Irradiation of heat- or dithiothreitol-activated CF1 or the alpha3beta3gamma subcomplex of CF1 in the presence of 12 microM dequalinium led to rapid photoinactivation . ATP and ADP, separately or in combination with Mg2+, protect against photoinactivation . After photoinactivating the alpha3beta3gamma subcomplex of CF1 with {14C}dequalinium, tryptic and peptic digests of the isolated, derivatized beta subunit were fractionated by high performance liquid chromatography . Sequencing of the isolated, radioactive tryptic and peptic peptides revealed that Metbeta183, which is at or near the catalytic site, is derivatized in a single beta subunit when CF1 is photoinactivated with {14C}dequalinium.

J Dairy Sci, 1997 Dec, 80(12), 3114 - 22
Manufacture and quality of iron-fortified yogurt; Hekmat S et al.; Yogurts (nonfat and low fat) were manufactured and fortified with 10, 20, and 40 mg of iron/kg of yogurt . Growth of starter culture bacteria and nonstarter culture bacteria as well as lipid oxidation of the yogurts were monitored over 30 d of storage at 4 degrees C . Sensory characteristics of the yogurts were determined during that time by a trained panel of judges, and consumer panels were used to test acceptability of iron-fortified yogurt . Counts of Lactobacillus delbrueckii ssp . bulgaricas and Streptococcus thermophilus after 1 d of storage in iron-fortified skim yogurts were 7.0 x 10(8) cfu/ml, which were not significantly different from numbers in unfortified yogurts . Counts decreased to 2.5 x 10(8) and 1.9 x 10(8) cfu/ml for L . delbrueckii ssp . bulgaricus and S . thermophilus, respectively, after 30 d of storage . Fortifying yogurt with iron did not support the growth of Pseudomonas fluorescens or Escherichia coli . No significant increases in chemical oxidation, as measured using the thiobarbituric acid assay, were detected as a consequence of iron fortification . Trained panelists scored all yogurts for oxidized, metallic, bitter, and other off-flavors in the range of "not perceptible" to "very slightly perceptible" . Iron-fortified yogurts had slightly higher oxidized flavor scores than did the control yogurt . There was no increase in metallic, bitter, or other off-flavors . The consumer panel did not detect any significant differences in the appearance, mouthfeel, flavor, or overall quality between fortified and unfortified flavored yogurts . All yogurt samples were liked by the consumer panelists, suggesting that yogurt is a suitable vehicle for iron fortification.

J Eukaryot Microbiol, 1997 Nov-Dec, 44(6), 535 - 9
Mini-exon gene sequences define six groups within the genus Crithidia; Fernandes O et al.; To develop molecular markers for lower trypanosmatids, we have examined the mini-exon gene repeats of 17 isolates that were classified as Crithidia by traditional methods . Representative repeats were amplified by polymerase chain reaction and the amplification products were cloned and used as hybridization probes against genomic DNA . Six hybridization groups of Crithidia were defined on the basis of the DNA blotting experiments . The three endosymbiont-bearing species (C . deanei, C . desouzai and C . oncopelti) and C . acanthocephali each belonged to single-member hybridization groups, while the C . fasciculata group contained additional named and undesignated species . The Crithidia lucilae thermophila probe hybridized to multiple undesignated isolates . The DNA sequence of the cloned products revealed that the specificity of the hybridization probes was due to substantial differences in the intron and the nontranscribed spacer regions . These data indicate substantial heterogeneity within the mini-exon gene locus of the taxon Crithidia.

Biochim Biophys Acta, 1997 Dec 5, 1343(2), 335 - 48
Cloning, characterization and functional analysis of groESL operon from thermophilic cyanobacterium Synechococcus vulcanus; Tanaka N et al.; Genes encoding 10914 Da and 58267 Da polypeptides homologous to groES and groEL of Escherichia coli were cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus . The deduced amino acid sequence of the GroEL protein was much more homologous to GroELs of other cyanobacteria which accompany GroES than another GroEL homolog of S . vulcanus (GroEL2) reported previously (M . Furuki, N . Tanaka, T . Hiyama, and H . Nakamoto, Biochim . Biophys . Acta 1294 (1996) 106-110) . We designate the gene as groEL1 to distinguish it from the non-operon forming groEL2 gene . A 9-base pair inverted repeat sequence (TTAGCACTC-N9-GAGTGCTAA) was located upstream of the promoter region of groEL1, which was absent in groEL2 . Southern blot analysis indicated that only one groESL1 operon was present in the genomic DNA of S . vulcanus . The amount of the bicistronic, 2.3 kb transcript of groESL1 operon increased 30-fold within 30 min upon heat shock . The increase was completely inhibited by chloramphenicol, suggesting the involvement of heat-induced production of a polypeptide . Introduction of the cloned groEL1 gene into a groEL defective mutant of E . coli resulted in the complementation of heat sensitivity, which contrasted with the previous result with groEL2.

Allergy Asthma Proc, 1997 Nov-Dec, 18(6), 355 - 7
Hypersensitivity pneumonitis due to inhalation of fungi-contaminated esparto dust in a plaster worker; Moreno-Ancillo A et al.; Hypersensitivity pneumonitis or extrinsic allergic alveolitis can be defined as a lung disease caused by a wide group of antigens that reach the lung by inhalation of organic and/or inorganic dust of various sources . The esparto (Stipa Tenacissima and Ligeum Spartum) is an herbaceous of the grass family used in the production of ropes, canvas, sandals, mats, baskets, and so forth . It is also used in the construction industry for the production of paper paste . Inhalation of esparto dust has been reported as cause of hypersensitivity pneumonitis . The existence of precipitating antibodies against esparto extract has been proved . During the esparto fiber manufacturing process, esparto grass can be contaminated by moulds and thermophilic actinomycetes, which have been described as the causing antigens of hypersensitivity pneumonitis in plaster workers . We present a case of occupational hypersensitivity pneumonitis in a plaster worker . Clinical findings, precipitating antibodies, and evolution, after having removed him from his work, confirmed the diagnosis . In our case, Aspergillus species contaminating esparto are probably the antigens that caused the disease.

J Muscle Res Cell Motil, 1997 Dec, 18(6), 697 - 709
Physical characterization and ATPase activity of 14S dynein fractions from Tetrahymena thermophila; Tharia HA et al.; Using anion-exchange fast protein liquid chromatography, 14S dynein was separated into four fractions (designated 1-4) . These fractions were distinguished with respect to polypeptide composition, and at least four unique heavy chains were identified . Each fraction was shown to exhibit ATPase activity . Fraction 2 has a specific activity 2-3 times greater than that of fractions 1, 3, and 4; the fractions showed a consistent trend of decreasing activity in the order 2 > 3 > 1 > 4 . In all cases, the specific ATPase activity was reduced by high ionic strength, in contrast to 22S dynein, which was previously shown to exhibit increased activity under identical conditions . Electron microscopy analysis revealed that the four fractions of 14S dynein were structurally distinct . Fraction 1 comprises two globular head domains interconnected via two stems; fraction 2 consists of at least two clearly different globular structures; fraction 3 is a single globular head; and fraction 4 comprises three globular head domains interconnected by three stems to a basal structure . Further structural characterization using hydrodynamic techniques enabled a determination of mass and sedimentation coefficient for each fraction . Fraction 1 had a mass of 654 kDa and a sedimentation coefficient of 20.1 S . Fraction 2 had a variable mass due to association (616-966 kDa), and a sedimentation coefficient of 16.6 S, whereas fractions 3 and 4 had variable sedimentation coefficients but were of mass 701 kDa and 527 kDa respectively . Where possible, hydrodynamic parameters were utilized, in conjunction with electron microscopy data, to construct low-resolution hydrodynamic bead models to represent the fractions . Optimal models, which were consistent with all the available data, were produced for fractions 1 and 4 . Bead modelling was also carried out for 22S dynein, using previously published data, to validate the 14S dynein modelling.

Eur J Biochem, 1997 Dec 1, 250(2), 332 - 41
Cytochrome ba3 from Natronobacterium pharaonis--an archaeal four-subunit cytochrome-c-type oxidase; Mattar S et al.; Cytochrome ba3, a terminal oxidase was isolated from the haloalkaliphilic archaeon Natronobacterium pharaonis . NH2-terminal sequence information of two subunits with apparent molecular masses of 40 and 36 kDa was used to generate a DNA probe by polymerase chain reaction . Cloning and sequencing of two overlapping genomic fragments revealed four genes forming a transcriptional unit . The policystronic messenger RNA of this cbaDBAC gene locus was identified by RNA analysis . The genes cbaC and cbaD code for small hydrophobic peptides with 81 and 54 amino acids . The genes cbaB and cbaA code for cytochrome oxidase subunit II (calculated molecular mass = 18.6 kDa) and I (calculated molecular mass = 63.8 kDa) respectively . Five potential CuA ligands for subunit II and six His residues for subunit I located in conserved positions indicate cytochrome ba3 to be a c-type oxidase . Sequence comparison and phylogenetic analysis place the natronobacterial enzyme together with the archaeal quinol oxidase SoxABCD from Sulfolobus acidocaldarius and the eubacterial ba3-type oxidase from Thermus thermophilus into a distinct evolutionary group . All three members are missing residues which are functionally important for vectorial proton translocation . The four-subunit enzyme complex was also identified on the protein level using chromatographic buffers containing ethylene glycol for purification.

Gene, 1997 Nov 20, 202(1-2), 83 - 8
The Tsp45I restriction-modification system is plasmid-borne within its thermophilic host; Wayne J et al.; Thermus species YS45 harbors two small cryptic plasmids of 5.8 (pTsp45s) and approximately 12 kb (pTsp45I) . Plasmid pTsp45s has been entirely sequenced, revealing three significant ORFs . In addition to a previously reported thermophilic plasmid-encoded replication protein (Rep), pTsp45s contains two genes for the Tsp45I methyltransferase (M.Tsp45I) and restriction endonuclease (Tsp45I) . These two converging genes (tsp45IM and tsp45IR) overlap by 4 bp at their stop codons within an XbaI site . M.Tsp45I (413 aa, 47.0 kDa, recognizing 5'-GTSAC-3') is highly homologous to other m6A-methyltransferases, especially M.EcaI (recognizing 5'-GGTNACC-3') . Tsp45I (332 aa, 37.4 kDa, cleaving 5'-/GTSAC-3') is not homologous to M.Tsp45I, or to other restriction endonucleases . Recombinant Tsp45I is stably produced in E . coli, and cleaves DNA at 65 degrees C with the same specificity as the native enzyme . Therefore, the thermophilic Tsp45I restriction-modification system is plasmid-borne within its native host.

Biol Chem, 1997 Nov, 378(11), 1313 - 29
Glutamyl-tRNA sythetase; Freist W et al.; Glutamyl-tRNA synthetase (GluRS) belongs to the class I aminoacyl-tRNA synthetases and shows several similarities with glutaminyl-tRNA synthetase concerning structure and catalytic properties . Phylogenetic studies suggested that both diverged from an ancestral glutamyl-tRNA synthetase responsible for the gluta-mylation of tRNA(Glu) and tRNA(Gln), and whose Glu-tRNA(Gln) product is transformed into Gln-tRNA(Gln) by a specific amidotransferase . This pathway is present in gram-positive and some gram-negative eubacteria, in some archae and in organelles, and was never found jointly with a glutaminyl-tRNA synthetase . Other gram-negative eubacteria and the cytoplasm of eukaryotes contain a glutamyl-tRNA synthetase specific for tRNA(Glu), and a glutaminyl-tRNA synthetase . Bacterial glutamyl-tRNA synthetases consist of about 500 amino acid residues, possess molecular masses of about 50 kDa, and act as monomers . In higher eukaryotes chimeric glutamyl-prolyl-tRNA synthetases were found, in a high molecular mass complex containing several other aminoacyl-tRNA synthetases . To date one crystal structure of a glutamyl-tRNA synthetase (Thermus thermophilus) has been solved . The molecule has the form of a bent cylinder and consists of four domains . The N-terminal half (domains 1 and 2) contains the 'Rossman fold' typical for class I synthetases and resembles the corresponding part of E . coli GlnRS, whereas the C-terminal half exhibits a GluRS-specific structure . As found for the other aminoacyl-tRNA synthetases the catalytic pathway of GluRS includes the formation of an aminoacyl adenylate in the first reaction step, but GluRS shares a special property with GlnRS and ArgRS: the ATP/PPi pyrophosphate exchange reaction is only catalyzed in the presence of the cognate tRNA . Compared with other aminoacyl-tRNA synthetases a relatively high number of investigations deals with recognition of tRNA(Glu) by GluRS . Besides interactions between the enzyme and the acceptor stem and the anticodon of tRNA(Glu), checking of the dihydrouridine arm and of the variable loop by GluRS are documented.

Protein Expr Purif, 1997 Dec, 11(3), 263 - 70
Thermostable tyrosine phenol-lyase of Symbiobacterium sp . SC-1: gene cloning, sequence determination, and overproduction in Escherichia coli; Lee SG et al.; During the screening for tyrosine phenol-lyase-producing thermophiles, we isolated an obligatory symbiotic thermophile, Symbiobacterium sp . SC-1, which grew only in coculture with Bacillus sp . SK-1 . A gene encoding thermostable tyrosine phenol-lyase (TPL) was cloned from the genomic DNA of the Symbiobacterium sp . SC-1 and the nucleotide sequence of the TPL structural gene was determined . The gene consists of 1374 base pairs encoding a polypeptide of 458 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 52,196 Da . The structural gene of TPL was amplified by PCR, blunt-ended, and ligated into the NcoI-HindIII site of plasmid pTrc99A to construct an expression vector for the overproduction of the thermostable TPL . The level of thermostable TPL production was about 15% of the total soluble proteins of Escherichia coli extract . The enzyme was purified to homogeneity from the E . coli extract with an overall yield of 48%.

Biochem Biophys Res Commun, 1997 Dec 18, 241(2), 565 - 9
The novel genes, cbbQ and cbbO, located downstream from the RubisCO genes of Pseudomonas hydrogenothermophila, affect the conformational states and activity of RubisCO; Hayashi NR et al.; The cbbQ and cbbO genes are located downstream from the RubisCO genes (cbbLS) in the thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila . Recombinant RubisCO enzymes were purified from E . coli cells which were transformed with plasmids expressing cbbLS, cbbLSQ, cbbLSO, or cbbLSQO . Co-expression of cbbQ and/or cbbO with cbbLS made the maximal rates of carboxylation (Vmax) of the recombinant RubisCOs about two-fold higher than that of the enzyme derived from only cbbLS . The RubisCOs with high Vmax also had a high stability when undergoing ultrasonic treatment . The results of the circular dichroism spectra and the 8-anilino-1-naphthalenesulfonate binding assay indicated that these recombinant RubisCOs were conformationally different to each other.

J Biotechnol, 1997 Oct 17, 58(2), 71 - 8
High level production of a cellulase-free xylanase in glucose-limited fed batch cultures of a thermophilic Bacillus strain; Samain E et al.; Bacillus sp . strain XE and its mutant derivative strain D3 produce a thermostable xylanase which is suitable for enzymatic bleaching of kraft pulp . Xylanase synthesis was shown to be induced by the soluble products of xylan hydrolysis (xylooligosaccharide) and equally, catabolically-repressed when these oligosaccharides accumulate in the medium . An optimal balance between these two antagonistic effects was obtained in a carbon-limited, fed-batch culture with continuous xylooligosaccharide feeding . To reduce substrate cost, the xylooligosaccharide could be 90% substituted by glucose without reduction of the xylanase production rate . Xylanase production ceased when the activity reached approximately 380 U ml-1 due to an amino acid shortage . A continuous supply of exogenous amino acids allowed the production to continue to more than 1000 U ml-1.

Mol Mar Biol Biotechnol, 1997 Dec, 6(4), 296 - 307
Cathepsin, a major protease of the marine sponge Geodia cydonium: purification of the enzyme and molecular cloning of cDNA; Krasko A et al.; Sponges are suspension-feeders that are devoid of body cavities . Phagocytosis is the major route of nutrition in these animals . In an attempt to understand protein digestion, cathepsin was identified in crude extracts from the sponge Geodia cydonium . This enzyme was purified from lysosomes by a two-step procedure--pH precipitation and FPLC separation--to apparent homogeneity; it showed an M(r) of 26,000 . Inhibitor as well as substrate studies showed that the sponge cathepsin belongs to the subfamily L of these cysteine proteases . The complete cDNA coding for cathepsin L was isolated and characterized . The deduced aa sequence contains 322 residues, has an M(r) of 36,085, and shows the characteristic signatures known from other cathepsins of the L subfamily: e.g., cleavage site for the proregion, the ERFNIN motif, and the conserved regions forming the catalytic triad of cysteine proteases . Phylogenetic analyses revealed that the sponge sequence groups with the cathepsin L subfamily and branches off first from the other metazoan members . The sponge sequence shows high homology to that isolated from Dictyostelium discoideum and only low similarity to the protozoan cathepsins L from Paramecium tetraurelia and Tetrahymena thermophila . From the data presented it is concluded that cathepsin L is the major digestive protease in sponges.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 73 - 9
Molecular cloning and sequence analysis of the lysR gene from the extremely thermophilic eubacterium, Thermus thermophilus HB27; Kosuge T et al.; We have isolated a lysine-auxotrophic and kanamycin-resistant mutant from an extreme thermophile, Thermus thermophilus HB27 . This mutant showed the lysA- or lysR- genotype since it could not grow on the minimal plate which contained diaminopimelic acid . Sequence analysis of the clones which could rescue the Lys- mutant indicated the lysR gene . The lysR gene overlapped with the rimK gene for the modification enzyme of ribosomal protein S6 . In the Lys- mutant, the lysR gene was disrupted and the C-terminus region of the RimK protein was different from that of the wild-type, which contributed to the Lys- and kanamycin-resistant phenotype . The deduced amino acid sequence of the lysR gene showed 20.9% identity with the LysR protein of Escherichia coli . The percentage of use of cytosine or guanine in the third letter of the codons in the lysR gene was only 67.4% . We also determined that the argC gene encoding N-acetyl-gamma-glutamyl phosphate reductase and the argB gene encoding acetylglutamate kinase were located immediately upstream of the lysR gene.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 39 - 45
Role of the COOH-terminal pro-sequence of aqualysin I (a heat-stable serine protease) in its extracellular secretion by Thermus thermophilus; Kim DW et al.; Aqualysin I is a subtilisin-type serine protease secreted into the medium by Thermus aquaticus YT-1 . Thermus thermophilus cells harboring a plasmid for the aqualysin I precursor secreted pro-aqualysin I with the C-terminal pro-sequence into the culture medium, and the precursor was then processed to the mature enzyme during the cultivation . However, the extracellular levels of aqualysin I in T . thermophilus cells harboring plasmids for deletion mutants as to the C-terminal pro-sequence were about 10-20% in comparison with the level of wild-type . Only the mature enzyme could be detected in the medium, while pro-aqualysin I with the C-terminal pro-sequence could not . These results suggest that the C-terminal pro-sequence of aqualysin I plays an important role in the extracellular secretion of aqualysin I.

Lett Appl Microbiol, 1997 Nov, 25(5), 345 - 8
Cell-wall protein profiles of dairy thermophilic lactobacilli; Gatti M et al.; SDS-PAGE fingerprinting of cell-wall proteins extracted from 119 strains belonging to different species of lactic acid bacteria have been compared . The method of extraction and electrophoretic separation utilized in this work was found to be a reliable and rapid way for characterizing thermophilic lactobacilli species and strains . A protein of approximately 50 kDa was found to be characteristic for all the Lact . helveticus strains, and two cell-wall proteins of about 20 and 30 kDa were typical of the species Lact . delbrueckii, but the discrimination between the subspecies lactis and bulgaricus was not possible by the electrophoretic technique used . The other thermophilic species studied in this work presented cell-wall protein patterns that permitted their differentiation from both Lact . helveticus and Lact . delbrueckii.

J Appl Microbiol, 1997 Nov, 83(5), 641 - 51
Detection of Campylobacter jejuni in food and poultry viscera using immunomagnetic separation and microtitre hybridization; Lamoureux M et al.; Thermophillic Campylobacter and Camp . jejuni were detected from samples of chicken liver, gall bladder, muscle and contaminated milk and chicken meat after an enrichment step by using immunomagnetic capture of cells with monoclonal antibody against a specific outer membrane protein of thermophilic Campylobacter . The detection of captured cells was achieved using two different hybridization methods . In one of the methods, the captured cells were lysed by guanidine isothiocyanate and the 23S rRNA was reacted with a microtitre plate-immobilized rDNA probe specific for thermophilic Campylobacter . In the other method, the captured cells were subjected to lysis by ultrasonication and the genomic DNA reacted with a microtitre plate-immobilized RNA probe specific for Camp.jejuni . Detection of the RNA-DNA hybrids formed in the wells was carried out using a monoclonal anti-RNA-DNA hybrid antibody.

J Appl Microbiol, 1997 Nov, 83(5), 619 - 26
The prevalence of campylobacters and arcobacters in broiler chickens; Atabay HI et al.; Chicken carcasses from a supermarket and from a poultry abattoir were examined using methods designed to isolate as many strains of campylobacters and related organisms as possible . Strains of arcobacter, but no campylobacters, were isolated from every carcass after enrichment . Campylobacter jejuni subsp . jejuni was isolated from all carcasses examined by direct plating and other Campylobacter-like strains were isolated from nine out of 15 abattoir carcasses by direct plating but not after enrichment . Only the Camp . jejuni subsp . jejuni strains could be identified to species level using a readily available identification scheme and/or a commercial identification kit . Examination of caecal contents from the 15 abattoir poultry yielded Camp . jejuni subsp . jejuni and Campylobacter-like strains from 15 and eight by direct plating, and from six and nine after enrichment, respectively . Four sites in the intestine of the abattoir birds (60 samples) were examined for arcobacters and only one strain was isolated . This indicates that arcobacters are probably not normal inhabitants of the poultry intestine . Poultry is a rich source of other campylobacteria besides the thermophilic Campylobacter spp.

Protein Sci, 1997 Dec, 6(12), 2659 - 62
Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold; Buss KA et al.; The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer . Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP . The crystals diffract to beyond 1.7 A resolution . Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees . Diffraction data have been collected from the crystals at 110 and 277 K . Data collected at 277 K extend to lower resolution, but are more reproducible . The orientation of a noncrystallographic two-fold axis of symmetry has been determined . Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.

