Biochemistry, 1999 Jul 13, 38(28), 9126 - 36 The oxidized (3,3) state of manganese catalase . Comparison of enzymes from Thermus thermophilus and Lactobacillus plantarum; Whittaker MM et al.; Manganese catalases contain a binuclear manganese cluster that catalyzes the redox dismutation of hydrogen peroxide, interconverting between dimanganese(II) {(2,2)} and dimanganese(III) {(3,3)} oxidation states during turnover . We have investigated the oxidized (3,3) states of the homologous enzymes from Thermus thermophilus and Lactobacillus plantarum using a combination of optical absorption, CD, MCD, and EPR spectroscopies as sensitive probes of the electronic structure and protein environment for the active site metal clusters . Comparison of results for these two enzymes allows the essential features of the active sites to be recognized and the differences identified . For both enzymes, preparations having the highest catalytic activity have diamagnetic ground states, consistent with the bis-mu-bridging dimanganese core structure that has been defined crystallographically . Oxidative damage and exogenous ligand binding perturb the core structure of LPC, converting the enzyme to a distinct form in which the cluster becomes paramagnetic as a result of altered exchange coupling mediated by the bridging ligands . The TTC cluster does not exhibit this sensitivity to ligand binding, implying a different reactivity for the bridges in that enzyme . A mechanism is proposed involving distinct coordination modes for peroxide substrate in each of the two half-reactions for enzyme turnover. Biochemistry, 1999 Jul 13, 38(28), 8981 - 91 Mössbauer and electron paramagnetic resonance studies of the cytochrome bf complex; Schunemann V et al.; The (57)Fe-enriched cytochrome bf complex has been isolated from hydrocultures of spinach . It has been studied at different redox states by optical, EPR, and Mossbauer spectroscopy . The Mossbauer spectrum of the native complex at 190 K with all iron centers in the oxidized state reveals the presence of four different iron sites: low-spin ferric iron in cytochrome b {with an isomer shift (delta) of 0.20 mm/s, a quadrupole splitting (DeltaE(Q)) of 1.77 mm/s, and a relative area of 40%}, low-spin ferric iron of cytochrome f (delta = 0.26 mm/s, DeltaE(Q) = 1.90 mm/s, and a relative area of 20%), and two high-spin ferric iron sites of the Rieske iron-sulfur protein (ISP) with a bis-cysteine and a bis-histidine ligated iron (delta(1) = 0.15 mm/s, DeltaE(Q1) = 0.70 mm/s, and a relative area of 20%, and delta(2) = 0.25 mm/s, DeltaE(Q2) = 0.90 mm/s, and a relative area of 20%, respectively) . EPR and magnetic Mossbauer measurements at low temperatures corroborate these results . A crystal-field analysis of the EPR data and of the magnetic Mossbauer data yields estimates for the g-tensors (g(z)(), g(y)(), and g(x)()) of cytochrome b (3.60, 1.35, and 1.1) and of cytochrome f (3.51, 1.69, and 0.9) . Addition of ascorbate reduces not only the iron of cytochrome f to the ferrous low-spin state (delta = 0.43 mm/s, DeltaE(Q) = 1.12 mm/s at 4.2 K) but also the bis-histidine coordinated iron of the Rieske 2Fe-2S center to the ferrous high-spin state (delta(2) = 0.73 mm/s, DeltaE(Q2) = -2.95 mm/s at 4.2 K) . At this redox step, the Mossbauer parameters of cytochrome b have not changed, indicating that the redox changes of cytochrome f and the Rieske protein did not change the first ligand sphere of the low-spin ferric iron in cytochrome b . Reduction with dithionite further reduces the two hemes of cytochrome b to the ferrous low-spin state (delta = 0.49 mm/s, DeltaE(Q) = 1.08 mm/s at 4.2 K) . The spin Hamiltonian analysis of the magnetic Mossbauer spectra at 4.2 K yields hyperfine parameters of the reduced Rieske 2Fe-2S center in the cytochrome bf complex which are very similar to those reported for the Rieske center from Thermus thermophilus {Fee, J . A., Findling, K . L., Yoshida, T., et al . (1984) J . Biol . Chem . 259, 124-133}. Plant Mol Biol, 1999 May, 40(2), 307 - 21 A thermophilic cyanobacterium Synechococcus elongatus has three different Class I prenyltransferase genes; Ohto C et al.; Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C5) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences . Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus . Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C20) diphosphate synthase, another encodes a farnesyl (C15) diphosphate synthase whose optimal reaction temperature is 60 degrees C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 degrees C . The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C25), hexaprenyl (C30), and heptaprenyl (C35) diphosphates from dimethylallyl (C5) diphosphate, geranyl (C10) diphosphate, or farnesyl diphosphate as the allylic substrates . The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates . The situations of these three S . elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al . (1996). Proteins, 1999 Aug 15, 36(3), 295 - 306 High resolution structure and sequence of T . aurantiacus xylanase I: implications for the evolution of thermostability in family 10 xylanases and enzymes with (beta)alpha-barrel architecture; Lo Leggio L et al.; Xylanase I is a thermostable xylanase from the fungus Thermoascus aurantiacus, which belongs to family 10 in the current classification of glycosyl hydrolases . We have determined the three-dimensional X-ray structure of this enzyme to near atomic resolution (1.14 A) by molecular replacement, and thereby corrected the chemically determined sequence previously published . Among the five members of family 10 enzymes for which the structure has been determined, Xylanase I from T . aurantiacus and Xylanase Z from C . thermocellum are from thermophilic organisms . A comparison with the three other available structures of the family 10 xylanases from mesophilic organisms suggests that thermostability is effected mainly by improvement of the hydrophobic packing, favorable interactions of charged side chains with the helix dipoles and introduction of prolines at the N-terminus of helices . In contrast to other classes of proteins, there is very little evidence for a contribution of salt bridges to thermostability in the family 10 xylanases from thermophiles . Further analysis of the structures of other proteins from thermophiles with eight-fold (beta)alpha-barrel architecture suggests that favorable interactions of charged side chains with the helix dipoles may be a common way in which thermophilic proteins with this fold are stabilized . As this is the most common type of protein architecture, this finding may provide a useful guide for site-directed mutagenesis aimed to improve the thermostability of (beta)alpha-barrel proteins . Proteins 1999;36:295-306 . Mol Cell Biol, 1999 Aug, 19(8), 5631 - 41 Flanking regulatory sequences of the Tetrahymena R deletion element determine the boundaries of DNA rearrangement; Chalker DL et al.; In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements . It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems . In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element . We found that rearrangement requires specific sequences flanking each side of the deletion element . The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary . When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region . Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition . Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence . We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity . These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away . Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries . No apparent sequence similarity exists between the M and R elements . The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymena rearrangement. J Biol Chem, 1999 Jul 23, 274(30), 21387 - 94 A comparison of eubacterial and archaeal structure-specific 5'-exonucleases; Kaiser MW et al.; The 5'-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structure-specific 5'-exonucleases with similar function but limited sequence similarity . Their physiological role is to remove the displaced 5' strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase . In this paper, we define the substrate requirements for the 5'-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum . The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair . That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap . The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently . The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand . Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity. J Pept Res, 1999 Jun, 53(6), 599 - 605 Purification and characterization of an acid proteinase from mesophilic Mucor sp . solid-state cultures; Fernandez-Lahore HM et al.; The fourth-day extract of a solid-state culture of the mesophilic Mucor sp . (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture . After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography . Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination . A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data . The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range . The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively . This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin . The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA . The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes. Can J Microbiol, 1999 Mar, 45(3), 217 - 22 Isolation and partial characterisation of extracellular keratinase from a wool degrading thermophilic actinomycete strain Thermoactinomyces candidus; Ignatova Z et al.; The keratinase production by the thermophilic actinomycete strain Thermoactinomyces candidus was induced by sheep wool as the sole source of carbon and nitrogen in the cultivation medium . For complete digestion of wool by the above strain, both keratinolytic serine proteinase and cellular reduction of disulfide bonds were involved . Evidence was presented that substrate induction was a major regulatory mechanism and the keratinase biosynthesis was not completely repressed by addition of other carbon (glucose) and nitrogen (NH4C1) sources . The enzyme was purified 62-fold by diethylaminoethyl-anion exchange and Sephadex G-75 gel permeation chromatographies . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 30 kDa . The pH and temperature optima were determined to be 8.6 and 70 degrees C, respectively . The purified thermophilic keratinase catalyses the hydrolysis of a broad range of substrates and displays higher proteolytic activity against native keratins than other proteinases . Ca2+ was found to have a stabilizing effect on the enzyme activity at elevated temperatures. