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.,