J Cell Biol, 1997 Dec 1, 139(5), 1197 - 207
In vivo analysis of the major exocytosis-sensitive phosphoprotein in Tetrahymena; Chilcoat ND et al.; Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism . A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism . In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity . The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules . Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion . To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo . The unique T . thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications . Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity . We evaluated the activity and function of PGM1 through gene disruption . Surprisingly, DeltaPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity . Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

Plant Physiol, 1997 Dec, 115(4), 1473 - 80
Thermal protection of the oxygen-evolving machinery by PsbU, an extrinsic protein of photosystem II, in Synechococcus species PCC 7002; Nishiyama Y et al.; The evolution of oxygen is the reaction that is the most susceptible to heat in photosynthesis . We showed previously that, in the cyanobacterium Synechococcus sp . PCC 7002, some protein factors located on the thylakoid membranes are involved in the stabilization of this reaction against heat-induced inactivation, and we identified cytochrome C550 as one such factor (Y . Nishiyama, H . Hayashi, T . Watanabe, N . Murata {1994} Plant Physiol 105: 1313-1319) . In the present study we purified another protein that appears to be essential for the stabilization of the oxygen-evolving machinery . The purified protein had an apparent molecular mass of 13 kD, and the gene encoding the 13-kD protein was cloned from Synechococcus sp . PCC 7002 and sequenced . The deduced amino acid sequence revealed that the protein was homologous to PsbU, an extrinsic protein of the photosystem II complex, which has been found in thermophilic species of cyanobacteria . Western analysis showed that the level of PsbU in thylakoid membranes was constant, regardless of the growth temperature . Our studies indicate that PsbU, a constituent of the photosystem II complex, protects the oxygen-evolving machinery against heat-induced inactivation.

Cell Mol Life Sci, 1997 Oct, 53(10), 830 - 41
Psychrophilic enzymes: molecular basis of cold adaptation; Feller G et al.; Psychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures . At the enzymatic level, such organisms have to cope with the reduction of chemical reaction rates induced by low temperatures in order to maintain adequate metabolic fluxes . Thermal compensation in cold-adapted enzymes is reached through improved turnover number and catalytic efficiency . This optimization of the catalytic parameters can originate from a highly flexible structure which provides enhanced abilities to undergo conformational changes during catalysis . Thermal instability of cold-adapted enzymes is therefore regarded as a consequence of their conformational flexibility . A survey of the psychrophilic enzymes studied so far reveals only minor alterations of the primary structure when compared to mesophilic or thermophilic homologues . However, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in order to increase their flexibility.

Zentralbl Hyg Umweltmed, 1996 Nov, 199(1), 38 - 50
{Emission of odors from composting of biological waste}; Pohle H et al.; Results of qualitative and quantitative detection of odour substances during the process of composting of biowaste are shown . Olfactometric measurements were carried out beside detection of odour substances by gaschromatography--mass spectroscopy . Some odour relevant substances were set in relation to odorant concentration . 158 volatile substances could be identified by GC/MS . The occurrence of odour substances demonstrated a characteristic dynamic . As a consequence of analysis of odour substances, detection of odorant concentration and odour quality the composting process could be divided in 3 main phases . For the start phase substances from anaerobic degradation like alcoholes and esters of carbonic acids played an important role in odour generation . Sulfur containing compounds dominated the characteristic odour during thermophilic phase of composting process . The importance of ammonia during later composting process could be shown . The concentration of dimethyl disulfide and limonene was set in relation to odorant concentration and both substances are discussed as indicator compounds.

Gene, 1997 Nov 12, 201(1-2), 63 - 8
Cloning and identification of the Sulfolobus solfataricus lrp gene encoding an archaeal homologue of the eubacterial leucine-responsive global transcriptional regulator Lrp; Charlier D et al.; The lrp gene of the extreme thermophilic archaeon Sulfolofus solfataricus, encoding a homologue of the eubacterial global leucine-responsive regulatory protein, was identified by DNA sequencing and sequence comparisons on a 6.9-kb genomic fragment cloned into Escherichia coli . The S . solfataricus Lrp subunit is a 155-aa polypeptide that bears between 24.5 and 29% sequence identity with eubacterial regulatory proteins of the Lrp/AsnC family and 30.6% and 25.8% with the archaeal homologues of respectively Methanococcus jannaschii and Pyrococcus furiosus . Transcription initiation from the strong S . solfataricus lrp promoter was analyzed by primer extension mapping . The abundance of the S . solfataricus lrp messenger strongly suggests that this protein might function in archaea as a global transcriptional regulator and genome organizer, as proposed for E . coli Lrp, rather than as a local, specific regulatory protein . Our findings suggest the presence of a eubacterial type of regulatory mechanism in archaea, a situation that is noteworthy indeed, since the transcriptional machinery of archaea is more closely related to that of eukaryotes, whereas these latter apparently do not possess a homologue of Lrp.

Biochim Biophys Acta, 1997 Oct 9, 1354(1), 35 - 9
Cloning and sequence of a type I pullulanase from an extremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus; Albertson GD et al.; A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli . It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95,732 Da and is flanked by two other open reading frames . A truncated version of the gene which lacks 381 bp of 5'-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability . Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.

Mol Cell Biol, 1997 Dec, 17(12), 7237 - 47
Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila; Blomberg P et al.; We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila . The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA . These are derived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth . We have developed a transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule . Using constructs containing progressively longer deletions, we show that only a small portion (approximately 900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule . This core region is very near but does not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription . It includes two nearly identical nuclease-sensitive domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously . Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required . In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.

J Clin Lab Anal, 1997, 11(6), 323 - 7
Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase, incorporating uracil N glycosylase (UNG) in a single tube reaction; Poddar SK et al.; An optimized reaction condition for amplification of influenza A virus RNA, by thermus thermophilus (Tth) DNA polymerase-based PCR, incorporating uracil N glycosylase (UNG) and dUTP in the reaction has been determined . dUTP could not be substituted for all dTTP sites when UNG was present in the reaction . The relative concentration of dUTP and dTTP has been optimized for allowing amplification of the target RNA . It has been verified that the amplified product DNA had sufficient dUTP and was digestable by UNG . Using the optimized reaction condition, influenza A virus-specific DNA fragment could be amplified and detected in 15 of 15 culture positive (for influenza A virus) nasopharyngeal specimens.

Comp Biochem Physiol A Physiol, 1997 Nov, 118(3), 463 - 73
Molecular physiology of carbamoylation under extreme conditions: what can we learn from extreme thermophilic microorganisms?
Van de Casteele M, Legrain C, Desmarez L, Chen PG, Pierard A, Glansdorff N.
The importance of protein-protein interactions in the physiology of extreme thermophiles was investigated by analyzing the enzymes involved in biosynthetic carbamoylation in Thermus ZO5 and by comparing the results obtained with already available or as yet unpublished information concerning other thermophilic eu- and archaebacteria such as Thermotoga, Sulfolobus, and Pyrococcus . Salient observations were that (i) the highly thermolabile and reactive carbamoylphosphate molecule appears to be protected from thermodegradation by channelling towards the synthesis of citrulline and carbamoylaspartate, respectively precursors of arginine and the pyrimidines; (ii) Thermus ornithine carbamoyltransferase is clearly a thermophilic enzyme, intrinsically thermostable and showing a biphasic Arrhenius plot, whereas aspartate carbamoyltransferase is inherently unstable and is stabilized by its association with dihydroorotase, another enzyme encoded by the Thermus pyrimidine operon . Possible implications of these results are discussed.

Comp Biochem Physiol A Physiol, 1997 Nov, 118(3), 439 - 51
Sequence and structural comparison of thermophilic phosphoglycerate kinases with a mesophilic equivalent; Fleming T et al.; The monomeric glycolytic enzyme phosphoglycerate kinase (PGK) has been used as a model system to study protein thermostability . The primary sequence of this enzyme has been elucidated from 47 species to date . Although only 42 amino acids are totally conserved, most of which line the active site cleft, the protein is structurally conserved . This is achieved by making conservative changes to maintain the same secondary and tertiary folds . The crystal structures of 5 PGK enzymes have been solved by X-ray diffraction methods . This paper seeks to use the available information to understand protein thermostability . Although some general mechanisms to increase stability can be determined, different species have adopted a variety of subtle additive changes to achieve greater protein stability . Comparisons have been directly made between the PGK enzyme from yeast, the moderate thermophilic bacterium Bacillus stearothermophilus, the hyperthermophilic bacteria Thermus thermophilus, Thermotoga maritima, and the hyperthermophilic archaea Sulpholobus solfataricus and Methanothermus fervidus.

Comp Biochem Physiol A Physiol, 1997 Nov, 118(3), 429 - 38
Thermophilic proteins: stability and function in aqueous and organic solvents; Cowan DA; The molecular stability of thermophilic and hyperthermophilic enzymes generally reflects the growth temperatures of the parent organisms . Extracellular enzymes from the hyperthermophilic Archaea typically show very high levels of thermal stability and a number of enzymes with Tm values of greater than 100 degrees C have been reported . The mechanisms responsible for high molecular stability are typically intrinsic characteristics of the protein, as shown by the comparative stabilities of many native and recombinant proteins . However, some extrinsic stabilisation mechanisms have been demonstrated . High levels of thermal stability are positively correlated with stability in the presence of other denaturing agents, including detergents and organic solvents . This correlation suggests a common denaturation pathway where molecular mobility/flexibility is the prime determinant of susceptibility to irreversible denaturation . In single phase organic-aqueous solvents, protein destabilisation occurs via solvent-induced alteration to the protein hydration shell . However, correlations between protein stability and solvent hydrophobicity are unreliable . In two-phase organic-aqueous systems, interfacial denaturation predominates and is a function of both interfacial tension and interfacial surface area . Intracellular enzymes are protected from interfacial denaturation but are potentially susceptible to direct organic solvent effects, possibly depending on the role of the cell wall and cell membrane in the partitioning of the organic solvent into the cell cytoplasm . Immobilisation of thermophilic enzymes provides a method for enhancing both the thermal and solvent stabilities of thermophilic and mesophilic enzymes . Multi-point covalent immobilisation to glyoxal-agarose enhances thermal stability and limits protein-protein inactivation mechanisms . Miscible organic solvents have a profound influence on the specificities of enzyme reactions . The presence of high concentrations of miscible organic solvents may induce gross changes in substrate specificity and/or more subtle alterations in chiral selectivity . Correlations between the variation in enantioselectivity and both solvent hydrophobicity and solvent dielectric constant have been demonstrated although some recent studies implicate the formation of specific solvent-enzyme complexes which directly affect reaction kinetics.

Comp Biochem Physiol A Physiol, 1997 Nov, 118(3), 423 - 8
Adaptation of microorganisms and their transport systems to high temperatures; Tolner B et al.; Growth of Bacteria and Archaea has been observed at temperatures up to 95 and 110 degrees C, respectively . These thermophiles are adapted to environments of high temperature by changes in the membrane lipid composition, higher thermostabilities of the (membrane) proteins, higher turnover rates of the energy transducing enzymes, and/or the (exclusive) use of sodium-ions rather than protons as coupling ion in energy transduction . The proton permeability of the cytoplasmic membrane of bacteria and archaea was observed to increase with the temperature . This increased proton permeability limits the maximum temperature of growth of bacteria . Higher growth temperatures can be reached by an increased proton pumping activity by using the less permeable sodium ions as coupling ions or by changing the lipid composition of the cytoplasmic membrane . The Na+/H+/glutamate transport proteins of the thermophiles Bacillus stearothermophilus (GltTBs) and Bacillus caldotenax (GltTBc) were studied extensively . These transportproteins have unique features . Transport of L-glutamate occurs in symport with 1 Na+ and 1 H+ when the transport proteins are expressed in their natural environment . The sodium ion dependency of the GltT transporters of these Bacillus strains was found to increase with temperature . However, when the GltT proteins are expressed in the mesophile Escherichia coli, electrogenic symport of L-glutamate occurs with > or = 2 H+ . These observations suggest that the conformation of the transport proteins in the E . coli and the Bacillus membranes differs, and that the conformation influences the coupling ion selectivity . The Na+/H+/glutamate transport proteins of B . stearothermophilus (GltTBs) and B . caldotenax (GltTBc) are homologous to transport systems of glutamate and structurally related compounds from mesophilic organisms . Both sodium, as well as proton coupled transporters, belong to this family of carboxylate transporters (FCT).

J Bacteriol, 1997 Dec, 179(24), 7803 - 11
Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum; Schleper C et al.; Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments . Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution . Here we report on the first protein to be characterized from one of these organisms . The DNA polymerase gene of C . symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig . Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases . It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively) . The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence . The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags . It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate . Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity . The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C . It was found to be less thermostable than polymerase I of E . coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes . Although phylogenetic studies suggest a thermophilic ancestry for C . symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.

J Bacteriol, 1997 Dec, 179(24), 7718 - 23
Characterization of nicotinamide mononucleotide adenylyltransferase from thermophilic archaea; Raffaelli N et al.; The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the synthesis of NAD+ and nicotinic acid adenine dinucleotide . It has been purified to homogeneity from cellular extracts of the thermophilic archaeon Sulfolobus solfataricus . Through a database search, a highly significant match was found between its N-terminal sequence and a hypothetical protein coded by the thermophilic archaeon Methanococcus jannaschii MJ0541 open reading frame (GenBank accession no . U67503) . The MJ0541 gene was isolated, cloned into a T7-based vector, and expressed in Escherichia coli cells, yielding a high level of thermophilic NMN adenylyltransferase activity . The expressed protein was purified to homogeneity by a single-step chromatographic procedure . Both the subunit molecular mass and the N-terminal sequence of the pure recombinant protein were as expected from the deduced amino acid sequence of the MJ0541 open reading frame-encoded protein . Molecular and kinetic properties of the enzymes from both archaea are reported and compared with those already known for the mesophilic eukaryotic NMN adenylyltransferase.

J Bacteriol, 1997 Dec, 179(24), 7712 - 7
Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila; Rasche ME et al.; Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A) . Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J . Henkin and R . H . Abeles, Biochemistry 15:3472-3479, 1976) . Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues . All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined . The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme . Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide . Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis . Five arginines were individually replaced with glutamine . Kinetic analysis of the altered enzymes identified R310 as essential for activity . Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.

J Bacteriol, 1997 Dec, 179(24), 7625 - 30
Nucleoid structure and distribution in thermophilic Archaea; Poplawski A et al.; Nucleoid structure and distribution in thermophilic organisms from the Archaea domain were studied . Combined phase-contrast and fluorescence microscopy of DAPI (4',6-diamidino-2-phenylindole)-stained Sulfolobus acidocaldarius and Sulfolobus solfataricus cells revealed that the nucleoids were highly structured . Different nucleoid distribution within the cells, representing different partition stages, was observed . The conformation of the nucleoids differed between exponentially growing and stationary-phase cells . Also, the stationary-phase cells contained two chromosomes, and the nucleoids occupied a larger part of the interior of the cells than in the exponentially growing cells . The part of the cell cycle during which fully separated nucleoids could be detected was short . Since the postreplication period is long in these organisms, there was a considerable time interval between termination of chromosome replication and completion of nucleoid separation, similar to the G2 phase in eukaryotic cells . The length of the visible cell constriction period was found to be in the same range as that of eubacteria . Finally, cell-cell connections were observed under certain conditions . Possible eubacterial, eukaryotic, and unique features of nucleoid processing and cell division in thermophilic archaea are discussed.

J Biochem (Tokyo), 1997 Oct, 122(4), 764 - 71
A novel cytochrome b(o/a)3-type oxidase from Bacillus stearothermophilus catalyzes cytochrome c-551 oxidation; Sakamoto J et al.; Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain . To identify alternative oxidases, we isolated several mutants from B . stearothermophilus defective in the caa3-type oxidase activity {Sakamoto, J . et al (1996) FEMS Microbiol . Lett . 143, 151-158} . A novel oxidase was isolated from membrane preparations of one of the mutants, K17 . The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme O, heme A, and Cu in a ratio of 1:0.7:0.2:3 . CO difference spectra indicate that the high-spin heme is mainly heme O . These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A . The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus . The turnover rate of the activity (Vmax = 190 s{-1}) and its affinity for cytochrome c-551 (Km = 0.15 microM) were much higher than those for yeast and equine heart cytochromes c . The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a Ki value of 19 microM . The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase . Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the heme-copper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.

Biochim Biophys Acta, 1997 Nov 1, 1354(2), 116 - 26
Expression of Tetrahymena histone H4 in yeast; Fogel GB et al.; Histone H4 is one of the most conserved proteins known . The very low rate of nonsynonymous substitution in H4 suggests that it fulfills an essential function in virtually all eukaryotes . While the majority of histone H4 sequences differ only slightly from the general consensus H4 sequence, yeast and Tetrahymena sequences diverge substantially from both the consensus and from each other . This study demonstrates that despite this divergence, when Saccharomyces cerevisiae cells are forced to use the Tetrahymena thermophila histone H4 protein, they are viable although they have a reduced growth rate, are temperature-sensitive relative to wild-type, have a lengthened G2 phase, and show a dramatic repression of mating . An amino acid replacement at position 33 in the protein improves the growth rate of these cells growing at temperatures above 28 degrees C . This replacement changes a proline to a serine and is a further divergence from both the Tetrahymena thermophila and Saccharomyces cerevisiae histone H4 sequences . Thus, the replacement and expression of a non wild-type histone H4 in yeast offers measurable effects on cell growth, identifying amino acids required for optimal yeast functioning.

J Bacteriol, 1997 Dec, 179(23), 7456 - 61
Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus; Delle Fratte S et al.; Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate . No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor . These modified folates are not readily available for use in assays for SHMT activity . This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus . The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay . The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S . solfataricus . Replacement of the modified folate with tetrahydrofolate did not support serine synthesis . In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate . The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs . The enzyme contains covalently bound pyridoxal phosphate . Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.

Biochem Biophys Res Commun, 1997 Nov 17, 240(2), 247 - 56
Intramolecular rotation in ATP synthase: dynamic and crystallographic studies on thermophilic F1; Kagawa Y et al.; A single molecule of ATP synthase (F0F1) is by itself a rotary motor, the smallest ever found, and this biomotor is driven by an electrochemical potential of H+ (delta microH+) . F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1) . The major breakthroughs in studies on the mechanochemical coupling have been the direct observation of the rotation of a stable alpha 3 beta 3 gamma complex of thermophilic F1 (TF1), and X-ray crystallography of the alpha 3 beta 3 gamma portion of mitochondrial F1 (MF1) and the alpha 3 beta 3 oligomer of TF1 . This review focuses on the dynamics of TF1, demonstrated by a crucial experiment . The torque of the rotation was estimated to be 42 pN.nm from the delta microH+ and frictional force . Important unsolved problems are the crystallography of F0, elastic energy conversion, and the stator and rotor of this biomotor.

Microbiology, 1997 Nov, 143 ( Pt 11), 3417 - 29
Sequence analysis and characterization of phi O1205, a temperate bacteriophage infecting Streptococcus thermophilus CNRZ1205; Stanley E et al.; The complete nucleotide sequence of phi O1205, a temperate bacteriophage infecting Streptococcus thermophilus strain CNRZ1205, was determined . The phage genome has a unit length of 43,075 bp and appears to be packaged by the so-called headful mechanism . The genomic organization and structure of phi O1205 resemble those of several temperate lactococcal phages that display a life-cycle-specific organization, where ORFs believed to be involved in the lysogenic life-cycle are clustered and arranged in an orientation opposite to the ORFs supposedly involved in the lytic life-cycle . Database searches revealed putative functions for several identified ORFs and further indicated that phi O1205 is genetically related to a particular group of lactococcal phages . Three genes encoding the major structural proteins were identified on the phi O1205 genome . The phage attachment site attP, the bacterial attachment site attB, and the two phage/chromosome junctions attL and attR were identified and found to contain a 40 bp common core sequence.

J Bacteriol, 1997 Nov, 179(22), 7135 - 55
Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics; Smith DR et al.; The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach . A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions . A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases . Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea . Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M . thermoautotrophicum ORFs encode sequences that are >50% identical to M . jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes . When the M . thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences . The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment . Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences . Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins . There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M . jannaschii . DNA replication initiation and chromosome packaging in M . thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation . The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides . The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location . There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters . Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location . There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids . The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M . jannaschii genome.

Eur J Biochem, 1997 Oct 15, 249(2), 443 - 9
Cloning and characterization of the arginine-specific carbamoyl-phosphate synthetase from Bacillus stearothermophilus; Yang H et al.; Bacillus stearothermophilus contains two carbamoyl-phosphate synthetases (CPS), one specific for pyrimidine biosynthesis and the other for arginine biosynthesis . The pyrimidine-specific CPS is repressed by exogenous pyrimidines, and its activity is inhibited by UMP and activated by 5-phospho-alpha-D-ribosyl diphosphate . The arginine-specific CPS is similarly repressed by exogenous arginine but its activity is not sensitive to these or other potential effectors . Each of the two enzymes consist of two unequal subunits, as is the case for other microbial CPS; however, the large subunit for the arginine-specific CPS is smaller than that for the pyrimidine-specific enzyme . Comparison of the derived amino acid sequence for the cloned large subunit of the arginine-specific CPS with those for subunits from pyrimidine-sensitive CPS showed significant similarity throughout the polypeptides except at the carboxy terminus, which was identified by other laboratories to contain the binding site for the pyrimidine effector . Unlike the results previously reported for CPS from an enteric mesophile, the kinetic properties of the arginine-specific CPS were not affected by growth of B . stearothermophilus at temperatures near the minimal growth temperature . Furthermore, calorimetric studies showed that the thermal stability of cloned CPS was identical regardless of the growth temperature of B . stearothermophilus between 42 degrees C and 63 degrees C . The thermal stability of cloned CPS was not affected by expression at 37 C in Bacillus subtilis or Escherichia coli . In contrast, the thermal stabilities for CPS and other proteins were higher in extracts of cells grown at higher temperatures . These results indicate that cellular factors, probably chaperonins, are necessary for thermal stability of proteins at and below the optimal temperature for this thermophile.

J Mol Biol, 1997 Oct 10, 272(5), 741 - 69
Photosystem I of Synechococcus elongatus at 4 A resolution: comprehensive structure analysis; Schubert WD et al.; An improved structural model of the photosystem I complex from the thermophilic cyanobacterium Synechococcus elongatus is described at 4 A resolution . This represents the most complete model of a photosystem presently available, uniting both a photosynthetic reaction centre domain and a core antenna system . Most constituent elements of the electron transfer system have been located and their relative centre-to-centre distances determined at an accuracy of approximately 1 A . These include three pseudosymmetric pairs of Chla and three iron-sulphur centres, FX, FA and FB . The first pair, a Chla dimer, has been assigned to the primary electron donor P700 . One or both Chla of the second pair, eC2 and eC'2, presumably functionally link P700 to the corresponding Chla of the third pair, eC3 and eC'3, which is assumed to constitute the spectroscopically-identified primary electron acceptor(s), A0, of PSI . A likely location of the subsequent phylloquinone electron acceptor, QK, in relation to the properties of the spectroscopically identified electron acceptor A1 is discussed . The positions of a total of 89 Chla, 83 of which constitute the core antenna system, are presented . The maximal centre-to-centre distance between antenna Chla is < or = 16 A; 81 Chla are grouped into four clusters comprising 21, 23, 17 and 20 Chla, respectively . Two "connecting" Chla are positioned to structurally (and possibly functionally) link the Chla of the core antenna to those of the electron transfer system . Thus the second and third Chla pairs of the electron transfer system may have a dual function both in energy transfer and electron transport . A total of 34 transmembrane and nine surface alpha-helices have been identified and assigned to the 11 subunits of the PSI complex . The connectivity of the nine C-terminal (seven transmembrane, two "surface") alpha-helices of each of the large core subunits PsaA and PsaB is described . The assignment of the amino acid sequence to the transmembrane alpha-helices is proposed and likely residues involved in co-ordinating the Chla of the electron transfer system discussed.