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 413 - 8 Nucleotide sequence, heterologous expression and novel purification of DNA ligase from Bacillus stearothermophilus(1); Brannigan JA et al.; The gene for DNA ligase (EC 6.5.1.2) from thermophilic bacterium Bacillus stearothermophilus NCA1503 has been cloned and the complete nucleotide sequence determined . The ligase gene encodes a protein 670 amino acids in length . The gene was overexpressed in Escherichia coli and the enzyme has been purified to homogeneity . Preliminary characterisation confirms that it is a thermostable, NAD(+)-dependent DNA ligase. Eur J Biochem, 1999 Jul, 263(2), 502 - 10 PepS from Streptococcus thermophilus . A new member of the aminopeptidase T family of thermophilic bacteria; Fernandez-Espla MD et al.; The proteolytic system of lactic acid bacteria is essential for bacterial growth in milk but also for the development of the organoleptic properties of dairy products . Streptococcus thermophilus is widely used in the dairy industry . In comparison with the model lactic acid bacteria Lactococcus lactis, S . thermophilus possesses two additional peptidases (an oligopeptidase and the aminopeptidase PepS) . To understand how S . thermophilus grows in milk, we purified and characterized this aminopeptidase . PepS is a monomeric metallopeptidase of approximately 45 kDa with optimal activity in the range pH 7.5-8.5 and at 55 degrees C on Arg-paranitroanilide as substrate . PepS exhibits a high specificity towards peptides possessing arginine or aromatic amino acids at the N-terminus . From the N-terminal protein sequence of PepS, we deduced degenerate oligonucleotides and amplified the corresponding gene by successive PCR reactions . The deduced amino-acid sequence of the PepS gene has high identity (40-50%) with the aminopeptidase T family from thermophilic and extremophilic bacteria; we thus propose the classification of PepS from S . thermophilus as a new member of this family . In view of its substrate specificity, PepS could be involved both in bacterial growth by supplying amino acids, and in the development of dairy products' flavour, by hydrolysing bitter peptides and liberating aromatic amino acids which are important precursors of aroma compounds. Biochem Biophys Res Commun, 1999 Jul 14, 260(3), 597 - 9 Rotation of Escherichia coli F(1)-ATPase; Noji H et al.; By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr . (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase . The torque value and the direction of the rotation were the same as those observed for TF(1) . F(1)-ATPases seem to share common properties of rotation irrespective of the sources . Curr Microbiol, 1999 Aug, 39(2), 99 - 102 Dissimilatory reduction of Fe(III) by thermophilic bacteria and archaea in deep subsurface petroleum reservoirs of western siberia Slobodkin AI, Jeanthon C, L'Haridon S, Nazina T, Miroshnichenko M, Bonch-Osmolovskaya E. Twenty-five samples of stratal fluids obtained from a high-temperature (60-84 degrees C) deep subsurface (1700-2500 m) petroleum reservoir of Western Siberia were investigated for the presence of dissimilatory Fe(III)-reducing microorganisms . Of the samples, 44% and 76% were positive for Fe(III) reduction with peptone and H2 respectively as electron donors . In most of these samples, the numbers of culturable thermophilic H2-utilizing iron reducers were in the order of 10-100 cells/ml . Nine strains of thermophilic anaerobic bacteria and archaea isolated from petroleum reservoirs were tested for their ability to reduce Fe(III) . Eight strains belonging to the genera Thermoanaerobacter, Thermotoga, and Thermococcus were found capable of dissimilatory Fe(III) reduction, with peptone or H2 as electron donor and amorphous Fe(III) oxide as electron acceptor . These results demonstrated that Fe(III) reduction may be a common feature shared by a wide range of anaerobic thermophiles and hyperthermophiles in deep subsurface petroleum reservoirs.com/link/service/journals/00284/bibs/39n2p99.html Appl Biochem Biotechnol, 1999 Spring, 77-79, 267 - 75 A high-copy-number plasmid capable of replication in thermophilic cyanobacteria; Miyake M et al.; A 2.5 kb high-copy-number plasmid, pMA4 in thermophilic cyanobacterium Synechococcus sp . MA4 was isolated and characterized to develop a genetic engineering system for thermophilic cyanobacteria . The copy number of pMA4 was determined to be by densitometry about 350/cell . The pMA4 may be a type of rolling-circle plasmid, because a possible rep gene encoding 34 kD-protein and a consensus sequence of a double-stranded origin nick site of rolling circle plasmids were found in the pMA4 sequence . The pMA4 was electro-introduced into another thermophile, Synechococcus sp . MA19, which is the strongest poly-beta-hydroxybutyrate (PHB) accumulator in photoautotrophic organisms . The pMA4 was incorporated and retained in MA19 . These results indicate that pMA4 could be developed as a useful vector for thermophilic cyanobacteria. J Nat Prod, 1999 Jun, 62(6), 859 - 63 Carotenoid glycoside esters from the thermophilic bacterium meiothermusruber Burgess ML, Barrow KD, Gao C, Heard GM, Glenn D. The thermophilic bacterium Meiothermus ruber produces a series of carotenoid glycoside esters . The major carotenoid has been identified as 1'-beta-glucopyranosyl-3,4,3',4'-tetradehydro-1', 2'-dihydro-beta,psi-caroten-2-one (1) . It is acylated at the 6' '-position of the glucose unit by a series of C10-C17 fatty acids . The structure of 1 was established by spectral means, including complete assignment of the 1H and 13C NMR resonances by inverse 2D NMR spectroscopy . These carotenoids are thought to play roles in stabilizing membranes of this thermophilic organism. Mol Cell Biochem, 1999 May, 195(1-2), 55 - 64 Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus; Pantazaki AA et al.; Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus . The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60 degrees C . The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity . The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP . When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition . The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity . Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC . Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T . thermophilus, and in a decrease of the specific activity of ornithine decarboxylase . The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az) . The T . thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E . coli antizyme . The antizyme of E . coli inhibited the ODC of T . thermophilus, and vice versa . The fragment of amino acids 56-292 of the E . coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E . coli or T . thermophilus. Acta Crystallogr D Biol Crystallogr, 1999 Jul, 55 ( Pt 7), 1348 - 9 Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius; De Simone G et al.; EST2, a thermophilic carboxylesterase from Alicyclobacillus acidocaldarius, belonging to the HSL group of the esterase/lipase superfamily, has been crystallized for the first time . Ammonium sulfate was used as a precipitant and the crystallization proceeded at pH 7.8 . The crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 78.8, c = 106 . 4 A . A complete data set has been collected at the synchrotron source Elettra in Trieste to 2.4 A resolution, using a single frozen crystal. J Mol Biol, 1999 Jul 9, 290(2), 595 - 604 Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability; Thompson MJ et al.; Understanding the molecular determinants of protein thermostability is of theoretical and practical importance . While numerous determinants have been suggested, no molecular feature has been judged of paramount importance, with the possible exception of ion-pair networks . The difficulty in identifying the main determinants may have been the limited structural information available on the thermostable proteins . Recently the complete genomes for mesophilic, thermophilic and hyperthermophilic organisms have been sequenced, vastly improving the potential for uncovering general trends in sequence and structure evolution related to thermostability and, thus, for isolating the more important determinants . From a comparative analysis of 20 complete genomes, we find a trend towards shortened thermophilic proteins relative to their mesophilic homologs . Moreover, sequence alignments to proteins of known structure indicate that thermophilic sequences are more likely than their mesophilic homologs to have deletions in exposed loop regions . The new genomes offer enough comparable sequences to compute meaningful statistics that point to loop deletion as a general evolutionary strategy for increasing thermostability . Appl Environ Microbiol, 1999 Jul, 65(7), 2863 - 70 Indication that the nitrogen source influences both amount and size of exopolysaccharides produced by streptococcus thermophilus LY03 and modelling of the bacterial growth and exopolysaccharide production in a complex medium Degeest B, De Vuyst L. Streptococcus thermophilus LY03 is a yogurt strain producing the same exopolysaccharide material in both milk and MRS broth . Actually, two types of exopolysaccharides are produced simultaneously . The two exopolysaccharides are identical in monomer composition (galactose and glucose in a 4:1 ratio) but differ in molecular size . Gel permeation chromatography revealed a high-molecular-mass exopolysaccharide (1.8 x 10(6)) and a low-molecular-mass exopolysaccharide (4.1 x 10(5)) . Both exopolysaccharides can be isolated from the fermentation broth separately . The proportion in which they are produced is strongly dependent on the carbon/nitrogen ratio of the fermentation broth . A shift from a high-molecular-mass exopolysaccharide to a low-molecular-mass exopolysaccharide was observed with increasing initial complex nitrogen concentrations . All necessary biokinetic parameters to study the kinetics of S . thermophilus LY03 fermentations were obtained from a mathematical model which describes both S . thermophilus LY03 growth and exopolysaccharide production and degradation . The model is valid with various initial complex nitrogen concentrations and can be applied to simulate exopolysaccharide production in a milk medium. J Appl Microbiol, 1999 Jun, 86(6), 1024 - 32 Evaluation of the effect of temperature and nutrients on the survival of Campylobacter spp . in water microcosms; Thomas C et al.; Batch microcosms containing various water types (de-ionized and river water with or without sediment), incubated at a range of temperatures (5-37 degrees C), were used to facilitate a comparative evaluation of the significance of such variables and their interactions upon the collective and individual survival of four species of thermophilic Campylobacter . All variables significantly influenced (P < = 0.031) population decay rates . Minimal decay for the group was identified at low temperatures (5 degrees C) in river water, i.e . nutrient-containing microcosms . Collective decay rates within river water microcosms were significantly decreased (P = 0.03) from those observed in de-ionized water, particularly at environmental temperatures (5 and 15 degrees C) . However, the increased nutrient levels observed in sediment-containing microcosms did not significantly (P = 0.41) reduce population decay rates . Overall, Camp . jejuni populations demonstrated the most resilience to the environmental stressors evaluated, with the exception of 15 degrees C where Camp . lari was the most persistent . Campylobacter coli and Camp . upsaliensis demonstrated comparable survival characteristics but were less resilient than Camp . jejuni and Camp . lari . These observations identify the suitability of water systems as a reservoir and medium for Campylobacter infection, and potentially identifies Camp . jejuni and Camp . lari as the main protagonists of water-mediated campylobacteriosis. Appl Environ Microbiol, 1999 Jul, 65(7), 3001 - 7 Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A; Chavagnat F et al.; The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies . The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C . It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase . The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+ . Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected . The N-terminal and short internal amino acid sequences of purified PepN were determined . By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252 . Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family . The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain . The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme. Protein Sci, 1999 Jun, 8(6), 1218 - 31 Sulfolobus acidocaldarius inorganic pyrophosphatase: structure, thermostability, and effect of metal ion in an archael pyrophosphatase; Leppanen VM et al.; The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 A . S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase) . In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+, while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 A . S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion . The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 A/Calpha . But the hexamer structures of S- and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization . In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E- and T-PPase . In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein . Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions. FEBS Lett, 1999 Jun 11, 452(3), 155 - 9 Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation; Martemyanov KA et al.; Two truncated variants of elongation factor G from Thermus thermophilus with deletion of its domain IV have been constructed and the mutated genes were expressed in Escherichia coli . The truncated factors were produced in a soluble form and retained a high thermostability . It was demonstrated that mutated factors possessed (1) a reduced affinity to the ribosomes with an uncleavable GTP analog and (2) a specific ribosome-dependent GTPase activity . At the same time, in contrast to the wild-type elongation factor G, they were incapable to promote translocation . The conclusions are drawn that (1) domain IV is not involved in the GTPase activity of elongation factor G, (2) it contributes to the binding of elongation factor G with the ribosome and (3) is strictly required for translocation . These results suggest that domain IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation. J Dairy Sci, 1999 Jun, 82(6), 1108 - 14 Study of the possible mechanisms involved in the mucosal immune system activation by lactic acid bacteria; Perdigon G et al.; The induction of a mucosal immune response is not easy due to the development of oral tolerance, but under some conditions, bacteria can activate this immune system . Antigens administered orally can interact with M cells of Peyer's patches or bind to the epithelial cells . We have demonstrated that certain lactic acid bacteria are able to induce specific secretory immunity, and others will enhance the gut inflammatory immune response . The aim of this work was to establish the reason for these different behaviors and to define possible mechanisms involved in the interaction of lactic acid bacteria at the intestinal level . We studied IgA+ and IgM+ B cells comparatively in bronchus and intestine and CD4+ T cells and IgA anti-lactic acid bacteria antibodies in the intestinal fluid, induced by oral administration of Lactobacillus casei, Lb . delbrueckii ssp . bulgaricus, Lb . acidophilus, Lb . plantarum, Lb . rhamnosus, Lactococcus lactis, and Streptococcus salivarius ssp . thermophilus . The increase in the IgA+ B cells in the bronchus means that these lactic acid bacteria were able to induce the IgA cycle by interaction with M cells from Peyer's patches or intestinal epithelial cells . The IgM+ cells increased when the stimulus did not induce the switch from IgM+ to IgA+ . The increase in the CD4+ cells suggests interaction of Peyer's patches and enhancement of the B- and T-cell migration . The anti-lactic acid bacteria antibody is related to the processing and presentation of the microorganisms to the immune cells . We demonstrated that Lb . casei and Lb . plantarum were able to interact with Peyer's patch cells and showed an increase in IgA-, CD4+ cells, and antibodies specific for the stimulating strain . Lactobacillus acidophilus induced gut mucosal activation by interaction with the epithelial cells without increase in the immune cells associated with the bronchus . Although Lb . rhamnosus and Strep . salivarius ssp . thermophilus interact with epithelial cells, they also induced an immune response against their epitopes . Lactococcus lactis and Lb . delbrueckii ssp . bulgaricus induced an increase of IgA+ cells entering the IgA cycle but not CD4+ cells; thus, these bacteria would have been bound to epithelial cells that activated B lymphocytes without processing and presenting of their epitopes . We did not determine specific antibodies against Lc . lactis or Lb . bulgaricus. Nature, 1999 Jun 17, 399(6737), 694 - 7 Regions of variant histone His2AvD required for Drosophila development; Clarkson MJ et al.; One way in which a distinct chromosomal domain could be established to carry out a specialized function is by the localized incorporation of specific histone variants into nucleosomes . H2AZ, one such variant of the histone protein H2A, is required for the survival of Drosophila melanogaster, Tetrahymena thermophila and mice (R . Faast et al., in preparation) . To search for the unique features of Drosophila H2AZ (His2AvD, also referred to as H2AvD) that are required for its essential function, we have performed amino-acid swap experiments in which residues unique to Drosophila His2AvD were replaced with equivalently positioned Drosophila H2A.1 residues . Mutated His2AvD genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null-mutant lethality . We show that the unique feature of His2AvD does not reside in its histone fold but in its carboxy-terminal domain . This C-terminal region maps to a short alpha-helix in H2A that is buried deep inside the nucleosome core. Mol Microbiol, 1999 Jun, 32(6), 1287 - 95 Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide; Stingele F et al.; Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) composed of glucose, galactose and N-acetylgalactosamine in the molar ratio of 1:2:1 . The genes responsible for the EPS biosynthesis have been isolated previously and found to be clustered in a 14.5 kb region encoding 13 genes . Transfer of this gene cluster into a non-EPS-producing heterologous host, Lactococcus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host . The structure of the recombinant EPS was determined by means of 1H homonuclear and 1H-13C heteronuclear two-dimensional nuclear magnetic resonance (NMR) spectra and was found to be --> 3)-beta-D-Glcp-(1 --> 3)-alpha-D-Galp-(1 --> 3)-beta-D-Galp-(1 --> as opposed to --> 3){alpha-D-Galp-(1 --> 6)}-beta-D-Glcp-(1 --> 3)-alpha-D-GalpNAc-(1 --> 3)-beta-D-Galp-(1 --> for the wild-type S . thermophilus Sfi6 . Furthermore, L . lactis MG1363 (pFS101) was also lacking a UDP-N-acetylglucosamine C4-epimerase activity, which would provide UDP-GalNAc for a GalNAc incorporation into the EPS and probably caused the substitution of GalNAc by Gal in the recombinant EPS . This modification implies that (i) bacterial glycosyltransferases could potentially have multiple specificities for the donor and the acceptor sugar molecule; and (ii) the repeating unit polymerase can recognize and polymerize a repeating unit that differs in the backbone as well as in the side-chain from its native substrate. J Biol Chem, 1999 Jul 2, 274(27), 19181 - 7 Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures; Alvarez M et al.; The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs . His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown . To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs . Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity . No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs . We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution . A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10 . This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature . Analysis of bTIM single mutants supports this assumption. DNA Res, 1999 Apr 30, 6(2), 83 - 101, 145-52 Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1; Kawarabayasi Y et al.; The complete sequence of the genome of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, which optimally grows at 95 degrees C, has been determined by the whole genome shotgun method with some modifications . The entire length of the genome was 1,669,695 bp . The authenticity of the entire sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA . As the potential protein-coding regions, a total of 2,694 open reading frames (ORFs) were assigned . By similarity search against public databases, 633 (23.5%) of the ORFs were related to genes with putative function and 523 (19.4%) to the sequences registered but with unknown function . All the genes in the TCA cycle except for that of alpha-ketoglutarate dehydrogenase were included, and instead of the alpha-ketoglutarate dehydrogenase gene, the genes coding for the two subunits of 2-oxoacid:ferredoxin oxidoreductase were identified . The remaining 1,538 ORFs (57.1%) did not show any significant similarity to the sequences in the databases . Sequence comparison among the assigned ORFs suggested that a considerable member of ORFs were generated by sequence duplication . The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 47 tRNA genes including 14 genes with intron structures . All the assigned ORFs and RNA coding regions occupied 89.12% of the whole genome . The data presented in this paper are available on the internet homepage . Biochemistry (Mosc), 1999 May, 64(5), 581 - 6 A site-specific endonuclease from the thermophilic strain Bacillus species F4; Zharmukhamedova TY et al.; A strain producing the site-specific endonuclease BspF4I was found during screening of thermophilic bacteria isolated from soil . The restriction endonuclease, free from contaminant nonspecific nucleases, was purified using three steps of column chromatography--on hydroxyapatite, blue agarose, and DEAE-Trisacryl . The enzyme is stable on storage and exhibits maximal activity at 48-56 degrees C in the presence of albumin in buffer containing 10 mM Tris-HCl (pH 7.5) and 10 mM MgCl2 . BspF4I recognizes the sequence 5;-GGNCC-3; on DNA and is an isomer and not an isoschizomer of the endonuclease Sau96I . Unlike the prototype, BspF4I does not cleave the site in a defined way . A strand with purine in the center of the sequence is cleaved after the first G, as in the case of the prototype, while the strand with pyrimidine is cleaved either before or after the first G. J Eukaryot Microbiol, 1999 May-Jun, 46(3), 239 - 47 Analysis of genomic G + C content, codon usage, initiator codon context and