Biochem Biophys Res Commun, 1997 Oct 29, 239(3), 810 - 5
Cloning and sequencing of aspartate aminotransferase from Thermus aquaticus YT1; O'Farrell PA et al.; A 39-base oligonucleotide "guessmer" probe, based on partial N-terminal sequence analysis of the aspartate aminotransferase purified from Thermus aquaticus strain YT1, was used to screen a genomic library prepared from T . aquaticus DNA . A 1842 bp DNA fragment was isolated that proved to contain the coding sequence for the aspartate aminotransferase . The gene is 1152 bases long and codes for a protein of 383 amino acid residues . The amino acid sequence obtained showed 88.7%, 45.1% and 32.9% identity of sequence with those of thermostable aspartate aminotransferases from T . thermophilus, Bacillus YM2, and Sulfolobus solfataricus, respectively . It showed 39.1% identity with one of the gene products tentatively identified as aspartate aminotransferase from the methanogenic archaebacterium Methanococcus jannaschii . Neither the amino acid compositions nor the aligned amino acid sequences provides any obvious clue as to the origin of thermal stability in this group of enzymes.

Proteins, 1997 Nov, 29(3), 309 - 20
Insights into thermal resistance of proteins from the intrinsic stability of their alpha-helices; Petukhov M et al.; To investigate the role of alpha helices in protein thermostability, we compared energy characteristics of alpha helices from thermophilic and mesophilic proteins belonging to four protein families of known three-dimensional structure, for at least one member of each family . The changes in intrinsic free energy of alpha-helix formation were estimated using the statistical mechanical theory for describing helix/coil transitions in peptide helices {Munoz, V., Serrano, L . Nature Struc . Biol . 1:399-409, 1994; Munoz, V., Serrano, L . J . Mol . Biol . 245:275-296, 1995; Munoz, V., Serrano, L . J . Mol . Biol . 245:297-308, 1995} . Based on known sequences of mesophilic and thermophilic RecA proteins we found that (1) a high stability of alpha helices is necessary but is not a sufficient condition for thermostability of RecA proteins, (2) the total helix stability, rather than that of individual helices, is the factor determining protein thermostability, and (3) two facets of intrahelical interactions, the intrinsic helical propensities of amino acids and the side chain-side chain interactions, are the main contributors to protein thermostability . Similar analysis applied to families of L-lactate dehydrogenases, seryl-tRNA synthetases, and aspartate amino transferases led us to conclude that an enhanced total stability of alpha helices is a general feature of many thermophilic proteins . The magnitude of the observed decrease in intrinsic free energy on alpha-helix formation of several thermoresistant proteins was found to be sufficient to explain the experimentally determined increase of their thermostability . Free energies of intrahelical interactions of different RecA proteins calculated at three temperatures that are thought to be close to its normal environmental conditions were found to be approximately equal . This indicates that certain flexibility of RecA protein structure is an essential factor for protein function . All RecA proteins analyzed fell into three temperature-dependent classes of similar alpha-helix stability (delta G(int) = 45.0 +/- 2.0 kcal/mol) . These classes were consistent with the natural origin of the proteins . Based on the sequences of protein alpha helices with optimized arrangement of stabilizing interactions, a natural reserve of RecA protein thermoresistance was estimated to be sufficient for conformational stability of the protein at nearly 200 degrees C.

Biosci Biotechnol Biochem, 1997 Oct, 61(10), 1710 - 7
Cloning of genes of the aminopeptidase T family from Thermus thermophilus HB8 and Bacillus stearothermophilus NCIB8924: apparent similarity to the leucyl aminopeptidase family; Motoshima H et al.; To obtain genes with sequence similarity to aminopeptidase T (AP-T) of Thermus aquaticus YT-1, we cloned the genes encoding aminopeptidase Th (AP-Th) from Thermus thermophilus HB8 and aminopeptidase II (APII) from Bacillus stearothermophilus NCIB8924 . The AP-Th gene encoded a polypeptide of 408 amino acid residues and the deduced molecular weight of this subunit was 45,015 . The APII gene encoded a polypeptide of 413 amino acid residues with a deduced molecular weight of 46,207 . The extent of amino acid sequence similarity between AP-Th and AP-T was 86%, and that between APII and AP-T was 43% . The substrate specificities of these expressed enzymes were similar, and each efficiently hydrolyzed leucyl- or phenyl-peptide substrates . Since the deduced amino acid sequence of these enzymes show no similarity to other known aminopeptidases, they appear to comprise an independent family of peptidases, designated the AP-T family . However, a conserved region within the enzymes of the AP-T family shows similarity to the active site signature of the leucyl aminopeptidase family, suggesting that these enzymes may belong to the leucyl aminopeptidase superfamily.

Appl Environ Microbiol, 1997 Nov, 63(11), 4593 - 6
Application of the extracellular alpha-amylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains; Satoh E et al.; Streptococcus thermophilus ATCC 19258, Lactobacillus delbrueckii subsp . bulgaricus T-11, and Lactococcus lactis subsp . lactis IL1403 were transformed with the alpha-amylase gene (amyA) from Streptococcus bovis 148 by using a wide host-range vector, and all the transformants secreted the alpha-amylase successfully . Since the promoter and the secretion signal of the amyA gene were functional in these strains, we constructed a secretion vector using the expression elements of amyA . Trials to secrete foreign enzymes in yogurt starter strains were performed using this novel secretion vector.

Biochemistry (Mosc), 1997 Aug, 62(8), 883 - 9
Site-specific endonuclease from thermophilic Bacillus species MK strain is isoschizomer of SalI; Kerzhner MA et al.; Screening of thermophilic bacterial strains revealed a strain containing site-specific endonuclease BspMKI . This endonuclease was purified to functional homogeneity during sequential chromatographic steps . The enzyme recognizes sequence 5'-G decreases TCGAC-3' on DNA molecule and is isoschizomer of endonuclease SalI . The molecular mass of BspMKI is about 45 kD . The enzyme is maximally active at 55 degrees C and MRB (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol) is the optimal buffer . The enzyme is highly stable and retains its activity during two weeks at room temperature.

Gene, 1997 Oct 15, 199(1-2), 77 - 82
Cloning and characterization of the uvrD gene from an extremely thermophilic bacterium, Thermus thermophilus HB8; Hiramatsu Y et al.; The uvrD gene encodes a DNA helicase which plays an important role in prokaryotic nucleotide (nt) excision repair, mismatch repair and DNA replication . A cosmid-based genomic DNA library for Thermus thermophilus (Tt) HB8 was constructed, and this was screened by Southern hybridization using a uvrD fragment amplified by PCR as the probe . The nt sequence of cloned Tt uvrD was then determined . Characteristic helicase motifs, made up of seven elements, were all conserved in the amino acid (aa) sequence of Tt UvrD . The aa sequence showed 41% homology with that of Escherichia coli (Ec) . In the aa composition of Tt UvrD, the number of Asn, Gln, Met and Cys residues was decreased, and the number of Pro residues was increased . The distribution of Pro residues and recent data on X-ray crystallographic structure suggested the importance of the structural dynamics of the protein . These changes are thought to stabilize the native protein conformation against heat denaturation . Tt uvrD complemented the UV sensitivity of a Ec uvrD mutant . Thus, the thermophilic bacterium has a UvrD helicase, whose function is common to Ec UvrD.

J Mol Biol, 1997 Oct 31, 273(3), 635 - 45
Structural and functional characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the hyperthermophilic archaeum Thermococcus strain KS-1; Yoshida T et al.; To elucidate the function of group II chaperonin, the gene for the chaperonin from the hyperthermophilic archaeum Thermococcus strain KS-1 was cloned and sequenced . Two distinct genes coding for chaperonin subunits, designated alpha and beta, were obtained, and their deduced amino acid sequences are highly homologous to those of group II chaperonins from other sources . The alpha and beta subunits were individually expressed in Escherichia coli . Both of the recombinant subunits assemble to constitute the homo-oligomeric double-ring complexes, which are prone to form large aggregates . The alpha aggregate is dissociated into the typical chaperonin ring complex by incubation in buffer containing 15% (v/v) methanol, while the beta aggregate cannot be dissociated . At high temperature, both of the recombinant complexes have weak ATPase activities . They are able to arrest refolding of a chemically denatured thermophilic enzyme in the absence of ATP, and refolding is resumed when ATP is supplemented . These results suggest that homo-oligomeric complexes of the archaeal chaperonin have activity .

J Biochem (Tokyo), 1997 Jun, 121(6), 1031 - 4
Screening of a mutant plasmid with high expression efficiency of GC-rich leuB gene of an extreme thermophile, Thermus thermophilus, in Escherichia coli; Suzuki T et al.; A mutant plasmid with elevated expression efficiency of GC-rich Thermus thermophilus leuB gene was screened in Escherichia coli . A wild-type plasmid pHB2 carrying T . thermophilus leuB gene was introduced into leuB-deficient E . coli C600 cells . During successive cultures of the transformant in leucine-free medium, the original plasmid was spontaneously replaced by a mutant plasmid . The expression efficiency of the leuB gene on the mutant plasmid was 4.8-fold higher than that of the wild-type plasmid . Sequencing of the mutant plasmid revealed that the open reading frame (ORF1) in front of the leuB gene was shortened from 822 to 306 bp . Several expression vectors were constructed to investigate the effect of the length of ORF1, and the optimal length for the expression of the following leuB gene was determined . It was also shown that the stop codon of ORF1 should be overlapped with the initiation codon of leuB gene for the highest efficiency.

J Appl Microbiol, 1997 Oct, 83(4), 508 - 17
The influence of cell surface properties of thermophilic streptococci on attachment to stainless steel; Flint SH et al.; The quality of milk products is threatened by the formation of biofilms of thermophilic streptococci on the internal surfaces of plate heat exchangers used in milk processing . Although attachment to stainless steel surfaces is one of the first stages in the development of a biofilm, the mechanisms involved in attachment have not been reported . The cell surface properties of 12 strains of thermophilic streptococci were examined to determine their importance in attachment to stainless steel surfaces . Hydrophobicity, extracellular polysaccharide production and cell surface charge varied between the different strains but could not be related to numbers attaching . Treating the cells with sodium metaperiodate, lysozyme or trichloroacetic acid to disrupt cell surface polysaccharide had no effect on attachment . Treatment with trypsin or sodium dodecyl sulphate to remove cell surface proteins resulted in a 100-fold reduction in the number of bacteria attaching . This result suggests that the surface proteins of the thermophilic streptococci are important in their attachment to stainless steel.

J Appl Microbiol, 1997 Sep, 83(3), 335 - 9
Use of the Malthus conductance growth analyser to determine numbers of thermophilic streptococci on stainless steel; Flint SH et al.; The use of the Malthus conductance growth analyser for the detection of Streptococcus bovis attached to stainless steel surface was evaluated . A comparison between the results from acridine orange epifluorescence direct counts, swab recovery viable count and conductance estimates of attached cell concentrations, based on calibrations for planktonic cells, showed that the conductance results were up to 2 log10 greater than the epifluorescence results and the swab counts . The growth rates of planktonic and attached cells were similar over 16 h using the Malthus technique . This suggests that the Malthus technique detects more attached cells of Strep . bovis than epifluorescence microscopy or swab recovery.

FEBS Lett, 1997 Sep 29, 415(2), 155 - 9
Preliminary NMR studies of Thermus thermophilus ribosomal protein S19 overproduced in Escherichia coli; Davydova NL et al.; The gene for the ribosomal protein S19 from Thermus thermophilus was cloned, sequenced and overexpressed in Escherichia coli . A simple procedure for isolating the recombinant protein was developed . Preliminary NMR studies revealed a high content of alpha-helical secondary structure in the protein.

Biochemistry, 1997 Oct 14, 36(41), 12477 - 85
Guanosine binds to the Tetrahymena ribozyme in more than one step, and its 2'-OH and the nonbridging pro-Sp phosphoryl oxygen at the cleavage site are required for productive docking; Profenno LA et al.; The dynamics of binding of various guanosine, or G, substrates to the Tetrahymena thermophila L-21 ScaI ribozyme have been investigated by fluorescence-detected stopped-flow experiments . Upon rapid mixing of various G substrates with a preformed complex of the ribozyme and the fluorescent 5' splice site analogue CCUCUepsilonA, fluorescence transients that provide rates for binding of G substrates and the rate-limiting step for transesterification are observed . The measured apparent bimolecular rate constant for binding of pG is 10(3) M-1 s-1, much slower than expected for diffusion . pG appears to bind to the preformed complex of the ribozyme and CCUCUepsilonA in at least two steps, a bimolecular step followed by at least one conformational change . This two-step binding of pG, involving a rapid pre-equilibrium, leads to the slow apparent rate constant for binding of pG . Furthermore, the 2'-OH of pG and of the 3' terminal G of the G substrate GUCG and the nonbridging pro-Sp phosphoryl oxygen atom at the site of phosphoryl transfer on CCUCUepsilonA appear to mediate formation of a properly conformed docked ternary complex of the G substrate, 5' splice site, and ribozyme which may represent an intermediate required for initiation of transesterification . It is possible that the 2'-OH of pG and this nonbridging pro-Sp phosphoryl oxygen interact, directly or indirectly, with one another.

Eur J Biochem, 1997 Sep 1, 248(2), 466 - 74
Ornithine carbamoyltransferase from the extreme thermophile Thermus thermophilus--analysis of the gene and characterisation of the protein; Sanchez R et al.; The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a lambda-ZAP genomic library . An ORF of 903 bp was found coding for a protein of Mr 33,200 . The coding region has a very high overall G+C content of 68.0% . T . thermophilus OTC displays 38-48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus furiosus and from Bacillus subtilis . The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on delta-N-phosphonoacetyl-L-ornithine-Sepharose . The native enzyme has an Mr of about 110,000, suggesting a trimeric structure, as for most anabolic OTC from various organisms . T . thermophilus OTC exhibits Michaelis-Menten kinetics for carbamoyl phosphate and ornithine with a Km(app) of 0.10 mM for both substrates . The pH optimum was dependent on ornithine concentration with an optimum at pH 8 for ornithine concentrations around Km values . Higher concentrations shift the optimum towards lower pH . The optimal temperature was above 65 degrees C and the activation energy 39.1 kJ/mol . The enzyme is highly thermostable . In the presence of its substrates the half-life time was several hours at 85 degrees C . Ionic and hydrophobic interactions contribute to the stability . The expression of T . thermophilus OTC was negatively regulated by arginine.

Virology, 1997 Oct 13, 237(1), 148 - 58
The site-specific integration system of the temperate Streptococcus thermophilus bacteriophage phiSfi21; Bruttin A et al.; The temperate bacteriophage phiSfi21 integrates its DNA into the chromosome of Streptococcus thermophilus strains via site-specific recombination . Nucleotide sequencing of the attachment sites identified a 40-bp identity region which surprisingly overlaps both the 18-terminal bp of the phage integrase gene and the 11-terminal bp of a host tRNAArg gene . A 2.4-kb phage DNA segment, covering attP, the phage integrase, and a likely immunity gene contained all the genetic information for faithful integration of a nonreplicative plasmid into the attB site . A deletion within the int gene led to the loss of integration proficiency . A number of spontaneous deletions were observed in plasmids containing the 2.4-kb phage DNA segment . The deletion sites were localized to the tRNA side of the identity region and to phage or vector DNA with 3- to 6-bp-long repeats from the border region . A similar type of deletion was previously observed in a spontaneous phage mutant .

Appl Environ Microbiol, 1997 Oct, 63(10), 3902 - 10
Genes encoding two different beta-glucosidases of Thermoanaerobacter brockii are clustered in a common operon; Breves R et al.; A 5.9-kb fragment of chromosomal DNA coding for beta-glucosidase activity of the thermophilic anaerobe Thermoanaerobacter brockii was sequenced . Two genes, cglT and xglS, encoding a cellodextrin-cleaving beta-glucosidase and a xylodextrin-degrading xylo-beta-glucosidase, respectively, were located directly adjacent to each other . The 5' region contained two additional genes, cglF and cglG, whose products exhibited similarity to integral membrane proteins of metabolite transport systems . The two beta-glucosidases, CglT and XglS, with deduced molecular masses of 52 and 81 kDa, belong to different families of glycosyl hydrolases . Both enzymes were overexpressed in Escherichia coli and could be detected after protein gel electrophoresis and activity staining . The enzyme CglT was purified by fast protein liquid chromatography and identified by N-terminal sequencing . The enzyme was thermostable at 60 degrees C for at least 24 h, and the temperature optimum was 75 degrees C . The ki for glucose inhibition was calculated to 200 mM . The enzyme released glucose from the nonreducing end of beta-1,4-cello oligomers as well as from various disaccharides . CglT was active on glucosides, galactosides and on fucosides, while XglS cleaved beta-glucosides and beta-xylosides as well . The cglT gene was also expressed in Bacillus subtilis, and the enzyme was mainly intracellular during exponential growth but was efficiently released into the supernatant after cultures entered the stationary phase.

Appl Environ Microbiol, 1997 Oct, 63(10), 3810 - 7
Streptococcus thermophilus and its biosurfactants inhibit adhesion by Candida spp . on silicone rubber; Busscher HJ et al.; The adhesion of yeasts, two Candida albicans and two Candida tropicalis strains isolated from naturally colonized voice prostheses, to silicone rubber with and without a salivary conditioning film in the absence and presence of adhering Streptococcus thermophilus B, a biosurfactant-releasing dairy isolate, was studied . Coverage of 1 to 4% of the surface of silicone rubber substrata with adhering S . thermophilus B gave significant reductions in the initial yeast adhesion regardless of the presence of a conditioning film . Mechanistically, this interference in yeast adhesion by S . thermophilus B was not due to direct physical effects but to biosurfactant release by the adhering bacteria, because experiments with S . thermophilus B cells that had released their biosurfactants prior to adhesion to silicone rubber and competition with yeasts did not show interference with initial yeast adhesion . The amounts of biosurfactants released were highest for mid-exponential- and early-stationary-phase bacteria (37 mg.g of cells-1 {dry weight}), but biosurfactants released by stationary-phase bacteria (14 mg.g of cells-1 {dry weight}) were the most surface active . The crude biosurfactants released were mixtures of various components, with a glycolipid-like component being the most surface active . A lipid-enriched biosurfactant fraction reduced the surface tension of an aqueous solution to about 35 mJ.m-2 at a concentration of only 0.5 mg.ml-1 . The amount of biosurfactant released per S . thermophilus B cell was estimated to be sufficient to cover approximately 12 times the area of the cross section of the bacterium, making biosurfactant release a powerful defense weapon in the postadhesion competition of the bacterium with microorganisms such as yeasts . Preadsorption of biosurfactants to the silicone rubber prior to allowing yeasts to adhere was as effective against C . albicans GB 1/2 adhesion as covering 1 to 2% of the silicone rubber surface with adhering S . thermophilus B, but a preadsorbed biosurfactant layer was less effective against C . tropicalis GB 9/9.

Mol Cell Biol, 1997 Nov, 17(11), 6394 - 401
Reprogramming of telomerase by expression of mutant telomerase RNA template in human cells leads to altered telomeres that correlate with reduced cell viability; Marusic L et al.; Telomerase synthesizes telomeric DNA by copying the template sequence of its own RNA component . In Tetrahymena thermophila and yeast (G . Yu, J . D . Bradley, L . D . Attardi, and E . H . Blackburn, Nature 344:126-131, 1990; M . McEachern and E . H . Blackburn, Nature 376:403-409, 1995), mutations in the template domain of this RNA result in synthesis of mutant telomeres and in impaired cell growth and survival . We have investigated whether mutant telomerase affects the proliferative potential and viability of immortal human cells . Plasmids encoding mutant or wild-type template RNAs (hTRs) of human telomerase and the neomycin resistance gene were transfected into human cells to generate stable transformants . Expression of mutant hTR resulted in the appearance of mutant telomerase activity and in the synthesis of mutant telomeres . Transformed cells were not visibly affected in their growth and viability when grown as mass populations . However, a reduction in plating efficiency and growth rate and an increase in the number of senescent cells were detected in populations with mutant telomeres by colony-forming assays . These results suggest that the presence of mutant telomerase, even if coexpressed with the wild-type enzyme, can be deleterious to cells, likely as a result of the impaired function of hybrid telomeres.

Mol Cell Biol, 1997 Nov, 17(11), 6303 - 10
Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila; Yu L et al.; Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown . Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells . Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes . This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants . Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena . Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable . Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth . In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not . These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.

J Biol Chem, 1997 Oct 24, 272(43), 27131 - 9
Thermus thermophilis dnaX homolog encoding gamma- and tau-like proteins of the chromosomal replicase; Yurieva O et al.; This report identifies the dnaX homolog from Thermus thermophilis . Replicases from bacteria to humans contain subunits that are homologous to one another . These homologs are subunits of a clamp loading apparatus that loads sliding clamps onto DNA, which in turn act as mobile tethers for the replication machinery . In Escherichia coli, two of these subunits (gamma and tau) are encoded by one gene (dnaX) in nearly equal amounts by way of an efficient translational frameshift . The gamma and tau subunits form the central touchpoint that holds together two DNA polymerases with one clamp loading apparatus to form the E . coli chromosomal replicase, DNA polymerase III holoenzyme . The E . coli holoenzyme is an efficient replication machine that simultaneously replicates both strands of duplex DNA . The T . thermophilis dnaX homolog also contains a frameshift signature and produces both tau- and gamma-like proteins . Recombinant T . thermophilis tau- and gamma-like proteins, expressed in E . coli, have an oligomeric state similar to that of their E . coli counterparts and display ATPase activity that is stimulated by DNA . These results imply that T . thermophilis utilizes a DNA polymerase III holoenzyme replication machinery similar to that of E . coli.

Bioelectromagnetics, 1997, 18(7), 491 - 8
Influence of extremely low frequency electromagnetic fields on the swimming behavior of ciliates; Hemmersbach R et al.; Different species of ciliates (Paramecium biaurelia, Loxodes striatus, Tetrahymena thermophila) have been taken as model systems to study the effects of extremely low-frequency electromagnetic fields (50 Hz, 0.5-2.0 mT) on the cellular level . A dose-dependent increase in the mean swimming velocity and a decrease in the linearity of cell tracks were observed in all wild-type cells . In contrast, field-exposure did not increase the number of directional turns of the Paramecium tetraurelia pawn mutant (d4-500r), which is characterized by defective Ca2+-channels . The described changes indicate a direct effect of low frequency electromagnetic fields on the transport mechanisms of the cell membrane for ions controlling the motile activity of cilia.

Int Arch Allergy Immunol, 1997 Oct, 114(2), 205 - 6
Hypersensitivity pneumonitis related to a covered and heated swimming pool environment; Moreno-Ancillo A et al.; Hypersensitivity pneumonitis (HP) or extrinsic allergic alveolitis is a lung disease caused by a large group of inhaled antigens of various sources . The most common HP occurring in the farm environment is classically caused by exposure to various thermophilic actinomycetes and fungi that can grow in the farm environment . Pullularia species and thermophilic actinomycetes have been involved in HP related to humidifier water and saunas . Our case illustrates the value of a site visit in the diagnosis of HP . During a visit to the covered and heated swimming-pool where our patient used to swim we could see that favourable conditions to fungal growth existed . To determine the possible aetiological agents of a suspected HP, cultures from several parts of the swimming-pool were taken . These cultures showed an intense growth of thermophilic actinomycetes, Neurospora and Aspergillus species . Precipitating antibodies against Neurospora species and Mycropolyspora faeni were detected . A case of HP related to a covered and heated swimming-pool environment is reported . Thermophilic actinomycetes and Neurospora species may be the causing agents.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1246 - 8
Rapid identification of heterotrophic, thermophilic, spore-forming bacteria isolated from hot composts; Blanc M et al.; The restriction enzyme profiles of 16S ribosomal DNAs (rDNAs) amplified by PCR from thermophilic heterotrophic bacterial strains isolated from composts were compared with those of reference strains . This allowed us to assign all but 1 of 16 strains to four different Bacillus species (namely, Bacillus stearothermophilus, Bacillus pallidus, Bacillus thermoglucosidasius, and "Bacillus thermodenitrificans") . This study showed that PCR restriction analysis of 16S rDNA contributes to rapid and reliable identification of newly isolated strains belonging to recognized species.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1225 - 30
Meiothermus cerbereus sp . nov., a new slightly thermophilic species with high levels of 3-hydroxy fatty acids; Chung AP et al.; Strains of Meiothermus cerbereus sp . nov . were isolated from the hot springs within the Geysir geothermal area of Iceland . The strains of Meiothermus cerbereus produce red-orange-pigmented colonies, have an optimum growth temperature of about 55 degrees C, and have higher levels of 3-OH fatty acids than the strains of the other species of the genus Meiothermus . These strains, unlike all other strains of the species of the genus Meiothermus examined previously, required cysteine, thiosulfate, or thioglycolate for growth in liquid Thermus medium, but not in the corresponding medium solidified with agar . Several strains belonging to Meiothermus silvanus, isolated from Geysir, also required reduced sulfur compounds for growth in liquid medium, leading to the hypothesis that this requirement is not a taxonomic characteristic of the new species . The new species represented by strains GY-1T and GY-5 can be distinguished from the other species of the genus Meiothermus by biochemical characteristics, fatty acid composition, DNA-DNA reassociation values, and 16S ribosomal DNA sequence . The type strain for Meiothermus cerbereus is GY-1 (= DSM 11376).

Int J Syst Bacteriol, 1997 Oct, 47(4), 1118 - 23
Thermosipho melanesiensis sp . nov., a new thermophilic anaerobic bacterium belonging to the order Thermotogales, isolated from deep-sea hydrothermal vents in the southwestern Pacific Ocean; Antoine E et al.; A new thermophilic, anaerobic rod-shaped bacterium, strain BI429T was isolated from the gills of a deep-sea vent hydrothermal mussel, Bathymodiolus brevior, from the Lau Basin (Southwestern Pacific Ocean) . Phenotypically, this isolate exhibited characteristics similar to those described for members of the order Thermotogales . This organism was identified as a member of the genus Thermosipho on the basis of the presence of the typical outer sheath-like structure (toga), its 16S rRNA sequence, and its ability to grow on carbohydrates (sucrose, starch, glucose, maltose, lactose, cellobiose, and galactose) . The cells of this organism were gram negative and rod shaped and generally occurred singly or in pairs, rarely occurring as chains with a maximum of five rods . At the optimum temperature for growth (70 degrees C), optimum pH (6.5), and optimum salinity (30 g of NaCl per liter), the doubling time was 100 min . In spite of the high percentage of similarity of its 16S rRNA sequence with that of Thermosipho africanus (98.6%), the weak level of DNA-DNA reassociation with this strain (2%) and particular physiological characteristics allowed us to differentiate this new organism from the sole species of the genus Thermosipho previously described (T . africanus) . On the basis of these observations, we propose that the new organism should be described as a new species, Thermosipho melanesiensis . The type strain of T . melanesiensis is BI429.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1013 - 9
Thermotoga hypogea sp . nov., a xylanolytic, thermophilic bacterium from an oil-producing well; Fardeau ML et al.; A new thermophilic, xylanolytic, strictly anaerobic, rod-shaped bacterium, strain SEBR 7054T, was isolated from an African oil-producing well . Based on the presence of an outer sheath (toga) and 16S rRNA sequence analysis data, this organism was identified as a member of the genus Thermotoga . Strain SEBR 7054T possessed lateral flagella, had a G + C content of 50 mol%, produced traces of ethanol from glucose but no lactate, and grew optimally in the presence of 0 to 0.2% NaCl at 70 degrees C . Its phenotypic and phylogenetic characteristics clearly differed from those reported for the five previously validly described Thermotoga species . Therefore, we propose that strain SEBR 7054T is a member of a new species of the genus Thermotoga, Thermotoga hypogea sp . nov . The type strain of T . hypogea is SEBR 7054 (= DSM 11164).

Int J Syst Bacteriol, 1997 Oct, 47(4), 969 - 74
Isolation and characterization of the homoacetogenic thermophilic bacterium Moorella glycerini sp . nov; Slobodkin A et al.; A thermophilic, anaerobic, spore-forming bacterium (strain JW/AS-Y6T) was isolated from a mixed sediment-water sample from a hot spring (Calcite Spring area) at Yellowstone National Park . The vegetative cells of this organism were straight rods, 0.4 to 0.6 by 3.0 to 6.5 microns . Cells occurred singly and exhibited a slight tumbling motility . They formed round refractile endospores in terminal swollen sporangia . Cells stained gram positive . The temperature range for growth at pH 6.8 was 43 to 65 degrees C, with optimum growth at 58 degrees C . The range for growth at 60 degrees C (pH60C; with the pH meter calibrated at 60 degrees C) was 5.9 to 7.8, with an optimum pH60C of 6.3 to 6.5 . The substrates utilized included glycerol, glucose, fructose, mannose, galactose, xylose, lactate, glycerate, pyruvate, and yeast extract . In the presence of CO2, acetate was the only organic product from glycerol and carbohydrate fermentation . No H2 was produced during growth . The strain was not able to grow chemolithotrophically at the expense of H2-CO2; however, suspensions of cells in the exponential growth phase consumed H2 . The bacterium reduced fumarate to succinate and thiosulfate to elemental sulfur . Growth was inhibited by ampicillin, chloramphenicol, erythromycin, rifampin, and tetracycline, but not by streptomycin . The G+C content of the DNA was 54.5 mol% (as determined by high-performance liquid chromatography) . The 16S ribosomal DNA sequence analysis placed the isolate in the Gram type-positive Bacillus-Clostridium subphylum . On the basis of physiological properties and phylogenetic analysis we propose that the isolated strain constitutes a new species, Moorella glycerini; the type strain is JW/AS-Y6 (= DSM 11254 = ATCC 700316).

Int J Syst Bacteriol, 1997 Oct, 47(4), 939 - 47
Deinococcus geothermalis sp . nov . and Deinococcus murrayi sp . nov., two extremely radiation-resistant and slightly thermophilic species from hot springs; Ferreira AC et al.; Strains of Deinococcus geothermalis sp . nov . were isolated from the hot spring and runoff at Agnano, Naples, Italy, and from the hot spring at Sao Pedro do Sul in central Portugal, while strains of Deinococcus murrayi sp . nov . were isolated from the hot springs at Sao Pedro do Sul, Sao Gemil, and Alcafache in central Portugal . The strains of D . geothermalis and D . murrayi produce orange-pigmented colonies and have an optimum growth temperature of about 45 to 50 degrees C . The type strains of the two new species are extremely gamma radiation resistant . The fatty acids of these new species are primarily branched-chain fatty acids . The two new species can be distinguished from each other by the lower pH range of D . geothermalis than of D . murrayi, by their fatty acid compositions, and by several biochemical parameters, including the ability of D . geothermalis to grow in minimal medium without yeast extract . 16S rRNA gene sequencing also showed that the isolates constitute two species and that these species are distinct from the other species of the genus Deinococcus . The type strain of D . geothermalis is AG-3a (= DSM 11300), and the type strain of D . murrayi is ALT-1b (= DSM 11303).

Nucleic Acids Res, 1997 Nov 1, 25(21), 4194 - 200
The tRNATyr-isoacceptors and their genes in the ciliate Tetrahymena thermophila: cytoplasmic tRNATyr has a QPsiA anticodon and is coded by multiple intron-containing genes; Junker V et al.; In the ciliated protozoa Tetrahymena thermophila introns have been detected in rRNA and mRNAs until now . We have isolated and sequenced seven tRNATyr genes from the T.thermophila nuclear genome . All of these genes contain introns of identical length and sequence . The 11 bp long intervening sequences are located 1 nt 3' to the anticodon as found in other eukaryotic nuclear tRNA genes . Tetrahymena tRNATyr genes are efficiently transcribed in HeLa cell nuclear extract . Moreover, processing and splicing occurred in HeLa as well as in wheat germ extracts, supporting the notion that Tetrahymena tRNATyr introns can be classified as authentic tRNA introns . We have also isolated cytoplasmic tRNATyr from Tetrahymena cells . This tRNATyr isoacceptor has a QPsiA anticodon and is not a UAG suppressor as shown in in vitro translation studies . Since UAG and UAA codons are used as glutamine codons in Tetrahymena macronuclear DNA, the presence of a strong natural UAG suppressor such as tRNATyr with GPsiA anticodon should cause misreading of the glutamine as tyrosine codons and the absence of the latter had thus been predicted . Furthermore we have studied the organization of tRNATyr genes in the genome of T.thermophila and have found two types of tRNATyr gene arrangement . A minimum of 12 tRNATyr genes are present as single copies in genomic DNA HindIII restriction fragments ranging in size from 0.6 to 7 kb . Additionally one cluster of tRNATyr genes consisting of six members has been detected in a 2.3 kb HindIII fragment.

J Bacteriol, 1997 Oct, 179(20), 6499 - 503
Characterization of a thermostable DNA photolyase from an extremely thermophilic bacterium, Thermus thermophilus HB27; Kato R et al.; The photolyase gene from Thermus thermophilus was cloned and sequenced . The characteristic absorption and fluorescence spectra of the purified T . thermophilus photolyase suggested that the protein has flavin adenine dinucleotide as a chromophore . The second chromophore binding site was not conserved in T . thermophilus photolyase . The purified enzyme showed light-dependent photoreactivation activity in vitro at 35 and 65 degrees C and was stable when subjected to heat and acidic pH.

J Biotechnol, 1997 Sep 16, 57(1-3), 3 - 14
The cellulolytic system of Clostridium cellulolyticum; Belaich JP et al.; Recent findings on the cellulolytic system of the mesophilic Clostridium cellulolyticum are reviewed . Six cellulases and the scaffolding protein, which are, at the present time, the known components of the cellulosome have been cloned . The catalytic and structural properties of the cloned enzymes CelA, CelC, CelD and CelF are described . It was shown that the grafting of the cellulases onto the scaffolding protein was performed using the dockerin-cohesin attachment device and was strictly dependent on the integrity of both components of the complex . The amino-acid sequences of dockerin and cohesin domains of C . cellulolyticum were compared to that of C . cellulovorans and C . thermocellum . This sequence analysis shows that domains belonging to the thermophilic or the mesophilic bacteria can be placed into two well defined groups . The genetic organization of the gene cluster of C . cellulolyticum is discussed.

Nat Struct Biol, 1997 Oct, 4(10), 801 - 4
Structure of a kinetic protein folding intermediate by equilibrium amide exchange; Hosszu LL et al.; A combination of equilibrium amide exchange and kinetic folding data show that the essential features of the complex topology of the N-terminal domain of a thermophilic phosphoglycerate kinase are established on a millisecond or faster timescale, before the rate-limiting step in the folding pathway commences.

Antimicrob Agents Chemother, 1997 Oct, 41(10), 2244 - 50
Antimicrobial susceptibility patterns of thermophilic Campylobacter spp . from humans, pigs, cattle, and broilers in Denmark; Aarestrup FM et al.; The MICs of 16 antimicrobial agents were determined for 202 Campylobacter jejuni isolates, 123 Campylobacter coli isolates, and 6 Campylobacter lari isolates from humans and food animals in Denmark . The C . jejuni isolates originated from humans (75), broilers (95), cattle (29), and pigs (3); the C . coli isolates originated from humans (7), broilers (17), and pigs (99); and the C . lari isolates originated from broilers (5) and cattle (1) . All isolates were susceptible to apramycin, neomycin, and gentamicin . Only a few C . jejuni isolates were resistant to one or more antimicrobial agents . Resistance to tetracycline was more common among C . jejuni isolates from humans (11%) than among C . jejuni isolates from animals (0 to 2%) . More resistance to streptomycin was found among C . jejuni isolates from cattle (10%) than among those from humans (4%) or broilers (1%) . A greater proportion of C . coli than of C . jejuni isolates were resistant to the other antimicrobial agents tested . Isolates were in most cases either coresistant to tylosin, spiramycin, and erythromycin or susceptible to all three antibiotics . More macrolide-resistant isolates were observed among C . coli isolates from swine (79%) than among C . coli isolates from broilers (18%) and humans (14%) . Twenty-four percent of C . coli isolates from pigs were resistant to enrofloxacin, whereas 29% of C . coli isolates from humans and none from broilers were resistant . More resistance to streptomycin was observed among C . coli isolates from swine (48%) than among C . coli isolates from broilers (6%) or humans (0%) . The six C . lari isolates were susceptible to all antimicrobial agents except ampicillin and nalidixic acid . This study showed that antimicrobial resistance was found only at relatively low frequencies among C . jejuni and C . lari isolates . Among C . coli isolates, especially from swine, there was a high level of resistance to macrolides and streptomycin . Furthermore, this study showed differences in the resistance to antimicrobial agents among Campylobacter isolates of different origins.

Gene, 1997 Sep 15, 197(1-2), 205 - 14
Nucleotide sequences and gene organization of TaqI endonuclease isoschizomers from Thermus sp . SM32 and Thermus filiformis Tok6A1; Cao W et al.; Eight TaqI isoschizomer genes, two from Yellowstone National Park, one from Japan, two from New Zealand, two from Portugal, and one from the Azores (1000 miles west of Portugal), were PCR-amplified and sequenced . Sequence alignment of isoschizomers isolated from close geographical locations shows identical or almost identical protein sequences, while isoschizomers from distant sites demonstrate considerable diversity, ranging from 54 to 75% in amino acid identity . Accordingly, these isoschizomers were arranged into four geographical groups, i.e., USA as represented by Thermus aquaticus YT1, Japan by Thermus thermophilus HB8, New Zealand by Thermus filiformis Tok6A1, Portugal by Thermus sp . SM32 . The complete ORFs of two new representative genes, tfiTok6A1I and tsp32IR, were obtained by bubble PCR . Unlike M . TaqI-R.TaqI and M . TthHB8I-R . TthHB8I which exhibit an unusual 13-codon overlap, the methylase and endonuclease genes are each separated by 15 nucleotides in the TfiTok6A1I and Tsp32IR restriction-modification systems . Phylogenetic analysis suggests that initially TfiTok6A1I diverged from a common ancestor, then Tsp32IR branched out, and finally TaqI and TthHB8I diverged from each other during evolution.

Structure, 1997 Sep 15, 5(9), 1187 - 98
The structure of ribosomal protein S7 at 1.9 A resolution reveals a beta-hairpin motif that binds double-stranded nucleic acids; Wimberly BT et al.; BACKGROUND: Ribosomal protein S7, a crucial RNA-binding component of the ribosome, is one of two proteins that initiates assembly of the 30S ribosomal subunit . It is required for proper folding of a large 3' domain of 16S ribosomal RNA . S7 regulates its own synthesis by binding to its own mRNA . This ability of S7 to bind both messenger and ribosomal RNAs makes determination of its mode of RNA recognition particularly interesting . RESULTS: The crystal structure of S7 from Thermus thermophilus was determined by a two-wavelength anomalous diffraction experiment using the LIII edge of mercury . The S7 structure consists of a bundle of six helices and an extended beta hairpin between helices 3 and 4, with two or more RNA-binding sites on its surface . The hairpin, along with portions of helices 1, 4 and 6, forms a large, positively charged, concave surface that has the appropriate curvature and dimensions to bind double-stranded RNA . A second putative RNA-binding site comprises parts of loop 2 and the helix 4-loop 5 turn . CONCLUSIONS: Structural similarity between S7 and the IHF/HU family of proteins strongly suggests that the beta hairpin of S7 binds to a groove of double-stranded RNA . The beta hairpin of S7 is also similar to those from other nucleic acid binding proteins, such as ribosomal protein L14 and BIV Tat, suggesting that it belongs to an extended family of such motifs, all of which bind to a groove of double-stranded nucleic acid . The residues in S7 loop 2 that belong to the second putative RNA-binding site may have a role analogous to the N-terminal residues of IHF/HU which grip an unbent portion of double helix.

FEMS Immunol Med Microbiol, 1997 Sep, 19(1), 47 - 56
Distribution of serotypes of Campylobacter jejuni and C . coli from Danish patients, poultry, cattle and swine; Nielsen EM et al.; The number of human cases of enteritis caused by Campylobacter jejuni and C . coli is increasing in Denmark and other European countries . No systemic typing has earlier been performed on Campylobacter isolates of Danish origin . The primary purpose of this study was to provide a serotype distribution of Campylobacter isolates from Danish patients and the major food production animals . In addition, the occurrence of intestinal carriers of thermophilic campylobacters among these food production animals was examined . In a nationwide survey, the individual isolation rate was 36% for broiler chickens, 47% for cattle and 46% for swine when sampled at the slaughterhouse . C . jejuni accounted for 83-91% of the thermophilic Campylobacter spp . in broiler chickens and cattle, whereas 95% of the isolates from swine was C . coli . In human patients with Campylobacter enteritis, 94% of the isolates were C . jejuni and 6% were C . coli . Heat-stable serotyping (the 'Penner scheme') was performed on a total of 398 isolates from the four sources: human patients (n = 145), broiler chickens (n = 94), swine (n = 111) and cattle (n = 48) . Among human isolates, serotype O:1,44, O:2 and the O:4-complex accounted for 62% of the C . jejuni isolates . These serotypes were also common in samples from broilers and cattle . In swine, C . coli O:30 and O:46 were most common . The serotype distribution of human clinical isolates showed large overlap with the serotype distribution of campylobacters in cattle and chickens, and on this basis both could be major sources of human campylobacteriosis.

Nucleic Acids Res, 1997 Oct 15, 25(20), 3969 - 73
Replication of DNA templates containing 5-formyluracil, a major oxidative lesion of thymine in DNA; Zhang QM et al.; 5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants . To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template . Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis . Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity . DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair . The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used . These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.

Biochemistry, 1997 Sep 30, 36(39), 11777 - 86
Investigation by electrospray ionization mass spectrometry of the extracellular hemoglobin from the polychaete annelid Alvinella pompejana: an unusual hexagonal bilayer hemoglobin; Zal F et al.; Alvinella pompejana inhabits deep-sea hydrothermal vent sites along the East-Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys . This worm is the most thermophilic metazoan known to date . In Alvinella, as in other alvinellids, oxygen transport is mainly achieved by an extracellular Hb dissolved in the vascular blood . This Hb has a molecular mass of 3833 +/- 14 kDa as revealed by multiangle laser light scattering (MALLS) . Native and derivative Hb (reduced, carbamidomethylated, and deglycosylated) were analyzed by electrospray ionization mass spectrometry (ESI-MS) . The data were processed by the maximum entropy deconvolution system (MaxEnt) . We identified three groups of peaks for Alvinella Hb, at ca . 16, 23-26, and 50 kDa corresponding to (i) four monomeric globin chains, a1 (16 633.4), a2(16 532.4), a3 (16 419.6), and a4(16 348.9); (ii) four linker chains, L1-L4 (22 887 . 1, 24 230.5, 26 233.6, and 25 974.4); and (iii) one disulfide-bonded trimer T (51 431.9) composed of globin chains b (16 477.5), c (16 916.1), and d (18 048.8) . These Hbs were also subjected to SDS-PAGE analysis for comparative purposes . In addition, using the ESI-MS data we propose two alternative models for the quaternary structure of Alvinella's Hb.

Indian J Exp Biol, 1997 May, 35(5), 511 - 5
Comparison of thermostable phosphatase and proteases from thermophilically disposed transition species and thermophilic actinomycetes; Lalwani L et al.; Thermoactinomycetes vulgaris is a thermophilic actinomycetes growing optimally at 50 degrees C and Streptomyces albus, S . coelicolor, S . fasciculus and S . olivochromogenes are thermophilically disposed transition species of actinomycetes, which have optimum biomass at 40 degrees C . The acid/alkaline phosphatase and acid/alkaline/neutral protease enzyme from Streptomycetes species showed enzyme activity up to 90 degrees C . In comparison to phosphatases and proteases from T . vulgaris it was concluded that these thermophilically disposed transition species showed macromolecular thermostability i.e . thermostable enzymes and protein.

FEBS Lett, 1997 Sep 8, 414(2), 243 - 6
S6 permutein shows that the unusual target topology is not responsible for the absence of rigid tertiary structure in de novo protein albebetin; Abdullaev ZK et al.; Ribosomal protein S6 from Thermus thermophilus was modified to form the unusual unique topology designed earlier for a de novo protein albebetin . The S6 gene was cloned, sequenced and circularly permutated by means of genetic engineering methods . The permutated gene was expressed in Escherichia coli and the permutein was isolated and investigated by means of circular dichroism, fluorescence spectroscopy and scanning microcalorimetry . The permutated protein revealed a pronounced secondary structure close to that of the wild type S6 protein and a rigid tertiary structure possessing cooperative temperature melting . It means that the unusual new topology of albebetin is compatible with a rigid tertiary structure, it may be realized in natural proteins and it is not responsible for the absence of rigid structure in albebetin.

Mol Cell Biol, 1997 Oct, 17(10), 6147 - 56
Developmental regulation of DNA replication: replication fork barriers and programmed gene amplification in Tetrahymena thermophila; Zhang Z et al.; The palindromic Tetrahymena ribosomal DNA (rDNA) minichromosome is amplified 10,000-fold during development . Subsequent vegetative replication is cell cycle regulated . rDNA replication differs fundamentally in cycling vegetative and nondividing amplifying cells . Using two-dimensional gel electrophoresis, we show for the first time that replication origins that direct gene amplification also function in normal dividing cells . Two classes of amplification intermediates were identified . The first class is indistinguishable from vegetative rDNA, initiating in just one of the two 5' nontranscribed spacer (NTS) copies in the rDNA palindrome at either of two closely spaced origins . Thus, these origins are active throughout the life cycle and their regulation changes at different developmental stages . The second, novel class of amplification intermediates is generated by multiple initiation events . Intermediates with mass greater than fully replicated DNA were observed, suggesting that onionskin replication occurs at this stage . Unlike amplified rDNA in Xenopus laevis, the novel Tetrahymena species are not produced by random initiation; replication also initiates in the 5' NTS . Surprisingly, a replication fork barrier which is activated only in these amplifying molecules blocks the progression of forks near the center of the palindrome . Whereas barriers have been previously described, this is the first instance in which programmed regulation of replication fork progression has been demonstrated in a eukaryote.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10675 - 80
Analysis of exocytosis mutants indicates close coupling between regulated secretion and transcription activation in Tetrahymena; Haddad A et al.; Stimulation of regulated secretory cells promotes protein release via the fusion of cytoplasmic storage vesicles with the plasma membrane . In Tetrahymena thermophila, brief exposure to secretagogue results in synchronous fusion of the entire set of docked dense-core granules with the plasma membrane . We show that stimulation is followed by rapid new dense-core granule synthesis involving gene induction . Two genes encoding granule matrix proteins, GRL1 and GRL4, are shown to undergo induction following stimulation, resulting in approximately 10-fold message accumulation within 1 h . The mechanism of induction involves transcriptional regulation, and the upstream region of GRL1 functions in vivo as an inducible promoter in a heterologous reporter construct using the gene encoding green fluorescent protein . Taking advantage of the characterized exocytosis (exo-) mutants available in this system, we asked whether the signals for regranulation were generated directly by the initial stimulation, or whether downstream events were required for transcription activation . Three mutants, with defects at three distinct stages in the regulated secretory pathway, failed to show induction of GRL1 and GRL4 after exposure to secretagogue . These results argue that regranulation depends upon signals generated by the final steps in exocytosis.