translation termination sites in Tetrahymena thermophila; Wuitschick JD et al.; In recent years, the amount of molecular sequencing data from Tetrahymena thermophila has dramatically increased . We analyzed G + C content, codon usage, initiator codon context and stop codon sites in the extremely A + T rich genome of this ciliate . Average G + C content was 38% for protein coding regions, 21% for 5' non-coding sequences, 19% for 3' non-coding sequences, 15% for introns, 19% for micronuclear limited sequences and 17% for macronuclear retained sequences flanking micronuclear specific regions . The 75 available T . thermophila protein coding sequences favored codons ending in T and, where possible, avoided those with G in the third position . Highly expressed genes were relatively G + C-rich and exhibited an extremely biased pattern of codon usage while developmentally regulated genes were more A + T-rich and showed less codon usage bias . Regions immediately preceding Tetrahymena translation initiator codons were generally A-rich . For the 60 stop codons examined, the frequency of G in the end + 1 site was much higher than expected whereas C never occupied this position. J Comp Physiol {A}, 1999 May, 184(5), 529 - 34 Chemosensory responses of Tetrahymena thermophila to CB2, a 24-amino-acid fragment of lysozyme; Kuruvilla HG et al.; While lysozyme is a depolarizing chemorepellent in Tetrahymena, the entire lysozyme molecule is not necessary to activate the lysozyme receptor . Reduced lysozyme was cut into three fragments by cyanogen bromide cleavage and the fragments (CB1, CB2 and CB3) were separated by HPLC . Behavioral bioassays showed that the carboxy-terminal 24-amino-acid fragment, which we call CB2, is 100 times more active than intact lysozyme as a chemorepellent . CB2 appears to activate the same receptor as lysozyme because behavioral cross-adaptation is seen between these two compounds and an antibody generated to the purified lysozyme receptor blocks responses to both lysozyme and CB2 . This is further supported by the observation that neomycin, which is a competitive inhibitor of lysozyme binding, also inhibits CB2 responses . This inhibition may be due to the fact that neomycin is highly positively charged (+5 at pH 7.0) and CB2 has a net charge of +4 at pH 7.0 . Intracellular electrophysiological recordings documented that CB2 elicits a transient, depolarizing receptor potential that is similar to the lysozyme-induced depolarizations except they are much smaller . CB2 is a more potent and specific ligand for use in studies of the lysozyme receptor of Tetrahymena. Proc Natl Acad Sci U S A, 1999 Jun 22, 96(13), 7184 - 9 Heat-inactivated proteins are rescued by the DnaK.J-GrpE set and ClpB chaperones; Motohashi K et al.; Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated in vitro . In a eubacterium Thermus thermophilus, DnaK and DnaJ exist as a stable trigonal ring complex (TDnaK.J complex) and the dnaK gene cluster contains a clpB gene . When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the TDnaK.J complex could suppress the aggregation of proteins in an ATP- and TGrpE-dependent manner . Subsequent incubation of these heated preparations at moderate temperature after addition of TClpB resulted in the efficient reactivation of the proteins . Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the TDnaK.J complex, TGrpE, TClpB, and ATP . Thus, all these components were necessary for the reactivation . Further, we found that TGroEL/ES could not substitute TClpB. J Mol Biol, 1999 Jun 25, 289(5), 1267 - 82 Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot; Gonzalez RL Jr et al.; Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies . The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus . Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins . The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+ . The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem . Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands . Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases . The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron . Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site . Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration . An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot . These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots . Gene, 1999 Jun 11, 233(1-2), 151 - 61 Are horizontal transfers involved in the evolution of the Streptococcus thermophilus exopolysaccharide synthesis loci? Bourgoin F, Pluvinet A, Gintz B, Decaris B, Guedon G. A 32.5kb variable locus of the Streptococcus thermophilus CNRZ368 chromosome, the eps locus, contains 25 ORF and seven insertion sequences (IS) . The putative products of 17 ORF are related to proteins involved in the synthesis of polysaccharides in various bacteria . The two distal regions and a small central region of the eps locus are constant and present in all or almost all of the S . thermophilus strains tested . The other regions are variable and present in only some S . thermophilus strains tested, particularly in the closely related strains CNRZ368 and A054 . A 13.6kb variable region of the eps locus of S . thermophilus CNRZ368 contains two ORF that are almost identical to epsL and orfY of the eps locus of Lactococcus lactis NIZOB40 and seven IS belonging to four different families, ISS1, IS981, IS1193 and IS1194 . Five of these sequences were probably acquired by horizontal transfer from L . lactis (Bourgoin, F., et al., 1996 . Gene 178, 15-23) . Three probes of this 13.6kb region hybridized with the DNA of several L . lactis strains tested . A specific probe for another sequence within the S . thermophilus eps locus, epsF, hybridized with the DNA of one of the L . lactis strains tested . Sequence comparisons also suggest that five ORF of the eps locus have a mosaic structure and probably result from recombinations between sequences that are 10 to 50% divergent . The chimeric structure of the eps locus suggests a very complex evolution . This evolution probably involves both the acquisition of the 13.6kb region from L . lactis by horizontal transfer and exchanges within the S . thermophilus species. EMBO J, 1999 Jun 15, 18(12), 3475 - 83 Structure-specific tRNA-binding protein from the extreme thermophile Aquifex aeolicus; Morales AJ et al.; The genome of the bacterium Aquifex aeolicus encodes a polypeptide which is related to a small portion of a sequence found in one prokaryotic and two eukaryotic tRNA synthetases . It also is related to a portion of Arc1p, a tRNA-binding protein believed to be important for nuclear trafficking of tRNAs . Here we cloned, expressed and purified the 111 amino acid polypeptide (designated Trbp111) and showed by ultracentrifugation analysis that it is a stable dimer in solution . The protein was also crystallized in a monoclinic lattice . X-ray diffraction analysis at 2.8 A resolution revealed a prominent non-crystallographic 2-fold axis, consistent with the presence of a symmetric homodimeric structure . Band-shift analysis with polyacrylamide gels showed that the dimer binds tRNAs, but not RNA duplexes, RNA hairpins, single-stranded RNA nor 5S rRNA . Complex formation with respect to tRNA is non-specific, with a single tRNA bound per dimer . Thus, Trbp111 is a structure-specific tRNA-binding protein . These results and other considerations raise the possibility that Trbp111 is a tRNA-specific chaperone which stabilizes the native L-shaped fold in the extreme thermophile and which has been incorporated into much larger tRNA-binding proteins of higher organisms. Nature, 1999 Jun 3, 399(6735), 496 - 9 Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase; Kohen A et al.; Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity . Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature . Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property . In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced . We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions . Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 degrees C; this is analogous to previous findings with mesophilic ADH at 25 degrees C . Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature . These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage. J Eukaryot Microbiol, 1999 Mar-Apr, 46(2), 147 - 54 New axonemal dynein heavy chains from Tetrahymena thermophila; Mobberley PS et al.; Two dyneins can be extracted from Tetrahymena ciliary axonemes . The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms . The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms . We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S . The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC . Antibodies against one of the 18S HCs do not recognize 22S dynein HCs . Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments . Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains. J Eukaryot Microbiol, 1999 Mar-Apr, 46(2), 142 - 6 Affinity-purification of concanavalin A-binding ciliary glycoconjugates of starved and feeding Tetrahymena thermophila; Driscoll C et al.; Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer . Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A) . To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T . thermophila . Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1 . Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation . In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells . However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells . This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T . thermophila . In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation . Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals. Fungal Genet Biol, 1999 Apr, 26(3), 178 - 89 Analysis of the heat-shock response displayed by two Chaetomium species originating from different thermal environments; Oberson J et al.; Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C . brasiliense . Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition . On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C . thermophile . This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions . It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response . C . thermophile contains less polyunsaturated fatty acids at normal temperature than C . brasiliense, a fact that can be directly related to thermophily . When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids . Nature, 1999 May 27, 399(6734), 323 - 9 Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima; Nelson KE et al.; The 1,860,725-base-pair genome of Thermotoga maritima MSB8 contains 1,877 predicted coding regions, 1,014 (54%) of which have functional assignments and 863 (46%) of which are of unknown function . Genome analysis reveals numerous pathways involved in degradation of sugars and plant polysaccharides, and 108 genes that have orthologues only in the genomes of other thermophilic Eubacteria and Archaea . Of the Eubacteria sequenced to date, T . maritima has the highest percentage (24%) of genes that are most similar to archaeal genes . Eighty-one archaeal-like genes are clustered in 15 regions of the T . maritima genome that range in size from 4 to 20 kilobases . Conservation of gene order between T . maritima and Archaea in many of the clustered regions suggests that lateral gene transfer may have occurred between thermophilic Eubacteria and Archaea. Nat Struct Biol, 1999 Jun, 6(6), 509 - 16 The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 A resolution; Williams PA et al.; The structure of the CuA-containing, extracellular domain of Thermus thermophilus ba3-type cytochrome c oxidase has been determined to 1.6 A resolution using multiple X-ray wavelength anomalous dispersion (MAD) . The Cu2S2 cluster forms a planar rhombus with a copper-copper distance of 2.51 +/- 0.03 A . X-ray absorption fine-structure (EXAFS) studies show that this distance is unchanged by crystallization . The CuA center is asymmetrical; one copper is tetrahedrally coordinated to two bridging cysteine thiolates, one histidine nitrogen and one methionine sulfur, while the other is trigonally coordinated by the two cysteine thiolates and a histidine nitrogen . Combined sequence-structure alignment of amino acid sequences reveals conserved interactions between cytochrome c oxidase subunits I and II. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6621 - 5 The telomerase RNA pseudoknot is critical for the stable assembly of a catalytically active ribonucleoprotein; Gilley D et al.; Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes telomeric DNA . A pseudoknot structure is phylogenetically conserved within the RNA component of telomerase in all ciliated protozoans examined . Here, we report that disruptions of the pseudoknot base pairing within the telomerase RNA from Tetrahymena thermophila prevent the stable assembly in vivo of an active telomerase . Restoring the base-pairing potential of the pseudoknot by compensatory changes restores telomerase activity to essentially wild-type levels . Therefore, the pseudoknot topology rather than sequence is critical for an active telomerase . Furthermore, we show that disruption of the pseudoknot prevents the association of the RNA with the reverse transcriptase protein subunit of telomerase . Thus, we provide an example of a structural motif within the telomerase RNA that is required for telomerase function and identify the domain that is required for telomerase complex formation . Hence, we identify a biological role for a pseudoknot: promoting the stable assembly of a catalytically active ribonucleoprotein. J Immunol, 1999 Jun 15, 162(12), 7397 - 401 Viral infection modulates expression of hypersensitivity pneumonitis; Gudmundsson G et al.; Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes that cause farmer's lung disease . The early response to Ag is an increase in neutrophils in the lung, whereas the late response is a typical Th1-type granulomatous disease . Many patients who develop disease report a recent viral respiratory infection . These studies were undertaken to determine whether viruses can augment the inflammatory responses in HP . C57BL/6 mice were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) for 3 consecutive days per wk for 3 wk . Some mice were exposed to SR at 2 wk after infection with respiratory syncytial virus (RSV), whereas others were exposed to SR after exposure to saline alone or to heat-inactivated RSV . SR-treated mice developed a typical, early neutrophil response and a late granulomatous inflammatory response . Up-regulation of IFN-gamma and IL-2 gene expression was also found during the late response . These responses were augmented by recent RSV infection but not by heat-inactivated RSV . Mice with a previous RSV infection also had a greater early neutrophil response to SR, with increased macrophage inflammatory protein-2 (MIP-2, murine equivalent of IL-8) release in bronchoalveolar lavage fluid . These studies suggest that viral infection can augment both the early and late inflammatory responses in HP. Extremophiles, 1999 May, 3(2), 131 - 7 Purification and properties of the pyrophosphate-dependent phosphofructokinase from Dictyoglomus thermophilum Rt46 B.1; Ding YH et al.; The distribution of phosphofructokinase phosphoryl donor subtypes (ATP-, ADP-, and pyrophosphate) in the deeply rooted phylogenetic lineages of thermophiles is of interest with regard to the evolution of phosphofructokinase activity and of the Embden-Meyerhof pathway . In this article we present the first biochemical description of a thermostable pyrophosphate-dependent phosphofructokinase from the hyperthermophilic bacterium Dictyoglomus thermophilum . The enzyme was not allosterically controlled by traditional modulators of phosphofructokinases and has significant activity with tripolyphosphate and polyphosphate . Kinetic parameters of the enzyme suggest it plays primarily a glycolytic role . The enzyme required Mg2+ for optimal activity, was partially activated by some monovalent and divalent cations, and was strongly inhibited by Cu2+ . The sequence of the 21 N-terminal residues suggests that the enzyme is most similar to the pyrophosphate-dependent phosphofructokinases from Amycolatopsis methanolica and the hyperthermophilic crenarchaeon Thermoproteus tenax, enzymes which have been suggested to represent an ancient lineage of phosphofructokinases (Siebers et al . 1998) . The unexpected finding of a pyrophosphate-dependent phosphofructokinase in Dictyoglomus thermophilum, which is phylogenetically related to Thermotoga maritima, previously shown to possess an ATP-dependent phosphofructokinase activity, is discussed. Extremophiles, 1999 May, 3(2), 103 - 11 Family 10 and 11 xylanase genes from Caldicellulosiruptor sp . strain Rt69B.1; Morris DD et al.; Three family 10 xylanase genes (xynA, xynB, and xynC) and a single family 11 xylanase gene (xynD) were identified from the extreme thermophile Caldicellulosiruptor strain Rt69B.1 through the use of consensus PCR in conjunction with sequencing and polyacrylamide gel electrophoresis . These genes appear to comprise the complete endoxylanase system of Rt69B.1 . The xynA gene was found to be homologous to the xynA gene of the closely related Caldicellulosiruptor strain Rt8B.4, and primers designed previously to amplify the Rt8B.4 xynA gene could amplify homologous full-length xynA gene fragments from Rt69B.1 . The complete nucleotide sequences of the Rt69B.1 xynB, xynC, and xynD genes were obtained using genomic walking PCR . The full-length xynB and xynC genes are more than 5 kb in length and encode highly modular enzymes that are the largest xylanases reported to date . XynB has an architecture similar to the family 10 xylanases from Thermoanaerobacterium saccharolyticum (XynA) and Clostridium thermocellum (XynX) and may be cell wall associated, while XynC is a bifunctional enzyme with an architecture similar to the bifunctional beta-glycanases from Caldicellulosiroptor saccharolyticus . The xynD gene encodes a two-domain family 11 xylanase that is identical in architecture to the XynB family 11 xylanase from the unrelated extreme thermophile Dictyoglomus thermophilum strain Rt46B.1 . The sequence similarities between the Rt69B.1 xylanases with respect to their evolution are discussed. FEBS Lett, 1999 May 14, 451(1), 51 - 5 N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E; Gongadze GM et al.; In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity . The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced . This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA . The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain . Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained. Microbiol Res, 1999 May, 154(1), 15 - 21 Survival of Enterobacter cloacae and Pseudomonas paucimobilis in yoghurts manufactured from cow's milk and soymilk during storage at different temperatures; Canganella F et al.; The survival of two microbial contaminants, Enterobacter cloacae and Pseudomonas paucimobilis, in yoghurts manufactured from cow's milk and soymilk was investigated during storage for 45 days at 4 and 12 degrees C . Sensory panel tests performed before microbiological investigation, showed that the flavor of soy-yoghurts made with cocoa powder or malt added did not have the beany taste of soy beans . Both contaminants were significantly resistant to low pH values during storage for 32 days at 4 degrees C . The survival at 4 degrees C was remarkable in both plain and flavored yoghurts and a population close to 10(2) C.F.U./ml was observed after 38 days of storage . Experiments performed with soymilk yoghurts showed an enhanced survival of P . paucimobilis at 4 degrees C compared to the storage in cow's milk yoghurts; microbial values were close to 7-8 x 10(6) C.F.U./ml after 16 days . Soymilk exhibited a protective effect on L . delbrueckii subsp . bulgaricus and S . thermophilus at 12 degrees C and, compared to the survival in cow's milk yoghurts, a larger number of viable cells of both probiotic microorganisms (10(6) and 10(8) C.F.U./ml, respectively) were observed after 36 days of storage. Biotechnol Prog, 1999 May-Jun, 15(3), 347 - 57 High-rate treatment of terephthalate in anaerobic hybrid reactors; Kleerebezem R et al.; The anaerobic degradation of terephthalate as sole substrate was studied in three anaerobic upflow reactors . Initially, the reactors were operated as upflow anaerobic sludge bed (UASB) reactors and seeded with suspended methanogenic biomass obtained from a full-scale down-flow fixed film reactor, treating wastewater generated during production of purified terephthalic acid . The reactors were operated at 30, 37, and 55 degrees C . The terephthalate removal capacities remained low in all three reactors (<4 mmolxL-1xday-1, or 1 g of chemical oxygen demand (COD)xL-1xday-1) due to limitations in biomass retention . Batch experiments with biomass from the UASB reactors revealed that, within the mesophilic temperature range, optimal terephthalate degradation is obtained at 37 degrees C . No thermophilic terephthalate-degrading culture could be obtained in either continuous or batch cultures . To enhance biomass retention, the reactors were modified to anaerobic hybrid reactors by introduction of two types of reticulated polyurethane (PUR) foam particles . The hybrid reactors were operated at 37 degrees C and seeded with a mixture of biomass from the UASB reactors operated at 30 and 37 degrees C . After a lag period of approximately 80 days, the terephthalate conversion capacity of the hybrid reactors increased exponentially at a specific rate of approximately 0.06 day-1, and high removal rates were obtained (40-70 mmolxL-1xday-1, or 10-17 g of CODxL-1xday-1) at hydraulic retention times between 5 and 8 h . These high removal capacities could be attributed to enhanced biomass retention by the development of biofilms on the PUR carrier material as well as the formation of granular biomass . Biomass balances over the hybrid reactors suggested that either bacterial decay or selective wash-out of the terephthalate fermenting biomass played an important role in the capacity limitations of the systems . The presented results suggest that terephthalate can be degraded at high volumetric rates if sufficiently long sludge ages can be maintained, and the reactor pH and temperature are close to their optima. Biochemistry, 1999 Jun 1, 38(22), 7075 - 84 Selenomethionine-substituted Thermus thermophilus cytochrome ba3: characterization of the CuA site by Se and Cu K-EXAFS; Blackburn NJ et al.; We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba3 subunit II {Keightley et al . (1995) J . Biol . Chem . 