Biochemistry (Mosc), 1997 Mar, 62(3), 237 - 46
Three site-specific endonucleases from thermophilic strain Bacillus species LA are isoschizomers of HhaI, AsuII, and HindIII; Shiryaev SA et al.; Screening of thermophilic bacterial strains revealed a strain containing three site-specific endonucleases: BspLAI, BspLAII, and BspLAIII . These endonucleases were purified to functional purity by sequential chromatography . Recognition sites, DNA cleavage sites, and some properties of the endonucleases were determined . BspLAI recognizes the sequence 5-GCG/C-3 on the DNA molecule and is an isoschizomer of endonuclease HhaI . BspLAII recognizes the sequence 5-TT/CGAA-3 and is an isoschizomer of AsuII . BspLAIII recognizes site 5-A/AGCTT-3 and is an isoschizomer of endonuclease HindIII . All the three enzymes exhibit maximal activity at 55 degrees C . The optimal buffer is MRB, pH 7.4 . They retain activity on storage for 3 weeks at room temperature and thus are highly stable.

J Biol Chem, 1997 Oct 3, 272(40), 24906 - 12
Thermophilic F1-ATPase is activated without dissociation of an endogenous inhibitor, epsilon subunit; Kato Y et al.; Subunit complexes (alpha3beta3gamma, alpha3beta3gammadelta, alpha3beta3gammaepsilon, and alpha3beta3gammadeltaepsilon) of thermophilic F1-ATPase were prepared, and their catalytic properties were compared to know the role of delta and epsilon subunits in catalysis . The presence of delta subunit in the complexes had slight inhibitory effect on the ATPase activity . The effect of epsilon subunit was more profound . The (-epsilon) complexes, alpha3beta3gamma and alpha3beta3gammadelta, initiated ATP hydrolysis without a lag . In contrast, the (+epsilon) complexes, alpha3beta3gammaepsilon and alpha3beta3gammadeltaepsilon, started hydrolysis of ATP (<700 microM) with a lag phase that was gradually activated during catalytic turnover . As ATP concentration increased, the lag phase of the (+epsilon) complexes became shorter, and it was not observed above 1 mM ATP . Analysis of binding and hydrolysis of the ATP analog, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, suggested that the (+epsilon) complexes bound substrate only slowly . Differing from Escherichia coli F1-ATPase, the activation of the (+epsilon) complexes from the lag phase was not due to dissociation of epsilon subunit since the re-isolated activated complex retained epsilon subunit . This indicates that there are two alternative forms of the (+epsilon) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

Eur J Biochem, 1997 Aug 15, 248(1), 171 - 8
4-alpha-glucanotransferase from the hyperthermophilic archaeon Thermococcus litoralis--enzyme purification and characterization, and gene cloning, sequencing and expression in Escherichia coli; Jeon BS et al.; 4-Alpha-Glucanotransferase was purified from cells of Thermococcus litoralis, a hyperthermophilic archaeon . The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration . The optimal temperature for its activity was 90 degrees C . The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides of various lengths and glucose . When maltoheptaose was used as the substrate, glucoamylase-resistant and glucoamylase-sensitive saccharides were produced . On incubation of amylose with the T . litoralis enzyme, glucoamylase-resistant but alpha-amylase-sensitive molecules were produced, but the amount of reducing sugar showed only slight increases . These results indicate that the T . litoralis enzyme catalyzes not only intermolecular transglycosylation to produce linear alpha-1,4-glucan, but also intramolecular transglycosylation to produce cyclic alpha-1,4-glucan (cycloamylose), similarly to potato 4-alpha-glucanotransferase (called disproportionating enzyme) . The gene encoding the T . litoralis 4-alpha-glucanotransferase was cloned, sequenced and expressed in Escherichia coli . The nucleotide sequence of the gene encoded a 659-amino acid protein with a calculated molecular mass of 77,883 Da . The amino acid sequence of the T . litoralis enzyme showed high similarity with those of alpha-amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4-alpha-glucanotransferases.

Arch Biochem Biophys, 1997 Sep 15, 345(2), 299 - 304
Biosynthesis of the peptide bond in the coenzyme N-(7-mercaptoheptanoyl)-L-threonine phosphate; Solow B et al.; The biochemical mechanism for the formation of the amide bond in N-(7-mercaptoheptanoyl)-L-threonine phosphate (HS-HTP) has been studied by measuring the incorporation of L-{3-(3)H}threonine into N-(7-mercaptoheptanoyl)-L-threonine (HS-HT) by cell extracts (CE) of Methanosarcina thermophila incubated with different precursors . Synthesis of HS-HT was observed from L-{3-(3)H}threonine and 7-mercaptoheptanoic acid (HS-H) when the incubations were conducted with either crude CE or Sephadex column-purified CE . In the presence of CE, the synthesis of HS-HT was found to be inhibited 66% by preincubation of the extract with ATPase, indicating that ATP was involved in the biosynthesis . In spite of this indication of ATP involvement in the coupling reaction, incubation of the crude CE with L-{3-(3)H}threonine, HS-H, and ATP was found to inhibit the formation of HS-HT . In contrast, the synthesis of HS-HT in the presence of Sephadex column-purified CE was found to be stimulated by the addition of ATP . Incubation of the crude CE with the CoA thioester of 7-mercaptoheptanoic acid (HS-HCoA) or the mixed disulfide formed between coenzyme M and 7-mercaptoheptanoic acid did not stimulate the biosynthesis . The biosynthesis of HS-HT was found to be strongly inhibited by an ethanol extract of the crude CE . This inhibition was found to be attributed to the HS-HTP present in the extract . Stimulation of HS-HT biosynthesis 300-fold was observed when the Sephadex column-purified CE was incubated with L-{3-(3)H}threonine and 7-mercaptoheptanoyl phosphate (HS-H-P) . Data indicate that HS-HT is produced by the phosphorylation of HS-H to HS-H-P with ATP, which then reacts with L-threonine to produce HS-HT.

Gene, 1997 Aug 22, 195(2), 321 - 8
Identification of a thermophilic plasmid origin and its cloning within a new Thermus-E . coli shuttle vector; Wayne J et al.; A pUC19-based vector has been generated for selecting functional thermophilic origins (oris) of Thermus ssp . Once combined with thermophilic DNA, the vector can be amplified in ampicillin resistant (Ap(R)) E . coli, prior to transformation and kanamycin (Km) selection in Thermus thermophilus . The Km(R) Thermus transformants replicate any newly-formed shuttle vectors via introduced thermophilic oris . Using this "ori-selecting" vector, three novel thermophilic oris were cloned from randomly digested Thermus cryptic plasmid DNA . These shuttle vectors are useful for genetic analyses, as well as protein engineering within thermophiles . The smallest ori-containing sequence of 4.2 kb has been subcloned, sequenced, and further refined to 2.3 kb . A significant ORF of 341 amino acids (aa), with a Thermus promoter and RBS, is found within the thermophilic ori . Deleting part of this ORF abolishes the shuttle vector's ability to replicate in T . thermophilus . Therefore, we postulate that this ORF encodes a replication protein (Rep) necessary for thermophilic plasmid replication . The thermophilic ori also contains two sequences which resemble DnaA boxes.

Gene, 1997 Aug 22, 195(2), 201 - 6
Sth132I, a novel class-IIS restriction endonuclease of Streptococcus thermophilus ST132; Poch MT et al.; The Sth132I restriction endonuclease (R.Sth132I) was detected in Streptococcus thermophilus ST132 and purified to near homogeneity by heparin Sepharose CL-6B affinity chromatography . Fragments from Sth132I digestion of plasmid DNA were subcloned into pUC19 in Escherichia coli DH5alpha and sequenced . Sequence analysis of inserts and their ligation junction sites revealed that Sth132I is a novel class-IIS restriction endonuclease, which recognizes the non-palindromic sequence 5'-CCCG(N)4-3', 3'-GGGC(N) 8-5'.

J Eukaryot Microbiol, 1997 Sep-Oct, 44(5), 518 - 22
Alternate junctions and microheterogeneity of Tlr1, a developmentally regulated DNA rearrangement in Tetrahymena thermophila; Patil NS et al.; A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila . Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA . Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells . We show here that C2 strain Tetrahymena also use alternate rearrangement junctions . We have mapped and sequenced two additional rearrangement variants and find that both the left and right junctions can vary over a range of approximately 200 bp . We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.

J Eukaryot Microbiol, 1997 Sep-Oct, 44(5), 435 - 7
Cloning and sequencing of a cDNA encoding glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena thermophila: growth-associated changes in its mRNA expression; Zhao Y et al.; Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway . Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis . We have cloned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T . thermophila cell . The open reading frame encoded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide-binding domain and a catalytic domain, which was highly similar to those of other organisms . Its mRNA levels at different growth stages were examined by northern blot analysis . The fragment of the isolated cDNA was hybridized to a 1.3-kb mRNA transcript . There was a marked increase in Tthgapdh mRNA level at the mid-exponential phase, followed by a gradual decrease . Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.

J Mol Biol, 1997 Aug 8, 271(1), 100 - 11
Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer; Lin Y et al.; Single-stranded DNA aptamers that recognize DNA polymerase from Thermus acquaticus (Taq pol) with high affinity have been described recently . These aptamers have been shown to efficiently inhibit the polymerase activity of Taq pol and are useful in enhancing the amplification efficiency of low copy number targets by the polymerase chain reaction (PCR) . Aptamers selected to bind to Taq pol fell into two different sequence families and inhibited several DNA polymerases isolated from the Thermus species, including that from Thermus thermophilus (Tth pol) . Aptamers from one sequence family inhibited the Stoffel fragment of Taq pol efficiently, whereas those from the other family did not . Truncated aptamers derived from two parent ligands from both families were combined to form a heterodimeric aptamer that effectively inhibited all three polymerases and were shown to be useful in detecting a low copy number target by PCR amplification . These data demonstrate that the combination of aptamers with different properties into a single molecule broadens their spectrum of utility.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 208 - 17
Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-beta-1,4-xylanase of Thermomonospora alba ULJB1 with cellulose-binding ability; Blanco J et al.; Several thermophilic actinomycetes were isolated from urban solid waste . One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers . Xylanase and cellulase activities were quantified and compared with those Thermomonospora fusca . Genes encoding two different endo-beta-1,4-xylanase were cloned from T . alba ULJB1 . One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans . It encodes a protein of 482 amino acids with a deduced molecular mass of 48,456 Da . The protein contains a 38-amino-acid leader peptide with six Arg+ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine . The XylA protein was purified to near homogeneity from S . lividans/XylA cultures . Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability . The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain . Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose . The major degradation product was xylobiose with traces of xylose . The xylanase activity was thermostable, showing a good activity up to 95 degrees C, and had broad pH stability in the range from pH 4.0 to pH 10.0.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 184 - 90
Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp . IHI-91 on olive oil; Becker P et al.; A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp . strain IHI-91 growing optimally at 65 degrees C . Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l-1 h-1 at a condition rate of 0.4 h-1 . Lipase productivity was increased by 50% compared to data from batch fermentations . Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate . Kinetic and stoichiometric parameters for the utilisation of olive oil were determined . The maximal biomass output method led to a saturation constant Ks of 0.88 g/l . Both batch growth data and a washout experiment yielded a maximal specific growth rate, mu max, of 1.0 h-1 . Oxygen uptake rates of up to 2.9 g l-1 h-1 were calculated and the yield coefficient, Y X/O, was determined to be 0.29 g dry cell weight/g O2 . From an overall material balance the yield coefficient, Y X/S, was estimated to be 0.60 g dry cell weight/g olive oil.

Biochem Biophys Res Commun, 1997 Aug 28, 237(3), 572 - 6
Cytochrome-c552 from Thermus thermophilus: a functional and crystallographic investigation; Soulimane T et al.; The eubacterium Thermus thermophilus expresses terminal oxidases of the ba3- and caa3-type . The soluble cytochrome-c552 of this organism has been isolated by a new method and characterized . In contrast to previous studies, but in line with coexpression at low aeration, the cytochrome was unambiguously identified as the substrate of the ba3-oxidase . In the presence of TMPD and ascorbate, biphasic Eadie-Hofstee plots with kmax = 250 s-1 at 25 degrees C are observed upon addition of cytochrome-c552 . Surprisingly, the caa3-oxidase with its single covalently bound cytochrome-c also exhibits a biphasic redox activity with kmax = 185 s-1 in the presence of TMPD and ascorbate only . Further addition of cytochrome-c552 does not lead to enhanced activity . Crystals of cytochrome-c552 were obtained by vapor diffusion using the sitting-drop method in the presence of ammonium sulfate as precipitant . They diffract to 1.28 A resolution using synchrotron radiation . The structure has been solved by MAD phasing.

J Mol Biol, 1997 Sep 19, 272(2), 178 - 89
A DNA polymerase III holoenzyme-like subassembly from an extreme thermophilic eubacterium; McHenry CS et al.; We have purified a novel DNA polymerase from Thermus thermophilus . This was enabled by use of general gap filling assays to monitor polymerase activity and cross-reactive monoclonal antibodies against the alpha catalytic subunit of E . coli DNA polymerase III holoenzyme to distinguish a novel polymerase from the well characterized DNA polymerase I-like Thermus thermophilus DNA polymerase . Two proteins migrating with the polymerase after three chromatographic steps were isolated and subjected to partial amino acid sequencing . The amino termini of both were homologous to the two products of the E . coli dnaX gene, the gamma and tau subunits of the DNA polymerase III holoenzyme . Using this information and sequences conserved among dnaX-like genes, we isolated a gene fragment by PCR and used it as a probe to isolate the full length Thermus thermophilus dnaX gene . The deduced amino acid sequence is highly homologous to the DnaX proteins of other bacteria . Examination of the sequence permitted identification of a frameshift site similar to the one used in E . coli to direct the synthesis of the shorter gamma DnaX-gene product . Based on this information, we conclude that a conventional replicase exists in extreme thermophilic eubacteria . The general biological and practical technological implications of this finding are discussed .

Dev Biol, 1997 Sep 15, 189(2), 233 - 45
A mutational analysis of conjugation in Tetrahymena thermophila . 2 . Phenotypes affecting middle and late development: third prezygotic nuclear division, pronuclear exchange, pronuclear fusion, and postzygotic development; Cole ES et al.; Conjugation following pair formation in Tetrahymena can be divided into three distinct sequences of events: prezygotic development, postzygotic development, and exconjugant development . The decision to proceed with postzygotic development is governed by a developmental checkpoint occurring sometime during the middle stages of conjugation . A second developmental decision is made to initiate pair separation and exconjugant development . This paper examines the phenotypes of five newly isolated conjugation mutants (cnj6-cnj10) which affect middle and late events within the conjugation program . cnj6 mutants exhibit normal nuclear behavior throughout development up to and including differentiation of new macronuclear anlagen . Pairs arrest at this developmental endpoint, unable to dissociate . cnj7 and cnj8 eliminate the third prezygotic nuclear division and the first postzygotic nuclear division . All subsequent developmental events appear normal . cnj9 eliminates the second postzygotic nuclear division, and subsequently, new macronuclei fail to develop despite parental macronuclear degradation . cnj10 results in a pleiotropic phenotype characterized by failure of numerous events which all appear to involve nuclear-cytoskeletal interactions . These defects include nuclear selection (anchoring nuclei to the exchange junction), pronuclear exchange, pronuclear fusion, and anchoring postzygotic nuclear division products to the posterior cell cortex . These mutant phenotypes are used to draw inferences regarding developmental dependencies that govern a cell's entry into the postzygotic and exconjugant developmental programs .

Dev Biol, 1997 Sep 15, 189(2), 215 - 32
A mutational analysis of conjugation in Tetrahymena thermophila . 1 . Phenotypes affecting early development: meiosis to nuclear selection; Cole ES et al.; Conjugation in the freshwater ciliate Tetrahymena thermophila involves a developmental program that models meiosis, fertilization, and early developmental events characteristic of multicellular eukaryotes . We describe a gallery of five early-acting conjugation mutations . These mutants, cnj1-5, exhibit phenotypes in which specific steps in the conjugal pathway have been altered or eliminated . Specifically, cnj1 and cnj2 fail to condense their micronuclear chromatin prior to each of the three prezygotic nuclear divisions . This results in nuclear division failure, failure to replicate DNA, and failure to initiate postzygotic development . The cnj3 mutant appears to exhibit a defect in chromosome separation during anaphase of mitosis . cnj4 mutants successfully carry out meiosis I, yet are unable to execute the second meiotic division and abort all further development . cnj5 mutants are unable to initiate either meiosis I or meiosis II, yet proceed to execute all subsequent developmental events . These mutant phenotypes are used to draw inferences regarding developmental dependencies that exist within the conjugation program .

Chem Phys Lipids, 1997 Aug 8, 88(1), 37 - 43
Slow fusion of liposomes composed of membrane-spanning lipids; Elferink MG et al.; The fusion characteristics of large unilamellar liposomes composed of bipolar tetraether lipids extracted from the thermophilic archaeon Sulfolobus acidocaldarius, was investigated . These lipids span the entire membrane and form single monolayer liposomes in aqueous media {Elferink, M.G.L., de Wit, J.G., Demel, R., Driessen, A.J.M . and Konings, W.N., (1992) J . Biol . Chem . 267, 1375-1381} . In the presence of calcium-phosphate, slow mixing of the aqueous liposome contents and membrane lipids occurred, demonstrating that these liposomes are fusion-competent . The fusion process was essentially nonleaky . The rate of fusion increased with the pH and the concentration of calcium and phosphate . Fusion resulted in an increase of the size of the liposomes . These data demonstrate that a monolayer organization of lipids in a membrane does not per se interfere with membrane fusion competence.

Chemosphere, 1997 Sep, 35(5), 1043 - 52
Toxicological comparisons of Tetrahymena species, end points and growth media: supplementary investigations to the pilot ring test; Pauli W et al.; Parallel to an international Pilot Ring Test to standardize a mutigeneration test protocol with the protozoon Tetrahymena pyriformis supplementary investigations were made . Effects on growth rate and 46 h population density were measured using an extended set of 12 chemicals, a further Tetrahymena-species (T . thermophila) and 4 different media (axenic and bacteria based) . Results are compared for sensitivity with effects on additional end points (chemosensory response, respiration and vitality) . Both species exhibit similar sensitivity towards the chemicals . In some cases the media composition influenced the toxic potential of chemicals . Both growth parameters proved to be a sensitive integral end point for toxicological studies, confirming the one point, photometric measurement of population density after 46 hours in axenic (preferably proteose peptone-) medium an obvious choice for a standard test procedure.

Arch Microbiol, 1997 Oct, 168(4), 270 - 6
New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1',2'-dihydro-gamma-carotene, 1',2'-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains; Takaichi S et al.; The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods . Major carotenoids were four kinds of carotenes: gamma-carotene, chlorobactene, and their 1',2'-dihydro derivatives (1',2'-dihydro-gamma-carotene and 1',2'-dihydrochlorobactene) . In lesser amounts, hydroxyl gamma-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found . The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid . In other strains of C . tepidum, the same carotenoids were found, but the composition varied from strain to strain . The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a . The effects of nicotine on carotenoid biosynthesis in C . tepidum differed from those in the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus.

Proteins, 1997 Sep, 29(1), 77 - 86
Structural basis of the properties of an industrially relevant thermophilic xylanase; Harris GW et al.; A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized . The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry . The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C . The pH optimum is 6, but the enzyme is active over a broad pH range . The xylanase has been cloned and sequenced, and the crystal structure has been determined . The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules . These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol . The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.

J Bacteriol, 1997 Sep, 179(18), 5693 - 8
Genetic responses of the thermophilic archaeon Sulfolobus acidocaldarius to short-wavelength UV light; Wood ER et al.; The archaea which populate geothermal environments are adapted to conditions that should greatly destabilize the primary structure of DNA, yet the basic biological aspects of DNA damage and repair remain unexplored for this group of prokaryotes . We used auxotrophic mutants of the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius to assess genetic and physiological effects of a well-characterized DNA-damaging agent, short-wavelength UV light . Simple genetic assays enabled quantitative dose-response relationships to be determined and correlated for survival, phenotypic reversion, and the formation of genetic recombinants . Dose-response relationships were also determined for survival and phenotypic reversion of the corresponding Escherichia coli auxotrophs with the same equipment and procedures . The results showed S . acidocaldarius to be about twice as UV sensitive as E . coli and to be equally UV mutable on a surviving-cell basis . Furthermore, UV irradiation significantly increased the frequency of recombinants recovered from genetic-exchange assays of S . acidocaldarius . The observed UV effects were due to the short-wavelength (i.e., UV-C) portion of the spectrum and were effectively reversed by subsequent illumination of S . acidocaldarius cells with visible light (photoreactivation) . Thus, the observed responses are probably initiated by the formation of pyrimidine dimers in the S . acidocaldarius chromosome . To our knowledge, these results provide the first evidence of error-prone DNA repair and genetic recombination induced by DNA damage in an archaeon from geothermal habitats.

Arch Environ Contam Toxicol, 1997 Aug, 33(2), 109 - 16
Bacillus stearothermophilus as a model to evaluate membrane toxicity of a lipophilic environmental pollutant (DDT); Donato MM et al.; The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify DDT-promoted events in biological membranes putatively related with the insecticide toxicity . Two strategies have been approached: a) bacterial growth and viability were followed and the effects of DDT (2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane) determined; b) biophysical studies with fluorescent probes were performed to elucidate the effects of DDT on the organization of the membrane lipid bilayer . The effects of DDT on growth and physical properties of the membrane were also determined in the presence of Ca2+ to further identify the interference of the insecticide at the membrane level and its putative contribution to cell toxicity . Growth inhibition by DDT is concentration-dependent, being attenuated or removed by the addition of 2.5-mM Ca2+ to bacterial cultures . Consistently, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its propionic acid derivative (DPH-PA) exhibited opposite effects of Ca2+ and DDT on the physical state of bacterial polar lipid dispersions . Growth and viability of bacterial cells are affected by DDT concentrations lower than those able to induce detectable bulk fluidity alterations, indicating high sensitivity of the intact bacterial system to alterations in limited membrane domains not directly probed by fluorescent probes that only report the average behavior of membrane lipid population.

Appl Environ Microbiol, 1997 Sep, 63(9), 3732 - 5
Characterization of superoxide dismutase in Streptococcus thermophilus; Chang SK et al.; Streptococcus thermophilus AO54 possesses a single manganese-containing superoxide dismutase (MnSOD) . The enzyme was found to be insensitive to cyanide or to a modified H2O2 treatment . The enzyme is expressed in a growth-phase-dependent fashion, increasing three- to fourfold upon entry into stationary phase . The specific activity for MnSOD was the same under anaerobic or aerobic conditions and was not induced by the presence of paraquat under aerobic conditions.

Appl Environ Microbiol, 1997 Sep, 63(9), 3577 - 84
Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme; Dong G et al.; The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli . The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide . The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family . In particular, none of the consensus regions present in the alpha-amylase family could be identified . P . furiosus APU showed similarity to three proteins, including the P . furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A . The mature protein had a molecular weight of 89,000 . The recombinant P . furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Denaturating temperatures of above 100 degrees C were required for complete unfolding . The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5 . Ca2+ increased the enzyme activity, thermostability, and substrate affinity . The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.

Appl Environ Microbiol, 1997 Sep, 63(9), 3512 - 8
Structural characterization of the exocellular polysaccharides produced by Streptococcus thermophilus SFi39 and SFi12; Lemoine J et al.; We investigated the structures of the exopolysaccharides (EPSs) produced by Streptococcus thermophilus SFi39 and SFi12 . Both polymers were found to have molecular masses of greater than 2 x 10(6) Da . The SFi39 EPS consisted of D-glucose and D-galactose in a molar ratio of 1:1, whereas the SFi12 EPS was composed of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1 . Methylation analysis of and nuclear magnetic resonance spectra recorded from the native polysaccharide, as well as oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of the SFi39 EPS, which consists of the following tetrasaccharide repeating unit: {formula: see text} Similar spectra recorded only from the native polysaccharide were sufficient to allow the structural determination of the SFi12 EPS, which consists of the following hexasaccharide repeating unit: {formula: see text} This study shows that the texturizing properties of different S . thermophilus ropy strains are based on the production of EPSs exhibiting chemical similarities but structural differences.

RNA, 1997 Sep, 3(9), 1037 - 51
Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes; Tanner MA et al.; Tertiary interactions are important in the higher-order folding of catalytic RNAs . Recently, a base triple, joining the two major domains of the catalytic core, was determined in group I introns from the cyanobacterium Anabaena PCC7120 and the eukaryote Tetrahymena thermophila . This base triple involves the fifth base pair of P4 and the fifth base of the single-stranded region J8/7 . We made base pair and single-nucleotide substitutions in the fifth base pair of P4, a G-C in the wild-type Anabaena intron, and tested them for self-splicing activity . The results suggest a hydrogen bonding model in which only the C of the base pair interacts directly with the fifth base of J8/7 . Comparative sequence analysis was used to determine the different combinations of base triples that occur in approximately 450 natural group I introns identified to date . About 94% of the base triples analyzed are compatible with the proposed hydrogen bonding model . Disrupting this base triple in the Tetrahymena intron resulted in the disappearance of splicing intermediates (intron 3' exon and 5' exon), even though the first step of splicing was not affected . Restoration of the base triple by a compensatory mutation reverted the intermediates to wild-type levels . These results suggest that disruption of the base triple increases the rate of the second step of splicing or of a conformational change preceding the second step . Repositioning of the base triple to form a new set of interactions may be required for the second step of splicing.

J Biol Chem, 1997 Sep 5, 272(36), 22703 - 13
Domain structure of Thermus thermophilus UvrB protein . Similarity in domain structure to a helicase; Nakagawa N et al.; UvrB protein plays an essential role in the prokaryotic excision repair system . UvrB protein shows cryptic ATPase activity, DNA binding, helicase-like activity, and incision activity by interacting with UvrA or UvrC proteins . To reveal the structure-function relationship of this multifunctional protein, the domain structure of Thermus thermophilus UvrB protein (ttUvrB) was studied by limited proteolysis and denaturation experiments . Proteolytic profiles indicated that ttUvrB consists of four domains: the N domain (residues 2-105), M domain (106-455), C1 domain (456-590), and C2 domain (591-665) . The properties of the proteolytic fragments indicated the involvement of the respective domains in the functions of the protein as follows: the N and C1 domains are necessary for ATPase activity, the C1 domain is indispensable for DNA binding, and the N and/or M domains are involved in UvrA binding . The structural stability of the C1 and C2 domains was higher than that of the N and M domains, which supports the proposed domain nature of ttUvrB . Based on these results and the crystal structure of PcrA helicase (Subramanya, H . S., Bird, L . E., Brannigan, J . A., and Wigley, D . B . (1996) Nature 384, 379-383), the domain organization of ttUvrB was proposed.

Nucleic Acids Res, 1997 Sep 1, 25(17), 3403 - 7
Effects of base mismatches on joining of short oligodeoxynucleotides by DNA ligases; Pritchard CE et al.; The requirement for Watson-Crick base pairing surrounding a nick in duplex DNA to be sealed by DNA ligase is the basis for oligonucleotide ligation assays that distinguish single base mutations in DNA targets . Experiments in a model system demonstrate that the minimum length of oligonucleotide that can be joined differs for different ligases . Thermus thermophilus (Tth) DNA ligase is unable to join any oligonucleotide of length six or less, while T4 DNA ligase and T7 DNA ligase are both able to join hexamers . The rate of oligonucleotide ligation by Tth DNA ligase increases between heptamer and nonamer . Mismatches which cause the duplex to be shortened by fraying, at the end distal to the join, slow the ligation reaction . In the case of Tth DNA ligase, mismatches at the seventh and eighth position 5'to the nick completely inhibit the ligation of octamers . The results are relevant to mechanisms of ligation.

Curr Microbiol, 1997 Sep, 35(3), 180 - 5
STP2201, a chromosomal promoter sequence of Streptococcus thermophilus; Somkuti GA et al.; Analysis of the structural and functional properties of chromosomal DNA fragments of Streptococcus thermophilus ST128 delineated the promoter sequence STP2201 and identified its -35, -10 and Shine-Dalgarno regions . STP2201 was used in cloning vectors derived from small resident plasmids pER8 (2094 bp) and pER371 (2672 bp) of S . thermophilus strains to facilitate expression of a Streptomyces sp . marker gene (cholesterol oxidase) in lactic acid bacteria . Cell extracts of ST128 transformants converted up to 75% of cholesterol into 4-cholesten-3-one during 8 h of incubation.

J Mol Biol, 1997 Aug 29, 271(4), 629 - 44
Thermus thermophilus cytochrome-c552: A new highly thermostable cytochrome-c structure obtained by MAD phasing; Than ME et al.; The three-dimensional structure of cytochrome-c552 from Thermus thermophilus has been determined by the multiple anomalous dispersion technique using synchrotron radiation and refined to a resolution of 1.28 A . Data collection at 90 K and the recording of three data sets (f'-minimum: 7125 eV, f"-maximum: 7138 eV and reference for scaling: 10,077 eV) resulted in an initial electron density of very high quality at 2.1 A, which was readily interpretable for model building . The model was refined to an R value of 19.1% (Rfree=22.4%) at 1.28 A resolution using a fourth data set collected at a photon energy of 11,810 eV . Comparison of this thermophilic cytochrome with its mesophilic mitochondrial or bacterial counterparts reveals significant structural differences which are discussed with respect to their importance for thermostability and binding between this cytochrome and its corresponding ba3-oxidase . Amino acid sequence similarities to other class I cytochromes are very weak and entirely limited to the region around the CXXCH motif close to the N terminus . The N-terminal two-thirds of cytochrome-c552 cover spatial regions around the heme prosthetic group that are similar to those observed for other cytochromes . The actual secondary structural elements that are responsible for that shielding do not, however, correlate well to other structures . Only the N-terminal helix (containing the heme binding cysteine residues) aligns reasonably well with other class I cytochromes . The most striking differences that distinguish the present structure from all other class I cytochromes is the C-terminal one-third of the molecule that wraps around the remainder of the structure as a stabilizing clamp, the existence of an extended beta-sheet covering one edge of the heme and the lack of any internal water molecule.

Nature, 1997 Aug 21, 388(6644), 805 - 8
A second catalytic metal ion in group I ribozyme; Weinstein LB et al.; Although only a subset of protein enzymes depend on the presence of a metal ion for their catalytic function, all naturally occurring RNA enzymes require metal ions to stabilize their structure and for catalytic competence . In the self-splicing group I intron from Tetrahymena thermophila, several divalent metals can serve structural roles, but only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation . A study of a ribozyme reaction analogous to 5'-splice-site cleavage by guanosine uncovered the first metal ion with a definitive role in catalysis . Substitution of the 3'-oxygen of the leaving group with sulphur resulted in a metal-specificity switch, indicating an interaction between the leaving group and the metal ion . Here we use 3'-(thioinosylyl)-(3'-->5')-uridine, IspU, as a substrate in a reaction that emulates exon ligation . Activity requires the addition of a thiophilic metal ion (Cd2+ or Mn2+), providing evidence for stabilization of the leaving group by a metal ion in that step of splicing . Based on the principle of microscopic reversibility, this metal ion activates the nucleophilic 3'-hydroxyl of guanosine in the first step of splicing, supporting the model of a two-metal-ion active site.

Chromosoma, 1997 Sep, 106(4), 233 - 42
The intranuclear organization of normal, hemizygous and excision-deficient rRNA genes during developmental amplification in Tetrahymena thermophila; Ward JG et al.; In the ciliated protozoan, Tetrahymena thermophila, the diploid germinal micronucleus contains two allelic copies of the gene for ribosomal RNA (rDNA) . During genesis of new somatic macronuclei the germline rDNA gene is excised by developmentally programmed chromosome breakage and preferentially amplified to approximately 9, 000 copies . We have studied this process by fluorescence in situ hybridization . We find that initially rDNA amplification is restricted to two separate and highly confined regions of the nucleus . Analysis of nuclei that are hemizygous for the rDNA locus reveals that each focus of hybridization is derived from a single allele of the rDNA . As rDNA amplification progresses these two foci of hybridization disperse and spread throughout the macronucleus, eventually forming approximately 100-500 new nucleoli . These events are correlated with morphologically distinct developmental stages . We investigated the amplification of the C3 allele of the rDNA that confers a replication advantage over the B allele during vegetative propagation, and find no evidence for preferential amplification of the C3 early in rDNA maturation . We also show that the rmm 11 rDNA mutant allele, which is defective for developmentally programmed rDNA excision, can be amplified during the two-foci stage in mutant homozygotes and heterozygotes, but fails to amplify further and disperse into multiple nucleoli . These data indicate that amplification of the rmm 11 allele is not delayed during the initial rounds of amplification, and suggest that efficient excision is not required for this amplification to occur . We propose that rDNA amplification is a two-step process . First, the two rDNA alleles are independently amplified, while allelic copies remain closely associated . Later, copies of the rDNA disperse and are further amplified, presumably because rDNA excision has occurred, generating fully mature rDNA minichromosomes that are able to replicate to high copy number.

Biochemistry, 1997 Aug 19, 36(33), 10246 - 55
Solution conformation of a five-nucleotide RNA bulge loop from a group I intron; Luebke KJ et al.; We present the solution conformation, determined by NMR spectroscopy, of a five-nucleotide RNA bulge loop . The bulge interrupts the stem of a 25-nucleotide RNA hairpin, and its sequence and flanking sequences are those of a conserved bulge from a Group I intron . The secondary structure of the bulge loop in the hairpin context is that predicted by the secondary structure prediction algorithm of Zuker . It differs, however, from the secondary structure deduced from sequence covariation of the bulge in the context of the functionally folded Group I introns and observed in the crystal structure of an independently folding domain of the Group I intron from Tetrahymena thermophila . This difference represents an exception to the heierarchical model of RNA folding in which preformed elements of secondary structure interact to form a tertiary structure . The three-dimensional structure of the bulge loop is characterized by discontinuous base stacking . Adjacent adenines stack with each other and with the flanking double helices . However, the position of the central uracil is not well defined by NOE distance constraints and is a point of discontinuity in the base stacking.

Biochemistry, 1997 Aug 19, 36(33), 9983 - 94
The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,; Russell RJ et al.; The crystal structure of the closed form of citrate synthase, with citrate and CoA bound, from the hyperthermophilic Archaeon Pyrococcus furiosus has been determined to 1.9 A . This has allowed direct structural comparisons between the same enzyme from organisms growing optimally at 37 degrees C (pig), 55 degrees C (Thermoplasma acidophilum) and now 100 degrees C (Pyrococcus furiosus) . The three enzymes are homodimers and share a similar overall fold, with the dimer interface comprising primarily an eight alpha-helical sandwich of four antiparallel pairs of helices . The active sites show similar modes of substrate binding; moreover, the structural equivalence of the amino acid residues implicated in catalysis implies that the mechanism proceeds via the same acid-base catalytic process . Given the overall structural and mechanistic similarities, it has been possible to make detailed structural comparisons between the three citrate synthases, and a number of differences can be identified in passing from the mesophilic to thermophilic to hyperthermophilic citrate synthases . The most significant of these are an increased compactness of the enzyme, a more intimate association of the subunits, an increase in intersubunit ion pairs, and a reduction in thermolabile residues . Compactness is achieved by the shortening of a number of loops, an increase in the number of atoms buried from solvent, an optimized packing of side chains in the interior, and an absence of cavities . The intimate subunit association in the dimeric P . furiosus enzyme is achieved by greater complementarity of the monomers and by the C-terminal region of each monomer folding over the surface of the other monomer, in contrast to the pig enzyme where the C-terminus has a very different fold . The increased number of intersubunit ion pairs is accompanied by an increase in the number involved in networks . Interestingly, all loop regions in the P . furiosus enzyme either are shorter or contain additional ion pairs compared with the pig enzyme . The possible relevance of these structural features to enzyme hyperthermostability is discussed.

FEBS Lett, 1997 Aug 18, 413(2), 194 - 6
Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters; Ledent P et al.; The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase . This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 387 - 92
Expression of small RNAs by Bacillus sp . strain PS3 and B . subtilis cells during sporulation; Fink PS et al.; A small RNA sequence identified in an rRNA-tRNA cluster from the thermophilic Bacillus sp . strain PS3 was examined . An oligonucleotide probe specific for the RNA bound to multiple restriction fragments in Bacillus sp . strain PS3 DNA, thus several copies of this sequence occur in its genome . Similar findings were observed using DNA from B . subtilis, B . stearothermophilus, Escherichia coli, Staphylococcus aureus, Haemophilus influenzae and Thermus thermophilus . This sequence apparently is widespread in the eubacteria . Northern analysis of RNA from sporulating Bacillus sp . strain PS3 and B . subtilis cells revealed RNA species homologous to the probe in both bacteria . Expression of the small RNA in B . subtilis depended on sigma H.

FEBS Lett, 1997 Aug 11, 413(1), 55 - 9
A single mutation at the catalytic site of TF1-alpha3beta3gamma complex switches the kinetics of ATP hydrolysis from negative to positive cooperativity; Muneyuki E et al.; Previously, we reported the substitution of Tyr341 of the F1-ATPase beta subunit from a thermophilic Bacillus strain PS3 with leucine, cysteine, or alanine (M . Odaka et al . J . Biochem., 115 (1994) 789-796) . These mutations resulted in a great decrease in the affinity of the isolated beta subunit for ATP-Mg and an increase in the apparent Km of the alpha3beta3gamma complex in ATP hydrolysis when examined above 0.1 mM ATP . Here, we examined the ATPase activity of the mutant complexes in a wide range of ATP concentration and found that the mutants exhibited apparent positive cooperativity in ATP hydrolysis . This is the first clear demonstration that a single mutation in the catalytic sites converts the kinetics from apparent negative cooperativity in the wild-type alpha3beta3gamma complex to apparent positive cooperativity . The conversion of apparent cooperativity could be explained in terms of a simple kinetic scheme based on the binding change model proposed by Boyer.

FEBS Lett, 1997 Aug 4, 412(3), 633 - 6
K+ is an indispensable cofactor for GrpE stimulation of ATPase activity of DnaK x DnaJ complex from Thermus thermophilus; Motohashi K et al.; K+ is an indispensable cofactor for ATPase activity of eukaryotic cytosolic Hsp70 chaperone systems which lack a GrpE homolog . In the case of the bacterial Hsp70 (DnaK) system, GrpE, a nucleotide exchange factor, stimulates ATPase activity but little is known about the effect of K+ . Here, we have cloned a grpE gene from a thermophile, Thermus thermophilus, and purified a homodimeric GrpE protein . Using proteins of this bacterium, we found that the GrpE stimulation of ATPase activity of DnaK x DnaJ complex was absolutely dependent on the presence of K+.

Virology, 1997 Aug 4, 234(2), 372 - 82
A highly conserved DNA replication module from Streptococcus thermophilus phages is similar in sequence and topology to a module from Lactococcus lactis phages; Desiere F et al.; A highly conserved DNA region extending over 5 kb was observed in Streptococcus thermophilus bacteriophages . Comparative sequencing of one temperate and 26 virulent phages demonstrated in the most extreme case an 18% aa difference for a predicted protein, while the majority of the phages showed fewer, if any aa changes . The relative degree of aa conservation was not homogeneous over the DNA segment investigated . Sequence analysis of the conserved segment revealed genes possibly involved in DNA transactions . Three predicted proteins (orf 233, 443, and 382 gene product (gp)) showed nucleoside triphosphate binding motifs . Orf 443 gp showed in addition a DEAH box motif, characteristically found in a subgroup of helicases, and a variant zinc finger motif known from a phage T7 helicase/primase . Tree analysis classified orf 443 gp as a distant member of the helicase superfamily . Orf 382 gp showed similarity to putative plasmid DNA primases . Downstream of orf 382 a noncoding repeat region was identified that showed similarity to a putative minus origin from a cryptic S . thermophilus plasmid . Four predicted proteins showed not only high degrees of aa identity (34 to 63%) with proteins from Lactococcus lactis phages, but their genes showed a similar topological organization . We interpret this as evidence for a horizontal gene transfer event between phages of the two bacterial genera in the distant past.

Eur J Biochem, 1997 Aug 1, 247(3), 1158 - 65
The regulatory functions of the gamma and epsilon subunits from chloroplast CF1 are transferred to the core complex, alpha3beta3, from thermophilic bacterial F1; Hisabori T et al.; The expression plasmids for the subunit gamma (gamma(c)) and the subunit epsilon (epsilon(c)) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli . Both expressed subunits were obtained as inclusion bodies . When recombinant gamma(c) was mixed with recombinant alpha and beta subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (alpha3beta3gamma(c)) was reconstituted and it showed significant ATP hydrolysis activity . The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in gamma(c) . Hence, this complex has similar modulation characteristics as CF1 . The effects of recombinant epsilon(c) and epsilon subunit from TF1 (epsilon(t)) on alpha3beta3gamma(c) were also investigated . Epsilon(c) strongly inhibited the ATP hydrolysis activity of chimeric alpha3beta3gamma(c) complex but epsilon(t) did not . The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium . The addition of epsilon(c) or epsilon(t) to the alpha3beta3gamma(t) complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity . These results suggest that (a) the specific regulatory function of gamma(c) can be transferred to the bacterial subunit complex; (b) the interaction between the gamma(c) subunit and epsilon(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the alpha3beta3 core.

Eur J Biochem, 1997 Aug 1, 247(3), 1046 - 55
Biochemical characterisation of ornithine carbamoyltransferase from Pyrococcus furiosus; Legrain C et al.; Ornithine carbamoyltransferase (OTCase) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus . The enzyme is a 400 +/- 20-kDa polymer of a 35-kDa subunit, in keeping with the corresponding gene sequence {Roovers, M., Hethke, C., Legrain, C., Thomm, M . & Glansdorff, N . (1997) Isolation of the gene encoding Pyrococcus furiosus ornithine cabamoyltransferase and study of its expression profile in vivo and in vitro, Eur . J . Biochem . 247, 1038-1045} . In contrast with the dodecameric catabolic OTCase of Pseudomonas aeruginosa, P . furiosus OTCase exhibits no substrate cooperativity . In keeping with other data discussed in the text, this suggests that the enzyme serves an anabolic function . Half-life estimates for the purified enzyme ranged over 21-65 min at 100 degrees C according to the experimental conditions and reached several hours in the presence of ornithine and phosphate . The stability was not markedly influenced by the protein concentration . Whereas comparative examination of OTCase sequences did not point to any outstanding feature possibly related to thermophily, modelling the enzyme on the X-ray structure of P . aeruginosa OTCase (constituted by four trimers assembled in a tetrahedral manner) suggests that the molecule is stabilized, at least in part, by a set of hydrophobic interactions at the interfaces between the trimers . The comparison between P . aeruginosa and P . furiosus OTCases suggests that two different properties, allostery and thermostability, have been engineered starting from a similar quaternary structure of high internal symmetry . Recombinant P . furiosus OTCase synthesised by Escherichia coli proved less stable than the native enzyme . In Saccharomyces cerevisiae, however, an enzyme apparently identical to the native one could be obtained.

Int J Biol Macromol, 1997 Aug, 21(1-2), 73 - 9
Structural characterization of three aldobiuronic acids derived from the capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus; Gloaguen V et al.; This study deals with the chemical characterization of a capsular polysaccharide (CPS) produced by a thermal biomass formed by the cyanobacterium Mastigocladus laminosus . Acid hydrolysis performed on the purified polysaccharide has led to the isolation of several acid-resistant oligosaccharides . Two of them have already been reported and assigned as: alpha - GlcA - (1 --> 2) - alpha - GalA - (1 --> 2) - Man . and alpha - GlcA - (1 --> 2) - alpha - GalA - (1 --> 2) - beta - Man - (1 --> 4) - beta - Gal(1 --> 2) - Rha . In this report, results on the isolation and partial purification of three supplementary oligosaccharidic units are presented . Gas chromatography-mass spectrometry (GC-MS) and H-nuclear magnetic resonance (NMR) spectroscopy investigations allowed them to be assigned as three aldobiuronic acids with the following structures: alpha - GlcA - (1 --> 3) - Gal alpha - GlcA - (1 --> 3) - Fuc alpha - GalA - (1 --> 3) - Fuc.

J Appl Microbiol, 1997 Aug, 83(2), 199 - 207
A study of the microflora of some recycled fibre pulps, boards and kitchen rolls; Suihko ML et al.; Current methodology used for studying the microflora of pulps and boards was assessed and some improvements are recommended . Microbiological quality of 37 samples including recycled fibre pulps, boards, kitchen rolls, virgin fibre sheets and circulating process water were investigated . The papermaking process had drastically reduced the total microbial counts . The dominant microflora in all the samples were aerobic bacteria . The amounts in boards were only 10(3)-10(6) cfu g-1 d.w., whereas the untreated pulps contained 10(8)-10(10) cfu g-1 d.w . Aerobic, anaerobic and facultatively anaerobic spore-forming bacteria formed a large group in the bacterial flora of pulp samples (10(3)-10(6) and 10(2)-10(4) cfu g-1 d.w., respectively) . In the boards the maximum numbers of aerobic spore-forming bacteria were about 10(4) cfu g-1 d.w . and the numbers of anaerobic and facultatively anaerobic spore formers were negligible . Moulds were common in the untreated pulp sampled at 10(2)-10(6) cfu g-1 d.w., but their occurrence in boards was close to the detection limit . Yeasts were common only in the pulps of one mill, and were found to be present in the circulation process water . Both mesophilic and thermophilic actinomycetes were detected in pulps at levels up to 10(2)-10(5) cfu g-1 d.w . However, no mesophilic actinomycetes were detected in boards, although some boards contained up to 10(2) cfu g-1 d.w . of thermophilic actinomycetes . The virgin fibre sheets were practically free of microbes . Only a few bacterial colonies were detected from the kitchen rolls.