270, 20345-20358} . The resulting ba3-CuAt10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A . When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the CuAt10 protein was quantitatively replaced with Se(Met) . Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties . The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 A in the mixed-valence form and 2.52 A in the fully reduced form of CuA . Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles . An expanded EXAFS structural model is presented. Exp Lung Res, 1999 Apr-May, 25(3), 217 - 28 Respiratory epithelial cells release interleukin-8 in response to a thermophilic bacteria that causes hypersensitivity pneumonitis; Gudmundsson G et al.; Hypersensitivity pneumonitis (HP) is a granulomatous inflammatory lung disease that is usually triggered by organic antigens . At early time points after inhalation of antigen, neutrophilic inflammation is prominent in the lungs . Interleukin (IL)-8 is a potent chemoattractant for neutrophils and it is known that alveolar macrophages can release IL-8 after exposure to organic antigens . However, the role of respiratory epithelial cells in the production of IL-8 in HP is unknown . We exposed A549 epithelial cells to the thermophilic bacteria Saccharopolyspora rectivirgula (SR), and measured IL-8 release via enzyme-linked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) induction via Northern analysis . We observed a dose- and time-dependent release of IL-8 in response to SR . The maximal release of IL-8 was measured at 24-48 hours after exposure . There was also an increase in release of IL-6 in a time-dependent fashion . SR induced a peak increase in IL-8 mRNA at 12-24 hours . SR also triggered expression of the DNA-binding activity of NF-kappa B, a transcription factor that mediates activation of the IL-8 gene . Both corticosteroids and IL-10 blocked the production of IL-8 . The release of IL8 was not mediated through IL-1 beta . These data suggest that SR-induced IL-8 production in airway epithelium may play a role in the initial inflammatory response in HP. Biochim Biophys Acta, 1999 May 18, 1431(2), 363 - 73 Pyruvate phosphate dikinase from a thermophilic actinomyces Microbispora rosea subsp . aerata: purification, characterization and molecular cloning of the gene; Eisaki N et al.; Various thermophilic bacteria were analyzed by Southern hybridization analysis using oligonucleotide probes coding for the pyruvate phosphate dikinase (PPDK) gene from Clostridium symbiosum, and positive hybridization signals were observed in the chromosomal DNAs from Microbispora rosea subsp . aerata (IFO 14047) . PPDK activity was detected in lactose induced cells and the enzyme was purified to homogeneity . The molecular mass of PPDK was estimated to be 230000 by gel filtration chromatography and 91000 by SDS-PAGE, suggesting that PPDK is a dimeric enzyme . This enzyme was specific for adenine nucleotide and the apparent Km values for AMP, PPi, and phosphoenolpyruvate were 5, 38, and 280 microM, respectively . It was stable in the pH range 6 to 11, and retained 80% activity after 60 min heat treatment at 60 degrees C . We cloned the PPDK gene from M . rosea . It consists of 878 amino acids with a molecular mass of 95514 . Sequence comparison indicates around 50% similarity with other PPDKs and it has all the highly conserved regions of the related enzymes . We expressed the PPDK gene in Escherichia coli and obtained enzymatically active protein. FEBS Lett, 1999 Apr 30, 450(1-2), 135 - 8 Inorganic Fe2+ formation upon Fe-S protein thermodestruction in the membranes of thermophilic cyanobacteria: Mössbauer spectroscopy study; Kaurov YuN et al.; A model description of the Mossbauer spectrum (80 K) of native membranes of the thermophilic cyanobacterium Synechococcus elongatus is suggested on the basis of the known values of quadrupole splitting (deltaE(Q)) and isomer shift (deltaFe) for the iron-containing components of the photosynthetic apparatus . Using this approach, we found that heating the membranes at 70-80 K results in a decrease of doublet amplitudes belonging to F(X), F(A), F(B) and ferredoxin and simultaneous formation of a new doublet with deltaE(Q) = 3.10 mm/s and delta-Fe = 1.28 mm/s, typical of inorganic hydrated forms of Fe2+ . The inhibition of electron transfer via photosystem I to oxygen, catalyzed by ferredoxin, occurs within the same range of temperatures . The data demonstrate that the processes of thermoinduced Fe2+ formation and distortions in the photosystem I electron transport in the membranes are interrelated and caused mainly by the degradation of ferredoxin . The possible role of Fe2+ formation in the damage of the photosynthetic apparatus resulting from heating and the action of other extreme factors is discussed. J Biochem (Tokyo), 1999 Jun, 125(6), 1016 - 21 Identification and designing of the S3 site of aqualysin I, a thermophilic subtilisin-related serine protease; Tanaka T et al.; Aqualysin I is a bacterial subtilisin-related alkaline serine protease, originating in Thermus aquaticus YT-1 . Based on computational analysis, we predicted that two residues, Ser102 and Gly131, form the S3 site of aqualysin I, and we proved that this prediction by site-directed mutagenesis . To alter the P3-specificity of the enzyme, we built a "wall" on the S3 site edge by introducing a bulky side chain at target sites . Six mutant proteins were prepared: S102H, S102K, S102E, G131H, G131K, and G131D . The mutant enzymes were examined with two kinetically typical peptides for aqualysin I, suc-X-Ala-Ala-pNA, where X is Ala or Phe . All mutations reduced the efficiency for the Phe-containing peptide, while they raised the k(cat) values for the Ala-containing peptide . Especially, the S102K mutant protein hydrolyzed the polyalanine peptide efficiently . The strategies we have adopted in this paper are applicable to all subtilisin-related enzymes. J Biol Chem, 1999 Jun 4, 274(23), 16363 - 9 Structure/function of the beta-barrel domain of F1-ATPase in the yeast Saccharomyces cerevisiae; Bakhtiari N et al.; The first 90 amino acids of the alpha- and beta-subunits of mitochondrial F1-ATPase are folded into beta-barrel domains and were postulated to be important for stabilizing the enzyme (Abrahams, J . P., Leslie, A . G., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628) . The role of the domains was studied by making chimeric enzymes, replacing the domains from the yeast Saccharomyces cerevisiae enzyme with the corresponding domains from the enzyme of the thermophilic bacterium Bacillus PS3 . The enzymes containing the chimeric alpha-, beta-, or alpha- and beta-subunits were not functional . However, gain-of-function mutations were obtained from the strain containing the enzyme with the chimeric PS3/yeast beta-subunit . The gain-of-function mutations were all in codons encoding the beta-barrel domain of the beta-subunit, and the residues appear to map out a region of subunit-subunit interactions . Gain-of-function mutations were also obtained that provided functional expression of the chimeric PS3/yeast alpha- and beta-subunits together . Biochemical analysis of this active chimeric enzyme indicated that it was not significantly more thermostable or labile than the wild type . The results of this study indicate that the beta-barrel domains form critical contacts (distinct from those between the alpha- and beta-subunits) that are important for the assembly of the ATP synthase. Appl Environ Microbiol, 1999 Jun, 65(6), 2654 - 60 Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound; Li K et al.; Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid . The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid . The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS {2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)} as a substrate) . During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed . Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL . Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT . The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase . Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT . The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation . The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases . We did not find any relationship between the carbohydrate content of the laccases and their inactivation . When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I. Appl Environ Microbiol, 1999 Jun, 65(6), 2577 - 84 Temperature dependence of inorganic nitrogen uptake: reduced affinity for nitrate at suboptimal temperatures in both algae and bacteria; Reay DS et al.; Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures . We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments . At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased . The specific affinity for nitrate was strongly dependent on temperature (Q10 approximately 3, where Q10 is the proportional change with a 10 degrees C temperature increase) and consistently decreased at temperatures below the optimum temperature . In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence . This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae . The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures. Appl Environ Microbiol, 1999 Jun, 65(6), 2312 - 6 Reductive dechlorination of tetrachloroethene to cis-1, 2-dichloroethene by a thermophilic anaerobic enrichment culture; Kengen SW et al.; Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas . Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis-1, 2-dichloroethene at the optimum temperature of 60 to 65 degrees C . After several transfers, methanogens were eliminated from the culture . Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H2, formate, or acetate . Fumarate and L-malate led to the highest dechlorination rate . In the absence of PCE, fumarate was fermented to acetate, H2, CO2, and succinate . With PCE, less H2 was formed, suggesting that PCE competed for the reducing equivalents leading to H2 . PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor . At the optimum dissolved PCE concentration of approximately 60 microM, a high dechlorination rate of 1.1 micromol h-1 mg-1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity . Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod . Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives . The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus, and with the genus Desulfotomaculum (86%). Appl Microbiol Biotechnol, 1999 Apr, 51(4), 447 - 55 Cultivation of Tetrahymena thermophila in a 1.5-m3 airlift bioreactor; Hellenbroich D et al.; A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium . Cell growth and the production of extracellular proteases were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors . Processes using defined and complex medium formulations were compared . After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and followed by cell lyophilization . Cell sedimentation was shown to be the more efficient extraction method as it enabled cell retention/separation while preserving the cell structure . Maximum cell growth was achieved in the stirred-tank bioreactor, supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized nutrient supply . The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present in complex medium formulations . The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations. Curr Biol, 1999 May 20, 9(10), 531 - 4 Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes; Sandigursky M et al.; Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in eukaryotes and prokaryotes {1}{2}{3} . This enzyme removes uracil bases that are present in DNA as a result of either deamination of cytosine or misincorporation of dUMP instead of dTMP {4} {5}, and it is the primary activity in the DNA base excision repair pathway . Although UDG activities have been shown to be present in several thermophiles {6}{7}{8}, no sequences have been found that are complementary to the Escherichia coli ung gene, which encodes UDG {9} . Here, we describe a UDG from the thermophile Thermotoga maritima . The T . maritima UDG gene has a low level of homology to the E . coli G-T/U mismatch-specific DNA glycosylase gene (mug) . The expressed protein is capable of removing uracil from DNA containing either a U-A or a U-G base pair and is heat-stable up to 75 degrees C . The enzyme is also active on single-stranded DNA containing uracil . Analogous genes appear to be present in several prokaryotic organisms, including thermophilic and mesophilic eubacteria as well as archaebacteria, the human-disease pathogens Treponema palladium and Rickettsia prowazekii, and the extremely radioresistant organism Deinococcus radiodurans . These findings suggest that the T . maritima UDG is a member of a new class of DNA repair enzymes. J Mol Biol, 1999 May 28, 289(1), 187 - 93 Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima; Wassenberg D et al.; Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities . Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems . Small single-domain proteins are suitable candidates to overcome these obstacles . Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry . The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process . Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C . The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K) . At pH 7, thermal denaturation occurs at 82 degrees C . The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol . At the optimal growth temperature of T . maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy . With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules . Protein Sci, 1999 May, 8(5), 1056 - 63 Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus; Sun MM et al.; In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt) . The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure . Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide . For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date . Stabilization of both native GDH and rGDHt was also achieved by adding glycerol . Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed. Protein Sci, 1999 May, 8(5), 947 - 57 Auracyanin A from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus represents an unusual class of small blue copper proteins; Van Driessche G et al.; The amino acid sequence of the small copper protein auracyanin A isolated from the thermophilic photosynthetic green bacterium Chloroflexus aurantiacus has been determined to be a polypeptide of 139 residues . His58, Cys123, His128, and Met132 are spaced in a way to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin . Secondary structure prediction also indicates that auracyanin has a general beta-barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves . However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes . The overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5% . We suggest that auracyanin A, together with the B forms, is the first example of a new class of small copper proteins that may be descendants of an ancestral sequence to both the azurin proteins occurring in prokaryotic nonphotosynthetic bacteria and the plastocyanin proteins occurring in both prokaryotic cyanobacteria and eukaryotic algae and plants . The N-terminal sequence region 1-18 of auracyanin is remarkably rich in glycine and hydroxy amino acids, and required mass spectrometric analysis to be determined . The nature of the blocking group X is not yet known, although its mass has been determined to be 220 Da . The auracyanins are the first small blue copper proteins found and studied in anoxygenic photosynthetic bacteria and are likely to mediate electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center. Protein Expr Purif, 1999 Jun, 16(1), 125 - 35 Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability; Cacciapuoti G et al.; The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme . The recombinant enzyme was purified to homogeneity by means of a heat treatment (10 min at 100 degrees C) and by a single affinity chromatography step . The appropriate expression vector and host strain were selected and the culture conditions were determined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture . The heterologously expressed enzyme is identical to the original S . solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide bonds . On the other hand, the recombinant 5'-methylthioadenosine phosphorylase is less thermophilic and thermostable than the S . solfataricus enzyme, since an incorrect positioning of disulfide bonds within the molecule generates structures less stable to thermal unfolding . Eur J Biochem, 1999 Jun, 262(2), 563 - 8 The noncatalytic site-deficient alpha3beta3gamma subcomplex and FoF1-ATP synthase can continuously catalyse ATP hydrolysis when Pi is present; Bald D et al.; We investigated ATP hydrolysis by a mutant (DeltaNC) alpha3beta3gamma subcomplex of F0F1-ATP synthase from the thermophilic Bacillus PS3 that is defective in the noncatalytic nucleotide binding sites . This mutant subcomplex was activated by inorganic phosphate ions (Pi) and did not show continuous ATP hydrolysis activity in the absence of Pi . Pi also activated the wild-type alpha3beta3gamma subcomplex in a similar manner . Sulphate activated wild-type alpha3beta3gamma but not DeltaNC alpha3beta3gamma, indicating that Pi activation did not involve noncatalytic sites but that sulphate activation did . Pi also activated ATP hydrolysis and coupled proton translocation by the wild-type and DeltaNC F0F1-ATP synthases reconstituted into vesicle membranes. Eur J Biochem, 1999 Jun, 262(2), 406 - 16 Purification and characterization of the 16-kDa heat-shock-responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an alpha-crystallin-related, small heat shock protein; Roy SK et al.; A 16-kDa protein, one of the major proteins that accumulates upon heat-shock treatment in the thermophilic cyanobacterium Synechococcus vulcanus, was purified to apparent homogeneity . The N-terminal and internal amino acid sequences of the protein exhibited a homology to the alpha-crystallin-related, small heat shock proteins from other organisms . The protein was designated HspA . Size-exclusion chromatography and nondenaturing gel electrophoresis demonstrated that HspA formed a large homo-oligomer consisting of 24 subunits . It prevented the aggregation of porcine malic dehydrogenase at 45 degrees C and 50 degrees C and citrate synthase at 50 degrees C . The activity of the malic dehydrogenase, however, was not protected under these heat-shock conditions or reactivated after a shift in temperature from 45 or 50 degrees C to 21 degrees C . HspA was able to enhance the refolding of chemically denatured rabbit muscle lactate dehydrogenase in an ATP-independent manner . A homologue to the 16-kDa protein was also found to be induced upon heat-shock treatment in the mesophilic cyanobacterium Synechocystis sp . PCC 6803. J Biol Chem, 1999 May 28, 274(22), 15655 - 61 Crystal structure of carboxylase reaction-oriented ribulose 1, 5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita; Sugawara H et al.; Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1 . 39) obtained from a thermophilic red alga Galdieria partita has the highest specificity factor of 238 among the Rubiscos hitherto reported . Crystal structure of activated Rubisco from G . partita complexed with the reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) has been determined at 2.4-A resolution . Compared with other Rubiscos, different amino residues bring the structural differences in active site, which are marked around the binding sites of P-2 phosphate of 2-CABP . Especially, side chains of His-327 and Arg-295 show the significant differences from those of spinach Rubisco . Moreover, the side chains of Asn-123 and His-294 which are reported to bind the substrate, ribulose 1,5-bisphosphate, form hydrogen bonds characteristic of Galdieria Rubisco . Small subunits of Galdieria Rubisco have more than 30 extra amino acid residues on the C terminus, which make up a hairpin-loop structure to form many interactions with the neighboring small subunits . When the structures of Galdieria and spinach Rubiscos are superimposed, the hairpin region of the neighboring small subunit in Galdieria enzyme and apical portion of insertion residues 52-63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other. Biotechnol Appl Biochem, 1999 Jun, 29 ( Pt 3), 235 - 9 Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus; Mori S et al.; Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined . The purified enzyme had specific activity of 4.43 micromol p-nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa . Its apparent Km for p-nitrophenyl phosphate was 1.114 mM . The enzyme exhibited an optimal pH of approx . 9.0 and exhibited its highest activity at 60-70 degrees C . It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+ . In addition, significant thermal stability was observed in comparison with counterparts from mesophiles . Its partial N-terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method . A search on the SwissProt database did not reveal any similar protein sequences from other sources. Arch Biochem Biophys, 1999 Jun 1, 366(1), 40 - 6 Purification and characterization of thermostable aspartase from Bacillus sp . YM55-1; Kawata Y et al.; A thermostable aspartase was purified from a thermophile Bacillus sp . YM55-1 and characterized in terms of activity and stability . The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies . The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH . At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively . The specific activity was four and three times higher than those of E . coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively . Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride . The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms . Nat Biotechnol, 1999 May, 17(5), 462 - 5 Surface display of a parasite antigen in the ciliate Tetrahymena thermophila; Gaertig J et al.; The ciliated protozoan, Tetrahymena thermophila, offers an attractive medium for the expression of heterologous proteins and could prove particularly useful for the display of foreign proteins on the cell surface . Although progress has been made in transformation of Tetrahymena with heterologous DNA, methods that permit reliable expression of foreign genes have been lacking . Using a mutant strain of T . thermophila carrying a negatively selectable allele of a beta-tubulin gene, we have been able to direct foreign genes to this locus by homologous recombination . Transformed cell lines producing foreign proteins were readily identified and, in at least one case, targeting of proteins to the plasma membrane was accomplished. J Protein Chem, 1999 Feb, 18(2), 215 - 23 Structural and functional studies on the overproduced L11 protein from Thermus thermophilus; Triantafillidou D et al.; The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol . The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli . Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide . Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus . The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms . Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion . The similar structural and biochemical properties as well as the significant homology between L11 from E . coli and B . stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function. Mol Cell Biochem, 1999 Mar, 193(1-2), 99 - 102 Identification of the archaeal NMN adenylytransferase gene; Raffaelli N et al.; Increasing evidence on the importance of fluctuations in NAD+ levels in the living cell is accumulating . Therefore a deeper knowledge on the regulation of coenzyme synthesis and recycling is required . In this context the study of NMN adenylyltransferase (EC 2.7.7.1), . a key enzyme in the NAD+ biosynthetic pathway, assumes a remarkable relevance . We have previously purified to homogeneity and characterized the protein from the thermophilic archaeon Sulfolobus solfataricus . The determination of partial sequence of the S . solfataricus enzyme, together with the recent availability of the genome sequence of the archaeon Methanococcus jannaschii, allowed us, based on sequence similarity, to identify the M . jannaschii NMN adenylyltransferase gene . As far as we know from literature, this is the first report on the NMN adenylyltransferase gene. Genetika, 1999 Jan, 35(1), 37 - 45 {The proline biosynthesis gene proA from the thermophilic bacterium Thermus ruber: its cloning, sequencing and heterologous expression}; Iaklichkin SIu et al.; The proA proline biosynthesis gene of thermophilic bacterium Thermus ruber was cloned and sequenced, and several properties of the encoded enzyme, gamma-glutamylphosphate reductase (GPR) were studied . The proA open reading frame (ORF) was of 1286 bp . Nucleotide sequence analysis revealed the ATG initiation codon in position 36 and the TTA termination codon in position 1304 . A deduced protein product of the gene was shown to be of 44,919 Da in molecular weight . The GC content was 66%, as is characteristic of various bacteria of the genus Thermus . An amino acid sequence encoded by the cloned gene showed the highest homology (up to 64%) with GPR of T . thermophilus . The maximum activity of GPR (8.2 x 10(-2) units/ml) was observed at 55 degrees C . A weak enzymatic activity was also detected at 70 degrees C . The enzyme can be used in biotechnological studies. J Biol Chem, 1999 May 21, 274(21), 14533 - 6 Identification and mutation of phosphorylation sites in a linker histone . Phosphorylation of macronuclear H1 is not essential for viability in tetrahymena; Mizzen CA et al.; Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation . In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase . Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation . Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila . Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps . Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences . Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences . Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34(cdc2) site was also phosphorylated . Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished . These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena. J Mol Biol, 1999 May 14, 288(4), 623 - 34 Crystal structures of thermostable xylose isomerases from Thermus caldophilus and Thermus thermophilus: possible structural determinants of thermostability; Chang C et al.; The crystal structures of highly thermostable xylose isomerases from Thermus thermophilus (TthXI) and Thermus caldophilus (TcaXI), both with the optimum reaction temperature of 90 degrees C, have been determined by X-ray crystallography . The model of TcaXI has been refined to an R-factor of 17.8 % for data extending to 2.3 A and that of TthXI to 17.1 % for data extending to 2.2 A . The tetrameric arrangement of subunits characterized by the 222-symmetry and the tertiary fold of each subunit in both TcaXI and TthXI are basically the same as in other xylose isomerases . Each monomer is composed of two domains . Domain I (residues 1 to 321) folds into the (beta/alpha)8-barrel . Domain II (residues 322 to 387), lacking beta-strands, makes extensive contacts with domain I of an adjacent subunit . Each monomer of TcaXI contains ten beta-strands, 15 alpha-helices, and six 310-helices, while that of TthXI contains ten beta-strands, 16 alpha-helices, and five 310-helices . Although the electron density does not indicate the presence of bound metal ions in the present models of both TcaXI and TthXI, the active site residues show the conserved structural features . In order to understand the structural basis for thermostability of these enzymes, their structures have been compared with less thermostable XIs from Arthrobacter B3728 and Actinoplanes missouriensis (AXI and AmiXI), with the optimum reaction temperatures of 80 degrees C and 75 degrees C, respectively . Analyses of various factors that may affect protein thermostability indicate that the possible structural determinants of the enhanced thermostability of TcaXI/TthXI over AXI/AmiXI are (i) an increase in ion pairs and ion-pair networks, (ii) a decrease in the large inter-subunit cavities, (iii) a removal of potential deamidation/isoaspartate formation sites, and (iv) a shortened loop . Arch Biochem Biophys, 1999 May 15, 365(2), 223 - 30 Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile; Subramaniam SS et al.; Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi . CDH contains one heme b and one FAD per molecule and oxidizes cellobiose to cellobionolactone in the presence of cytochrome c . In this report, a thermostable CDH from the thermophilic ascomycete Sporotrichum thermophile has been purified, cloned, and characterized . The temperature optimum for this CDH reaction was 60 degrees C, and the activation energy for the reaction was 26.3 kJ/mol . The Km and kcat were temperature-dependent and increased as reaction temperature increased . These kinetic properties prove that this CDH is truly thermophilic . A 2.8-kb cDNA was isolated by screening an expression library of S . thermophile with a polyclonal antisera raised against Phanerochaete chrysosporium CDH . The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Da . S . thermophile CDH is organized into three domains, an N-terminal flavin domain, a middle heme domain, and a C-terminal cellulose-binding domain, which shows sequence similarity with the cellulose-binding domains of endoglucanases and cellobiohydrolases from Trichoderma reesei . Comparison with the CDH sequences of P . chrysosporium and Trametes versicolor identified Met 95 and His 143 as potential heme coordinations . EFIG, LGGPM, and VNSTH motifs in the heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were identified as CDH-specific motifs . With regard to the amino acid composition, S . thermophile CDH has more disulfide linkages and acidic and basic amino acids compared to CDHs from P . chrysosporium and T . versicolor . J, Mar . Biotechnol. . 1998 Dec, 6(4), 201 - 205 Isoprenoid synthesis gene for geranylgeranyl diphosphate synthase from a hyperthermophile, Pyrococcus sp . strain OT3; Masuchi Y et al.; Pyrococcus sp . strain OT3, a hyperthermophilic archaeon that was isolated by the authors was found to contain tetraether lipid mainly in the membrane lipid, which was quite different from the other hyperthermophiles (Masuchi et al . 1997) . Those isoprenoids are synthesized by a family of isoprenyl diphosphate synthases from isopentenyl diphosphate to allylic diphosphates . The gene that encodes one of these families, geranylgeranyl diphosphate synthase (GGPPSase), from this strain was cloned and sequenced . This coding gene has a 960-bp (320aa) sequence . The putative Shine-Dalgarno sequence was six bases upper of start codon, exactly the same as Methanobacterium thermoautotrophicum, a methnogenic thermophile . Comparison of the amino acid sequence of 13 organisms including Eukarya, Bacteria, and Archaea showed that Archaea strains including Pyrococcus sp . strain OT3 consisted of a separate group from the others, but five conservative regions are very homologous. Genes Dev, 1999 May 1, 13(9), 1116 - 25 Telomerase RNA function in recombinant Tetrahymena telomerase; Licht JD et al.; Telomerase is a ribonucleoprotein reverse transcriptase specialized for use of a sequence within its integral RNA component as the template for DNA synthesis . Telomerase adds telomeric simple sequence repeats to single-stranded primers in vitro or chromosome ends in vivo . We have investigated the sequences and structures of recombinant Tetrahymena thermophila telomerase RNA necessary for physical association and activity with the catalytic protein subunit expressed in rabbit reticulocyte lysate . In contrast with previous results using another reconstitution method, we find that phylogenetically conserved primary sequences and a phylogenetically nonconserved secondary structure are essential for telomerase RNA function . Telomerase RNA binding to the catalytic protein subunit requires sequences 5' of the template and is highly sequence specific . Other telomerase RNA sequences are required for enzyme activity and proper template use but not for protein interaction affinity . In addition, we demonstrate that the production of active recombinant telomerase requires a factor in rabbit reticulocyte lysate that promotes ribonucleoprotein assembly . These studies demonstrate multiple functions for the telomerase RNA and indicate that recombinant telomerase activity requires more than the catalytic protein and RNA components of the enzyme that have been identified to date. J Bacteriol, 1999 May, 181(10), 3172 - 7 Sodium-dependent glutamate uptake by an alkaliphilic, thermophilic Bacillus strain, TA2.A1; Peddie CJ et al.; A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70 degrees C . Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth . Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (DeltapNa) across the cell membrane . N, N-Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F1F0-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions . Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1 . Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (DeltaPsi), but only when sodium was present . In the absence of sodium, the rate of DeltaPsi-driven glutamate uptake was fourfold lower . No glutamate transport was observed in the presence of DeltapNa alone (i.e., no DeltaPsi) . Glutamate uptake was specific