Protein Expr Purif, 1997 Aug, 10(3), 356 - 64
Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, purification, and partial characterization; Jensen HK et al.; The upp gene encoding the major uracil phosphoribosyltransferase (UPRT) of the thermophile Bacillus caldolyticus was cloned by complementation of an Escherichia coli upp mutation . The nucleotide sequence of the cloned DNA revealed an open reading frame of 630 bp encoding a polypeptide of 209 amino acids (M(r) 22,817) with 84% amino acid sequence identity to the deduced upp gene product of Bacillus subtilis . Primer extension analysis indicated that the transcriptional start site of the cloned gene was positioned 37 or 38 bp upstream of the coding region . When over-expressed in E . coli, the recombinant UPRT represented approximately 18% of the soluble cellular proteins . The enzyme was purified to homogeneity by two sequential precipitations with 50 mM Na-phosphate, pH 7.0 . Gel filtration chromatography indicated that the native enzyme existed as a dimer at high protein concentrations but that it dissociated to a monomeric form on dilution . In dilute solutions the enzyme is highly unstable but can be stabilized by addition of bovine serum albumin . In concentrated solution (> 5 mg/ml) the enzyme is stable for months at 4 degrees C, even in the absence of bovine serum albumin . By comparing the UPRT activity of crude extracts of B . subtilis and B . caldolyticus it was found that the enzyme from B . caldolyticus was considerably more stable toward thermal inactivation than the homologous enzyme from B . subtilis.

Curr Opin Biotechnol, 1997 Aug, 8(4), 423 - 8
Stabilization of protein structures; Lee B et al.; The technique of protein stabilization has been improving steadily in recent years, but it is only in the past year or two that the stability of some protein molecules has been improved to the level of those from extreme thermophilic organisms . This was achieved by multiple mutations and often by utilizing the knowledge gained from the homologous protein structures from extreme thermophiles.

J Bacteriol, 1997 Aug, 179(16), 5165 - 70
D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH; Howell DM et al.; The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea . In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion . In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes . A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate . Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M . thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.

J Bacteriol, 1997 Aug, 179(16), 5072 - 5
Gene cloning and expression and characterization of a toxin-sensitive protein phosphatase from the methanogenic archaeon Methanosarcina thermophila TM-1; Solow B et al.; With oligonucleotides modelled after conserved regions within the protein-serine/threonine phosphatases (PPs) of the PP1/2A/2B superfamily, the gene for the archaeal protein phosphatase PP1-arch2 was identified, cloned, and sequenced from the methanogenic archaeon Methanosarcina thermophila TM-1 . The DNA-derived amino acid sequence of PP1-arch2 exhibited a high degree of sequence identity, 27 to 31%, with members of the PP1/2A/2B superfamily such as PP1-arch1 from Sulfolobus solfataricus, PP1alpha from rats, PP2A from Saccharomyces cerevisiae, and PP2B from humans . The activity of the recombinant PP1-arch2 was sensitive to several naturally occurring microbial toxins known to potently inhibit eucaryal PP1 and PP2A, including microcystin-LR, okadaic acid, tautomycin, and calyculin A.

J Bacteriol, 1997 Aug, 179(16), 4963 - 9
Cell cycle characteristics of thermophilic archaea; Bernander R et al.; We have performed a cell cycle analysis of organisms from the Archaea domain . Exponentially growing cells of the thermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius were analyzed by flow cytometry, and several unusual cell cycle characteristics were found . The cells initiated chromosome replication shortly after cell division such that the proportion of cells with a single chromosome equivalent was low in the population . The postreplication period was found to be long; i.e., there was a considerable time interval from termination of chromosome replication until cell division . A further unusual feature was that cells in stationary phase contained two genome equivalents, showing that they entered the resting stage during the postreplication period . Also, a reduction in cellular light scatter was observed during entry into stationary phase, which appeared to reflect changes not only in cell size but also in morphology and/or composition . Finally, the in vivo organization of the chromosome DNA appeared to be different from that of eubacteria, as revealed by variation in the relative binding efficiency of different DNA stains.

Nat Struct Biol, 1997 Aug, 4(8), 650 - 6
Crystal structure of the EF-Tu.EF-Ts complex from Thermus thermophilus; Wang Y et al.; In order to study nucleotide exchange mechanisms in GTP-binding proteins, we have determined the crystal structure of the complex formed by the elongation factor Tu (EF-Tu) and its exchange factor Ts (EF-Ts) from Thermus thermophilus . The complex is a dyad symmetrical heterotetramer in which each EF-Tu, through a bipartite interface, interacts with two subunits of EF-Ts, explaining the need for a dimeric exchange factor . The architecture of the assembly is distinctly different from that of the corresponding heterodimeric E . coli complex, in which the monomeric E . coli EF-Ts remarkably forms essentially the same bipartite interface with EF-Tu through a sequence/structural repeat . GDP is released primarily by a Ts-induced peptide flip in the nucleotide binding pocket that disrupts hydrogen bonds to the phosphates and repositions the peptide carbonyl so as to sterically and electrostatically eject the GDP . The exchange mechanism may have useful implications for receptor-induced exchange in heterotrimeric G proteins.

Appl Environ Microbiol, 1997 Aug, 63(8), 3297 - 300
Expression and secretion of a thermostable bacterial xylanase in Kluyveromyces lactis; Walsh DJ et al.; The xynA structural gene from the extremely thermophilic anaerobe Dictyoglomus thermophilum Rt46B.1 was fused in frame with the secretion signal of the Kluyveromyces lactis killer toxin in episomal expression vectors based on the Kluyveromyces plasmid pKD1 . XynA was secreted predominantly as an unglycosylated 35-kDa protein which comprised up to 90% of the total extracellular proteins and reached a concentration of 130 micrograms/ml in shake-flask cultures grown under selective conditions.

Appl Environ Microbiol, 1997 Aug, 63(8), 3246 - 53
Two groups of bacteriophages infecting Streptococcus thermophilus can be distinguished on the basis of mode of packaging and genetic determinants for major structural proteins; Le Marrec C et al.; A comparative study of 30 phages of Streptococcus thermophilus was performed based on DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and host range data . All phages exhibited distinct DNA restriction profiles, with some phages displaying similarly sized restriction fragments . DNA homology was shown to be present among all 30 phages . The phages could be divided into two groups on the basis of their packaging mechanism as was derived from the appearance of submolar DNA fragments in restriction enzyme digests and the presence (cos-containing phages) or absence (pac-containing phages) of cohesive genomic extremities . Interestingly, the 19 identified cos-containing phages possessed two major structural proteins (32 and 26 kDa) in contrast to the remaining 11 pac-containing phages, which possessed three major structural proteins (41, 25, and 13 kDa) . Southern hybridization demonstrated that all pac-containing phages tested contain homologs of the genes encoding the three major structural proteins of the pac-containing phage O1205, whereas all cos-containing phages tested exhibit homology to the gene specifying one of the structural components of the cos-containing phage phi 7201 . Fifty-seven percent of the phages (both cos and pac containing) possessed the previously identified 2.2-kb EcoRI fragment of the temperate S . thermophilus phage Sfi18 (H . Brussow, A . Probst, M . Fremont, and J . Sidoti, Virology 200:854-857, 1994) . No obvious correlation was detected between grouping based on packaging mechanism and host range data obtained with 39 industrial S . thermophilus strains.

Appl Environ Microbiol, 1997 Aug, 63(8), 3151 - 7
Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae; Berka RM et al.; A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined . The deduced amino acid sequence of M . thermophila laccase (MtL) shows homology to laccases from diverse fungal genera . A vector containing the M . thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A . oryzae . The recombinant laccase expressed in A . oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography . Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme . The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation . The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit . With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min . This is the first report of the cloning and heterologous expression of a thermostable laccase.

Appl Environ Microbiol, 1997 Aug, 63(8), 3144 - 50
Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory; Bruttin A et al.; A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory . The two hypotheses led to different predictions about the genetic diversity of the phages . With respect to host range, 12 distinct phage types were observed . With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns . Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types . This diversity is as large as that between the most different sequences from phages in our collection . These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory . In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population . Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated . Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial . Apparently, all of the genetic diversity observed in the S . thermophilus phages isolated during our survey was already created in their natural environment . A better understanding of the raw-milk ecology of S . thermophilus phages is thus essential for successful practical phage control.

J Struct Biol, 1997 Aug, 119(3), 273 - 83
Structural Analysis of Photosystem II: Comparative Study of Cyanobacterial and Higher Plant Photosystem II Complexes
Hasler L, Ghanotakis D, Fedtke B, Spyridaki A, Miller M, Müller SA, Engel A, Tsiotis G.
Oxygen evolving photosystem II (PSII-OEC) complexes and PSII core complexes were isolated from spinach and the thermophilic cyanobacterium Synechococcus sp . OD24 and characterized by gel electrophoresis, immunoblotting, and absorbance spectroscopy . The mass of the core complexes was determined by scanning transmission electron microscopy (STEM) and found to be 281 ± 65 kDa for spinach and 313 ± 52 kDa for Synechococcus sp . OD24 . The mass of the spinach PSII-OEC complex was 327 ± 64 kDa . Digital images of negatively stained PSII-OEC and PSII core complexes were recorded by STEM and analyzed by single particle averaging . All monomeric complexes showed similar morphologies and were of comparable length (14 nm) and width (10 nm) . The averages revealed a pseudo-twofold symmetry axis, which is a prominent structural element of the monomeric form . Difference maps between the averaged projections of the oxygen evolving complexes and the core complexes from both species indicated where the 33-kDa extrinsic manganese stabilizing protein is bound . A symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex, is proposed.

J Bacteriol, 1997 Aug, 179(15), 4859 - 67
The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum; Wahlund TM et al.; Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria . One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum . The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000 . Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase . In addition to apparent structural similarities, the catalytic properties of C . tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola . Phosphorylation of C . tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme . In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C . tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity . Given the apparent structural and catalytic similarities of the enzyme from C . tepidum and its eukaryotic counterpart, the C . tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.

J Bacteriol, 1997 Aug, 179(15), 4811 - 4
A new Thermus-Escherichia coli shuttle integration vector system; Tamakoshi M et al.; We established a Thermus thermophilus strain in which the pyrE gene (coding for orotate phosphoribosyltransferase of the pyrimidine biosynthetic pathway) was totally deleted . We also constructed an integration vector, which consisted of the Escherichia coli plasmid vector pBluescript and a 2.1-kb segment of the T . thermophilus leu operon sequence, for the integration of a foreign gene into a chromosome of the thermophile . pyrE and leuB genes were used as probes to test the integration vector . The integration vector pINV, bearing the pyrE gene, transformed the delta pyrE strain at a frequency of 6 x 10(-5) through a single crossover event . The leuB gene could also be used as another marker of the integration vector system . The vector could be integrated at the expected site . By digesting the chromosomal DNA of the T . thermophilus transformants with a unique restriction enzyme, the vector could be recovered into E . coli after the recircularization in vitro . The kanamycin nucleotidyltransferase gene could be successfully expressed in the thermophile by using pINV.

Arch Microbiol, 1997 Aug, 168(2), 114 - 9
Thermoanaerobacter mathranii sp . nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland; Larsen L et al.; The extremely thermophilic ethanol-producing strain A3 was isolated from a hot spring in Iceland . The cells were rod-shaped, motile, and had terminal spores; cells from the mid-to-late exponential growth phase stained gram-variable but had a gram-positive cell wall structure when viewed by transmission electron microscopy . Strain A3 used a number of carbohydrates as carbon sources, including xylan, but did not utilize microcrystalline cellulose . Fermentation end products were ethanol, acetate, lactate, CO2, and H2 . The temperature optimum for growth was between 70 and 75 degrees C, and growth occurred in the range of 50-75 degrees C . The pH range for growth was 4.7-8.8, with an optimum at pH 7.0 . Strain A3 was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, and vancomycin at 100 mg/l but was not sensitive to chloramphenicol and neomycin at 10 mg/l, which indicates that strain A3 belongs to the eubacteria . Addition of 50.66 kPa H2 or 2% NaCl did not affect growth . The isolate grew in the presence of exogenously added 4% (w/v) ethanol . The G+C ratio was 37 mol% . 16S rDNA studies revealed that strain A3 belongs to the genus Thermoanaerobacter . Genotypic and phenotypic differences between strain A3 and other related species indicate that strain A3 can be assigned to a new species, and the name Thermoanaerobacter mathranii is proposed.

Arch Microbiol, 1997 Aug, 168(2), 92 - 101
Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping; Moreira LM et al.; Fifty isolates belonging to the six validly described species of the genus Thermus (T . aquaticus, T . filiformis, T . thermophilus, T . scotoductus, T . brockianus, and T . oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping . With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct . However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring . The estimated genomic size of strains of the Thermus spp . varied between approximately 1.8 and 2.5 Mbp . Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively . In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring.

Arch Microbiol, 1997 Aug, 168(2), 73 - 80
Recent advances in genetic analyses of hyperthermophilic archaea and bacteria; Noll KM et al.; Hyperthermophilic Archaea and Bacteria are an extraordinarily important class of organisms for which genetic tools remain to be developed . Unique technological obstacles to this goal are posed by the thermophilic and, in some cases, strictly anaerobic nature of these organisms . However, recent advances in the cultivation of hyperthermophiles, in the discovery of genetic elements for vector development, and in the construction of genetic markers point toward the achievement of this goal in the near future . Transformation protocols have already been reported for Sulfolobus and Pyrococcus, and plasmid-mediated conjugation was recently found in Sulfolobus . Plasmids are available for Sulfolobus, Pyrococcus, and the bacterial hyperthermophile Thermotoga, and these provide the bases for vector construction in these hosts . A Desulfurococcus mobile intron may provide a novel means to introduce genes into a variety of archaeal hosts . With full genome sequences of several hyperthermophiles available soon, genetic tools will allow full exploitation of this information to study these organisms in depth and to utilize their unique properties in biotechnological applications.

Mol Cell Biol, 1997 Aug, 17(8), 4517 - 25
Type I elements mediate replication fork pausing at conserved upstream sites in the Tetrahymena thermophila ribosomal DNA minichromosome; MacAlpine DM et al.; Two-dimensional gel electrophoresis was used to study replication of the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome . During vegetative growth, the rDNA is replicated exclusively from origins in the 5' nontranscribed spacer (NTS) . Whereas replication fork movement through the rest of the chromosome appears to be continuous, movement through the 5' NTS is not . Replication forks arrest transiently at three prominent replication fork pausing sites (RFPs) located in or immediately adjacent to nucleosome-free regions of the 5' NTS . Pausing at these sites is dramatically diminished during replication in Escherichia coli, suggesting that chromatin organization or Tetrahymena-specific proteins may be required . A conserved tripartite sequence was identified at each pausing site . Mutations in type I elements diminish pausing at proximal RFPs . Hence, type I elements, previously shown to control replication initiation, also regulate elongation of existing replication forks . Studies with rDNA transformants revealed a strong directional bias for fork pausing . Strong pausing only occurred in forks moving toward the rRNA-coding region . We propose that fork pausing in the 5' NTS evolved to synchronize replication and transcription of the downstream rRNA genes.

Curr Microbiol, 1997 Aug, 35(2), 116 - 21
Heat-shock response in Methanosarcina mazei S-6; Lange M et al.; The dnaK locus of Methanosarcina mazei S-6, a mesophilic organism of the phylogenetic domain Archaea, contains the heat-shock genes 5'-grpE-dnaK-dnaJ-3' . Parameters known to affect the response of these genes in organisms of the other two domains, Bacteria and Eucarya, were tested to determine their effects on the archaeal homologs . The mRNA from the three genes increased after heat shock more in lamina than in single cells (these S-6 morphologic stages can be grown in the same substrate) . Single cells in early stationary phase showed the highest levels of dnaK mRNA after heat shock, as compared with cells in exponential, or in late stationary, phase . The dnaK mRNA always had the size of a monocistronic transcript . dnaK was also found in the thermophileMethanosarcina thermophila TM-1, and its response to heat shock showed distinctive characteristics . However, dnaK was not revealed in other archaea: three hyperthermophiles (Methanothermus fervidus,Methanococcus jannaschii, and Sulfolobus sp.), and one mesophilic methanogen (Methanospirillum hungateii).

Biochem Biophys Res Commun, 1997 Jul 30, 236(3), 727 - 32
Purification and molecular cloning of the group II chaperonin from the acidothermophilic archaeon, Sulfolobus sp . strain 7; Nakamura N et al.; To elucidate the structure and functional mechanism of the group II chaperonin, molecular cloning of the gene for and purification of the group II chaperonin from the thermoacidophilic archaeon Sulfolobus sp . strain 7 were performed . The purified Sulfolobus chaperonin exhibited weak ATPase activity and arrested the spontaneous refolding of the thermophilic lactate dehydrogenase . However, the refolding could not be resumed by addition of ATP . The chaperonin consists of two kinds of subunits, alpha and beta, the deduced amino acid sequences of which were highly homologous to those of TF56 and TF55 from Sulfolobus shibatae, respectively.

J Biotechnol, 1997 Jul 23, 56(1), 1 - 24
Biological conversion of lignocellulosic biomass to ethanol; Lee J; The important key technologies required for the successful biological conversion of lignocellulosic biomass to ethanol have been extensively reviewed . The biological process of ethanol fuel production utilizing lignocellulose as substrate requires: (1) delignification to liberate cellulose and hemicellulose from their complex with lignin, (2) depolymerization of the carbohydrate polymers (cellulose and hemicellulose) to produce free sugars, and (3) fermentation of mixed hexose and pentose sugars to produce ethanol . The development of the feasible biological delignification process should be possible if lignin-degrading microorganisms, their echophysiological requirements, and optimal bioreactor design are effectively coordinated . Some thermophilic anaerobes and recently-developed recombinant bacteria have advantageous features for direct microbial conversion of cellulose to ethanol, i.e . the simultaneous depolymerization of cellulosic carbohydrate polymers with ethanol production . The new fermentation technology converting xylose to ethanol needs also to be developed to make the overall conversion process more cost-effective . The bioconversion process of lignocellulosics to ethanol could be successfully developed and optimized by aggressively applying the related novel science and technologies to solve the known key problems of conversion process.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7851 - 6
A DnaB intein in Rhodothermus marinus: indication of recent intein homing across remotely related organisms; Liu XQ et al.; A dnaB gene encoding a homologue of the Escherichia coli DNA helicase DnaB was cloned and sequenced in the thermophilic eubacterium Rhodothermus marinus, predicting a DnaB protein that harbors an intein . This DnaB intein is 428 amino acid residues long, has several putative intein sequence motifs (including two putative endonuclease motifs), and is capable of protein splicing when produced in E . coli cells . The R . marinus DnaB intein is a close homologue of a DnaB intein in the cyanobacterium Synechocystis sp . strain PCC6803 . The two inteins are positioned identically in their respective DnaB proteins . They also share a 54% sequence identity (74% sequence similarity) that is markedly higher than the 37% sequence identity shared by the extein sequences of the two DnaB proteins . Horizontal intein transfer (homing) is therefore invoked to relate these two DnaB inteins . The codon usage of R . marinus DnaB intein coding sequence differs markedly from the codon usages of its flanking extein coding sequences and other genes in the same genome, suggesting more recent acquisition of the DnaB intein in this organism.

Biochemistry, 1997 Jul 22, 36(29), 8904 - 13
Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H2O or D2O; Karge M et al.; Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C </= vartheta </= 25 degrees C in dark-adapted PS II core complexes from spinach {O2 evolution rate: 1500 +/- 100 micromol of O2 (mg of Chl)-1 h-1} that were dissolved either in H2O- or in D2O-containing buffer . Comparative measurements were performed at 20 degrees C in H2O- or D2O-containing suspensions of PS II membrane fragments {O2 evolution rate: 600 +/- 40 micromol of O2 (mg of Chl)-1 h-1} . The results obtained reveal the following: (a) The activation energies of the individual redox steps in the water oxidizing complex (WOC) are dependent on the redox state Si with EA(S1-->S2) = 14 kJ/mol, EA(S2-->S3) = 35 kJ/mol, and EA(S3-->-->S0 + O2) = 21 kJ/mol for vartheta > 11 degrees C, 67 kJ/mol for vartheta < 11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes (</=15%) of the activation energies; and (c) the rate constants of these reactions in PS II core complexes are characterized by H/D isotope ratios, ki(H)/ki(D), of 1.6, 2.3, and 1.5 for the transitions S1 --> S2, S2 --> S3, and S3 -->--> S0 + O2, respectively . The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1 . 4 . Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach {Renger, G., & Hanssum, B . (1992) FEBS Lett . 299, 28-32} and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland {Koike, H., Hanssum, B., Inoue, Y., & Renger, G . (1987) Biochim . Biophys . Acta 893, 524-533}, the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants . Furthermore, the rather high activation energy of the S2 --> S3 transition provides evidence for a significant structural change coupled with this reaction . Implications for the mechanism of photosynthetic water oxidation are discussed.

Biochemistry, 1997 Jul 22, 36(29), 8785 - 97
Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus . Structural and biochemical properties of the two enzymes; Becker HD et al.; Two aspartyl-tRNA synthetases (AspRSs) were isolated from Thermus thermophilus HB8 . Both are alpha2 dimers but differ in the length of their polypeptide chains (AspRS1, 68 kDa; and AspRS2, 51 kDa) . Both chains start with Met and are deprived of common sequences to a significant extent . This rules out the possibility that AspRS2 is derived from AspRS1 by proteolysis, in agreement with specific recognition of each AspRS by the homologous antibodies . DNA probes derived from N-terminal amino acid sequences hybridize specifically to different genomic DNA fragments, revealing that the two AspRSs are encoded by distinct genes . Both enzymes are present in various strains from T . thermophilus and along the growth cycle of the bacteria, suggesting that they are constitutive . Kinetic investigations show that the two enzymes are specific for aspartic acid activation and tRNAAsp charging . tRNA aspartylation by the thermostable AspRSs is governed by thermodynamic parameters which values are similar to those measured for mesophilic aspartylation systems . Both thermophilic AspRSs are deprived of species specificity for tRNA aspartylation and exhibit N-terminal sequence signatures found in other AspRSs, suggesting that they are evolutionarily related to AspRSs from mesophilic prokaryotes and eukaryotes . Comparison of the efficiency of tRNA aspartylation by each enzyme under conditions approaching the physiological ones suggests that in vivo tRNAAsp charging is essentially ensured by AspRS1, although AspRS2 is the major species . The physiological significance of the two different AspRSs in T . thermophilus is discussed.

Biochemistry, 1997 Jul 22, 36(29), 8733 - 42
The role of phenylalanine 31 in maintaining the conformational stability of ribonuclease P2 from Sulfolobus solfataricus under extreme conditions of temperature and pressure; Mombelli E et al.; Ribonuclease P2 from the thermophilic archaebacterium Sulfolobus solfataricus is a small protein (7 kDa) with a known three-dimensional structure . Inspection of the structure and molecular dynamics simulation reveal that three aromatic residues (Phe5, Phe31, and Tyr33) from the hydrophobic core have a strong van der Waals interaction energy . We studied the thermodynamics of the heat, cold, and pressure-induced protein conformational changes of the wild type and of the F31A and F31Y mutants by analyzing the protein UV absorbance in the fourth derivative mode . The wild-type protein was extremely stable under all conditions of temperature and pressure . Heat and cold denaturation of both mutants, as well as denaturation by pressure of the F31A mutant, led to significant blue shifts of the derivative spectrum, indicating increased solvent exposure of Tyr33 . For the F31Y mutant, high pressure (400 MPa) protected the protein against thermal denaturation . This study, probing the properties of the hydrophobic aromatic core, complements a thermal unfolding study which probes the overall structural changes {Knapp, S., Karshikoff, A., Berndt, K . D., Christova, P., Atanasov, B., & Ladenstein, R . (1996) J . Mol . Biol . 264,1132-1144} . The differences observed in response to extremes of temperature, pressure, and pH may be rationalized by an unfolding mechanism involving larger parts of the peripheral protein while the integrity of the hydrophobic core is maintained.

Gene, 1997 Jul 18, 194(1), 133 - 6
Cloning and sequencing of the hrcA gene of Bacillus stearothermophilus; Mogk A et al.; We report on cloning and sequencing of a 2.0-kb PCR fragment of chromosomal DNA from thermophilic Bacillus stearothermophilus carrying the complete hrcA gene . In addition, this amplicon contains the 3' end of an open reading frame exhibiting significant homology to the hemN gene of Bacillus subtilis (Bs) and other bacterial species . The hrcA gene could complement an Bs hrcA deletion mutant by repressing expression of class I heat shock (HS) genes . Furthermore, we could show that the HrcA protein derived from the thermophilic microorganism responds to HS in a similar way as reported for the Bs HrcA protein.

Biochim Biophys Acta, 1997 Jul 18, 1340(2), 170 - 7
Nucleotide sequence of a gene cluster encoding NusG and the L11-L1-L10-L12 ribosomal proteins from the thermophilic archaeon Sulfolobus solfataricus; Geiger M et al.; The complete nucleotide sequence of a gene cluster encoding the NusG and the L 11-L1-L10-L12 ribosomal proteins from the thermophilic crenarchaeon Sulfolobus solfataricus has been determined . The genes are arranged in the same order as the equivalent genes in the rif region of Escherichia coli . The ribosomal proteins exhibit between 66% (L10) and 80% (L12) identity with their respective equivalents from Sulfolobus acidocaldarius . The short distance (5 nucleotides) between the nusG stop codon and the L11 start codon suggests that nusG and the genes for the ribosomal proteins are transcribed as a single unit.

J Biol Chem, 1997 Jul 18, 272(29), 18155 - 60
ATP-, K+-dependent heptamer exchange reaction produces hybrids between GroEL and chaperonin from Thermus thermophilus; Taguchi H et al.; Chaperonin from Thermus thermophilus (Tcpn6014.Tcpn107) splits at the plane between two Tcpn607 rings into two parts in a solution containing ATP and K+ (Ishii, N., Taguchi, H., Sasabe, H., and Yoshida, M . (1995) FEBS Lett . 362, 121-125) . When Escherichia coli GroEL14 was additionally included in the solution described above, hybrid chaperonins GroEL7.Tcpn607 and GroEL7 . Tcpn607.Tcpn107 were formed rapidly (<20 s) at 37 degrees C . The hybrid was also formed from Tcpn6014 and GroEL14 but not from a mutant GroEL14 lacking ATPase activity . The hybrid formation was saturated at approximately 300 microM ATP and approximately 300 mM K+ . These results imply that GroEL14 also splits and undergoes a heptamer exchange reaction with Thermus chaperonin under nearly physiological conditions . Similar to parent chaperonins, the isolated hybrid chaperonins exhibited ATPase activity that was susceptible to inhibition by Tcpn107 or GroES7 and mediated folding of other proteins . Once formed, the hybrid chaperonins were stable, and the parent chaperonins were not regenerated from the isolated hybrids under the same conditions in which the hybrids had been formed . Only under conditions in which GroEL in the hybrids was selectively destroyed, such as incubation at 70 degrees C, Thermus chaperonin, but not GroEL14, was regenerated from the hybrid . Therefore, the split reaction may not be an obligatory event repeated in each turnover of the chaperonin functional cycles but an event that occurs only when chaperonin is first exposed to ATP/K+.

FEMS Microbiol Lett, 1997 Jul 15, 152(2), 279 - 85
Microbial flora in the deepest sea mud of the Mariana Trench; Takami H et al.; In an attempt to characterize the microbial flora on the deepest sea floor, we isolated thousands of microbes from a mud sample collected from the Mariana Trench . The microbial flora found at a depth of 10897 m was composed of actinomycetes, fungi, non-extremophilic bacteria, and various extremophilic bacteria such as alkaliphiles, thermophiles, and psychrophiles . Phylogenetic analysis of Mariana isolates based on 16S rDNA sequences revealed that a wide range of taxa were represented.

Nucleic Acids Res, 1997 Jul 15, 25(14), 2737 - 44
A biologically active 53 kDa fragment of overproduced alanyl-tRNA synthetase from Thermus thermophilus HB8 specifically interacts with tRNA Ala acceptor helix; Lechler A et al.; The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced . The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T . thermophilus AlaRS was overproduced in E.coli , purified and characterized . It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization . The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP . By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified . It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair . The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition.

J Mol Biol, 1997 Jul 11, 270(2), 259 - 74
The crystal structure of an Fe-superoxide dismutase from the hyperthermophile Aquifex pyrophilus at 1.9 A resolution: structural basis for thermostability; Lim JH et al.; Superoxide dismutase (SOD) from Aquifex pyrophilus, a hyperthermophilic bacterium, is an extremely heat-stable enzyme that maintains about 70% of its activity after heat treatment for 60 minutes at 100 degrees C . To understand the molecular basis of thermostability of this enzyme, we have determined the crystal structure of A . pyrophilus superoxide dismutase (Ap SOD), an Fe containing homotetrameric enzyme, at 1.9 A resolution, and compared it with SOD structures from a mesophile and a thermophile, and other enzyme structures from other hyperthermophiles . The structure has been refined to a crystallographic R-factor (I > 2sigma) of 17.0% and R-free (I > 2sigma) of 19.9% . While the overall structure of the Ap SOD monomer is similar to the other SODs, significant conformational differences are observed in a highly variable loop region and the C-terminal helix . The conformational differences in these regions alter the subunit arrangement of this enzyme and generate a very compact tetramer . Structural comparisons of three SODs have revealed that Ap SOD has some stabilizing features at both the tertiary and the quaternary structural level: The Ap SOD monomer contains a large number of ion-pairs and the Ap SOD tetramer has a dramatically increased buried surface area per monomer . Comparisons of the Ap SOD structure with that of other known enzymes from hyperthermophiles reveal that the increased number of intrasubunit ion-pairs is a common feature.

Biochemistry, 1997 Jul 8, 36(27), 8293 - 303
Effects of divalent metal ions on individual steps of the Tetrahymena ribozyme reaction; McConnell TS et al.; The Tetrahymena thermophila L-21 ScaI ribozyme utilizes Mg2+ to catalyze a site-specific endonuclease reaction analogous to the first step of self-splicing . To better understand the contribution of Mg2+ to ribozyme activity, the Mg2+ concentration dependence of individual rate constants was examined at concentrations greater than those required for ribozyme folding (>2 mM; at 50 degrees C and pH 6.7) . Analysis of metal ion inhibition of the chemical step of the reaction indicated that two Ca2+ ions compete with two Mg2+ ions involved in active site chemistry . These Mg2+ ions are bound tightly to the E.S complex (Kd < 2 mM) . The rate constant for association of the oligoribonucleotide substrate (S) increased 12-fold from 2 to 100 mM Mg2+ and exhibited saturation behavior, consistent with a single Mg2+ ion involved in S association that binds to the free ribozyme with a Kd for Mg2+ of 15 mM . The preference for the divalent metal ion (Mg2+ congruent with Ca2+ > Ba2+ >> Sr2+) suggested that enhancing the rate constant of S association is not simply a function of ionic strength, but is due to a distinct metal ion binding site . Even though Ca2+ does not support reaction, the RNA substrate S was able to bind in the presence of Ca2+ . Upon addition of Mg2+, S was cleaved without first dissociating . A model is proposed in which the inactive Ca2+ form of E.S is structurally equivalent to the open complex along the reaction pathway, which has the RNA substrate bound but not docked into the active site . Weaker binding of S in Ca2+ was shown to result from an increase in the rate constant of S dissociation, leading to the proposal that a tight Mg2+ binding site or sites in the E.S complex contribute to the strong binding of S . In summary, the data provide evidence for four functions for bound Mg2+ ions in the catalytic cycle: one increases the rate of RNA substrate binding, one or more decrease the rate of dissociation of S, and two are involved in the chemical step.

FEBS Lett, 1997 Jul 7, 411(1), 53 - 9
A mutant form of the ribosomal protein L1 reveals conformational flexibility; Unge J et al.; The crystal structure of the mutant S179C of the ribosomal protein L1 from Thermus thermophilus has been determined at 1.9 A resolution . The mutant molecule displays a small but significant opening of the cavity between the two domains . The domain movement seems to be facilitated by the flexibility of at least two conserved glycines . These glycines may be necessary for the larger conformational change needed for an induced fit mechanism upon binding RNA . The domain movement makes a disulfide bridge possible between the incorporated cysteines in two monomers of the mutant L1.

Mol Microbiol, 1997 Jul, 25(2), 385 - 98
The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor-operator interactions; Dion M et al.; We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product . The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis . Sequence analysis of B . stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region . B . stearothermophilus ArgR has been overexpressed in E . coli and purified as a 48.0-kDa trimeric protein . The repressor inhibits the expression of a B . stearothermophilus argC-lacZ fusion in E . coli cells . In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter . The purified B . stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90 degrees C, whereas B . subtilis AhrC was largely inactivated at 65 degrees C . Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85 degrees C, well above the optimal growth temperature of the moderate thermophile B . stearothermophilus . This pronounced resistance of the repressor-operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.

J Biochem (Tokyo), 1997 Jul, 122(1), 32 - 40
Extremely thermostable phosphoenolpyruvate carboxylase from an extreme thermophile, Rhodothermus obamensis; Takai K et al.; Phosphoenolpyruvate carboxylase (PEPC) was purified from an extremely thermophilic bacterium, Rhodothermus obamensis, growing optimally at 80 degrees C, which had recently been isolated from a shallow marine hydrothermal vent in Japan . The native enzyme was a homotetramer of 400 kDa in molecular mass, as estimated by gel filtration chromatography, and the subunit exhibited an apparent molecular mass of 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The optimum temperature for enzyme activity was 75 degrees C . The enzyme exhibited an absolute requirement for divalent cations and a pH optimum of 8.0 . The enzyme was extremely thermostable and there was no loss of enzyme activity on incubation for 2 h at 85 degrees C . The enzyme exhibited a positive allosteric property with acetyl-CoA and fructose 1,6-bisphosphate, and a negative one with L-aspartate and L-malate . These effectors affected not only the thermophilicity but also the thermostability of the enzyme, and the substrate, co-factors, and salts increased the thermostability as well . The extrinsic thermostabilization might be a possible mechanism for adaptation of the enzyme to high temperature.

Appl Microbiol Biotechnol, 1997 Jul, 48(1), 121 - 8
Thermophilic biodegradation of BTEX by two consortia of anaerobic bacteria; Chen CI et al.; Two thermophilic anaerobic bacterial consortia (ALK-1 and LLNL-1), capable of degrading the aromatic fuel hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (BTEX compounds), were developed at 60 degrees C from the produced water of ARCO'S Kuparuk oil field at Alaska and the subsurface water at the Lawrence Livermore National Laboratory gasoline-spill site, respectively . Both consortia were found to grow at 45-75 degrees C on BTEX compounds as their sole carbon and energy sources with 50 degrees C being the optimal temperature . With 3.5 mg total BTEX added to sealed 50-ml serum bottles, which contained 30 ml mineral salts medium and the consortium, benzene, toluene, ethylbenze, m-xylene, and an unresolved mixture of o- and p-xylenes were biodegraded by 22%, 38%, 42%, 40%, and 38%, respectively, by ALK-1 after 14 days of incubation at 50 degrees C . Somewhat lower, but significant, percentages of the BTEX compounds also were biodegraded at 60 degrees C and 70 degrees C . The extent of biodegradation of these BTEX compounds by LLNL-1 at each of these three temperatures was slightly less than that achieved by ALK-1 . Use of {ring-14C}toluene in the BTEX mixture incubated at 50 degrees C verified that 41% and 31% of the biodegraded toluene was metabolized within 14 days to water-soluble products by ALK-1 and LLNL-1, respectively . A small fraction of it was mineralized to 14CO2 . The use of {U-14C}benzene revealed that 2.6%-4.3% of the biodegraded benzene was metabolized at 50 degrees C to water-soluble products by the two consortia; however, no mineralization of the degraded {U-14C}benzene to 14CO2 was observed . The biodegradation of BTEX at all three temperatures by both consortia was tightly coupled to sulfate reduction as well as H2S generation . None was observed when sulfate was omitted from the serum bottles . This suggests that sulfate-reducing bacteria are most likely responsible for the observed thermophilic biodegradation of BTEX in both consortial cultures.

Gene, 1997 Jul 1, 193(1), 23 - 30
Sequencing and analysis of the Thermus thermophilus ribosomal protein gene cluster equivalent to the spectinomycin operon; Vysotskaya VS et al.; To assess the organization of the Thermus thermophilus ribosomal protein genes, a fragment of DNA containing the complete S10 region and ten ribosomal protein genes of the spc region was cloned, using an oligonucleotide coding for the N-terminal amino acid (aa) sequence of T . thermophilus S8 protein as hybridization probe . The nucleotide sequence of a 4290 bp region between the rps17 and rpl15 genes was determined . Comparative analysis of this gene cluster showed that the gene arrangement (S17, L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15) is identical to that of eubacteria . However, T . thermophilus ribosomal protein genes corresponding to the Escherichia coli S10 and spc operons are not resolved into two clusters: the stop codon of the rps17 gene (the last gene of the S10 operon in E . coli) and the start codon of the rpl14 gene (the first gene of the spc operon in E . coli) overlap . Most genes, except the rps14-rps8 intergenic spacer (69 bp), are separated by very short (only 3-7 bp) spacer regions or partially overlapped . The deduced aa sequences of T . thermophilus proteins share about 51-100% identities with the sequences of homologous proteins from thermophile Thermus aquaticus and Thermotoga maritima and 27-70% identities with the sequences of their mesophile counterparts.

Eur J Biochem, 1997 Jul 1, 247(1), 262 - 7
Purification and properties of a cellobiose phosphorylase (CepA) and a cellodextrin phosphorylase (CepB) from the cellulolytic thermophile Clostridium stercorarium; Reichenbecher M et al.; Two phosphorolytic enzymes displaying activity towards the soluble cellulose degradation products cellobiose and cellodextrins were purified from the crude extract of the cellulolytic thermophile Clostridium stercorarium . Both phosphorylases have monomeric structures with molecular masses of 93 and 91 kDa, respectively . Although the N-terminal amino acid sequences are highly similar, a clear distinction of the two enzymes could be made on the basis of their substrate specificities: the enzyme designated cellobiose phosphorylase cleaved exclusively the disaccharide substrate, whereas the enzyme designated cellodextrin phosphorylase accepted only oligosaccharides as substrates . Kinetic constants were determined for the cleavage of cellobiose and cellodextrins . Maximal activity was observed at 65 degrees C in the pH range 6.0-7.0 for both enzymes . The sequences of the genes cepA and cepB encoding the cellobiose phosphorylase and the cellodextrin phosphorylase, respectively, have been submitted to the GenBank database.

Eur J Biochem, 1997 Jul 1, 247(1), 114 - 20
Influence of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling capacity; Van Kraaij C et al.; Three mutants of the antibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties . 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine . Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification . Cleavage of {Glu32,Ser33}nisin Z by endoproteinase Glu-C yielded {Glu32}nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z . The activity of {Lys32,Ser33}nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while {Trp32,Ser33}nisin Z, {Glu32,Ser33}nisin Z and {Glu32}nisin Z(1-32)-peptide exhibited 3-5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity . All variants showed significant loss of activity against Streptococcus thermophilus . The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced . To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

Eur J Biochem, 1997 Jul 1, 247(1), 59 - 65
Interaction of N-tosyl-L-phenylalanylchloromethane with Thermus thermophilus elongation factor Tu; Nawrot B et al.; The interaction of N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF-Tu) was studied by affinity labelling and NMR spectroscopy . TosPheCH2Cl binds to GDP and GTP conformers of EF-Tu . The interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF-Tu x GTP does not lead to covalent labelling . {A82}EF-Tu, in which the Cys82 is replaced by Ala, has similar properties to wild-type EF-Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF-Ts) and interaction with ribosomes . This structural change did not lead to changes, compared with wild-type EF-Tu in the functionality of {A82}EF-Tu, either in the GTP or in the GDP conformation . TosPheCH2Cl binds to EF-Tu x GTP with a dissociation constant of 10 microM . The interaction of TosPheCH2Cl with EF-Tu promotes the hydration of the carbonyl group of TosPheCH2Cl . TosPheCH2Cl competes with aminoacyl-tRNA for its binding site on EF-Tu x GTP . Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl-tRNA . TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl-tRNA and binds to its binding site on EF-Tu x GTP . This rather specific interaction with EF-Tu x GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of this residue.

Lett Appl Microbiol, 1997 Jul, 25(1), 8 - 12
Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population dynamics in natural whey culture; Cocconcelli PS et al.; The development of communities of the thermophilic microflora of natural whey culture for Parmigiano Reggiano cheese production was studied by means of molecular techniques . RAPD analysis facilitates the identification of the Lactobacillus strains involved in this microbial association and permitted the study of population dynamics during two cycles of whey fermentation . Analysis of RAPD fingerprints revealed the presence of four biotypes that dominate the whey fermentation process . Sequence analysis of 16S rDNA demonstrated that the strains isolated from whey belong to Lact . helveticus and Lact . delbrueckii ssp . lactis species.

Int J Syst Bacteriol, 1997 Jul, 47(3), 651 - 6
Caloramator proteoclasticus sp . nov., a new moderately thermophilic anaerobic proteolytic bacterium; Tarlera S et al.; A new moderately thermophilic proteolytic anaerobe, strain UT, was isolated from mesophilic granular methanogenic sludge . The cells were spore-forming, motile rods that were 0.4 micron wide and 2.4 to 4 microns long and stained gram negative . Electron micrographs of thin sections revealed the presence of an atypical gram-positive cell wall . Optimum growth occurred at 55 degrees C and at pH values between 7.0 and 7.5, with a doubling time of 30 min . The DNA base ratio of guanine plus cytosine was 31 mol% . The bacterium fermented proteins mainly to acetate, hydrogen, formate, and branched-chain fatty acids . Several amino acids, including glutamate, aspartate, arginine, histidine, threonine, methionine, and branched-chain amino acids, were also utilized . Glutamate was degraded to acetate, formate, hydrogen, and alanine . In addition, the strain degraded carbohydrates, including glucose, fructose, mannose, cellobiose, and starch, to acetate, ethanol, formate, lactate, and hydrogen . The results of a 16S rRNA sequence analysis phylogenetically placed strain UT in the low-guanine-plus-cytosine-content subgroup of the gram-positive phylum . We propose to classify the described strain in the genus Caloramator as a new species, Caloramator proteoclasticus . The type strain of C . proteoclasticus, strain U, has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 10124.

Int J Syst Bacteriol, 1997 Jul, 47(3), 622 - 6
Thermococcus hydrothermalis sp . nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent; Godfroy A et al.; An extremely thermophilic archaeon, strain AL662T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at a latitude of 21 degrees N . This strain is a strictly anaerobic coccus, and its cells range from 0.8 to 2 microns in diameter . The optimum temperature, pH, and Sea Salt concentration for growth are 85 degrees C, 6, and 20 to 40 g/liter, respectively . Strain AL662T grows preferentially on proteolysis products, on a mixture of 20 amino acids, and on maltose in the presence of elemental sulfur . The membrane lipids consist of di- and tetraether glycerol lipids . The DNA G+C content is 58 mol% . Sequencing of the 16S rRNA gene showed that strain AL662T belongs to the genus Thermococcus . On the basis of hybridization results, we propose that this strain should be placed in a new species, Thermococcus hydrothermalis.

J Cell Biochem, 1997 Jul 1, 66(1), 37 - 42
ADPribosylation reaction by free ADPribose in Sulfolobus solfataricus, a thermophilic archaeon; Faraone-Mennella MR et al.; In the archaeon Sulfolobus solfataricus, protein ADPribosylation by free ADPribose was demonstrated by testing both {adenine-14C(U)}ADPR and {adenine-14C(U)}NAD as substrates . The occurrence of this process was shown by using specific experimental conditions . Increasing the incubation time and lowering the pH of the reaction mixture enhanced the protein glycation by free ADPribose . At pH 7.5 and 10 min incubation, the incorporation of free ADPribose into proteins was highly reduced . Under these conditions, the autoradiographic pattern showed that, among the targets of ADPribose electrophoresed after incubation with 32P-NAD, the proteins modified by free 32P-ADPribose mostly corresponded to high molecular mass components . Among the compounds known to inhibit the eukaryotic poly-ADPribose polymerase, only ZnCl2 highly reduced the ADPribose incorporation from NAD into the ammonium sulphate precipitate . A 20% inhibition was measured in the presence of nicotinamide or 3-aminobenzamide . No inhibition was observed replacing NAD with ADPR as substrate.

Appl Environ Microbiol, 1997 Jul, 63(7), 2949 - 51
Clostridium thermocellum JW20 (ATCC 31549) is a coculture with Thermoanaerobacter ethanolicus; Erbeznik M et al.; A PCR assay based on 16S rRNA sequence differences among four thermophilic anaerobic bacterial strains was used to demonstrate contamination of Clostridium thermocellum JW20 (ATCC 31549) with a Thermoanaerobacter ethanolicus strain . Therefore, we suggest that interpretation of experimental results with C . thermocellum JW20 be viewed with caution.

Appl Environ Microbiol, 1997 Jul, 63(7), 2802 - 13
Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis; Godon JJ et al.; The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed . After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established . The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long) . A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively . A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively . The other 16 bacterial OTUs represent 13% of the clones . They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree . A large number of bacterial OTUs are not closely related to any other hitherto determined sequences . The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences . However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences . Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum . In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp . Five percent of the sequences analyzed were chimeras and were removed from the analysis.

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