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| 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 specifically inhibited by monensin, and the Km for sodium was 5.6 mM . The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium . Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport . The rate of glutamate transport increased with increasing glutamate concentration; the Km for glutamate was 2.90 microM, and the Vmax was 0.7 nmol . min-1 mg of protein . Glutamate transport was specifically inhibited by glutamate analogues. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 783 - 6 Hydrogenophilus thermoluteolus gen . nov., sp . nov., a thermophilic, facultatively chemolithoautotrophic, hydrogen-oxidizing bacterium; Hayashi NR et al.; The taxonomic positions of 'Pseudomonas hydrogenothermophila' strain TH-1 and 'Flavobacterium autothermophilum' strain TH-4 were studied by 16S rDNA sequencing . These organisms are Gram-negative, strictly aerobic, thermophilic, facultatively chemolithoautotrophic hydrogen-oxidizing rods and have a DNA G + C content of 63-65 mol% . The major isoprenoid quinone is ubiquinone-8 and 3-hydroxy decanoic acid (3-OH C10:0) is the major 3-hydroxy cellular fatty acid . A phylogenetic analysis based on 16S rDNA sequences placed strains TH-1T and TH-4 in the beta-subclass of the Proteobacteria . The taxonomic characteristics of these organisms are different from those of previously described aerobic, facultatively chemolithoautotrophic, hydrogen-oxidizing bacteria that belong to the beta-subclass of Proteobacteria . On the basis of the information described above, a new genus and species, Hydrogenophilus thermoluteolus gen . nov., sp . nov., is described to include both strains . The type strain is strain TH-1T (= IFO 14978T). Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 759 - 67 Streptococcus waius sp . nov., a thermophilic Streptococcus from a biofilm; Flint SH et al.; Thermophilic streptococci were isolated from biofilms on stainless steel samples exposed to pasteurized skimmed milk and from dairy products from a dairy manufacturing plant . The phenotypic characters of these isolates were distinct from those of other thermophilic streptococci of dairy origin (Streptococcus thermophilus and Streptococcus bovis) . Genotypic data {restriction endonuclease analysis, ribotyping, random amplified polymorphic DNA (RAPD) profiles, DNA-DNA hybridization and G + C contents} support the classification of these isolates as a new species . The sequence of the 16S rRNA was compared with that of 29 species of streptococci and shown to be significantly different . The sequence of the 16S-23S rRNA intergenic spacer region also differed from published sequences of closely related species . A fluorescent in situ hybridization probe prepared to a specific part of the 16S rRNA gene sequence was able to distinguish the unknown isolates from reference isolates of S . thermophilus and S . bovis . It is proposed that these thermophilic streptococcal isolates from a dairy environment be classified in the genus Streptococcus as a new species, Streptococcus waius (from waiu, the New Zealand Maori word for milk) . The type strain is 3/1T (= NZRCC 20100T). Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 619 - 28 Thermaerobacter marianensis gen . nov., sp . nov., an aerobic extremely thermophilic marine bacterium from the 11,000 m deep Mariana Trench; Takai K et al.; A novel extremely thermophilic bacterium was isolated from the world's deepest sea-floor, the Mariana Trench Challenger Deep at a depth of 10,897 m . Cells were Gram-reaction variable, non-spore-forming and non-motile rods without flagella . Growth was observed between 50 and 80 degrees C (optimum: 74-76 degrees C; 90 min doubling time), pH 5.4 and 9.5 (optimum: pH 7.0-7.5) and 0.5 and 5% sea salts (optimum: 2% sea salts) . The isolate was a strictly aerobic heterotroph capable of utilizing as sole energy and carbon source: yeast extract, peptone, cellulose, starch, chitin, casein, Casamino acids, a variety of sugars, carboxylic acids and amino acids . The G + C content of the genomic DNA was 72.5 mol% . Phylogenetic analysis based on 16S rRNA sequences placed this aerobic, high-G + C-content bacterium among the members of the Gram-positive, low-G + C-content anaerobic thermophilic bacteria within the Bacillus-Clostridium subphylum . On the basis of the physiological and molecular properties of the new isolate, the name Thermaerobacter marianensis gen . nov., sp . nov . (type strain 7p75aT = JCM 10246T) is proposed. FEMS Microbiol Rev, 1999 Apr, 23(2), 153 - 77 Heteropolysaccharides from lactic acid bacteria; De Vuyst L et al.; Microbial exopolysaccharides are biothickeners that can be added to a wide variety of food products, where they serve as viscosifying, stabilizing, emulsifying or gelling agents . Numerous exopolysaccharides with different composition, size and structure are synthesized by lactic acid bacteria . The heteropolysaccharides from both mesophilic and thermophilic lactic acid bacteria have received renewed interest recently . Structural analysis combined with rheological studies revealed that there is considerable variation among the different exopolysaccharides; some of them exhibit remarkable thickening and shear-thinning properties and display high intrinsic viscosities . Hence, several slime-producing lactic acid bacterium strains and their biopolymers have interesting functional and technological properties, which may be exploited towards different products, in particular, natural fermented milks . However, information on the biosynthesis, molecular organization and fermentation conditions is rather scarce, and the kinetics of exopolysaccharide formation are poorly described . Moreover, the production of exopolysaccharides is low and often unstable, and their downstream processing is difficult . This review particularly deals with microbiological, biochemical and technological aspects of heteropolysaccharides from, and their production by, lactic acid bacteria . The chemical composition and structure, the biosynthesis, genetics and molecular organization, the nutritional and physiological aspects, the process technology, and both food additive and in situ applications (in particular in yogurt) of heterotype exopolysaccharides from lactic acid bacteria are described . Where appropriate, suggestions are made for strain improvement, enhanced productivities and advanced modification and production processes (involving enzyme and/or fermentation technology) that may contribute to the economic soundness of applications with this promising group of biomolecules. Mikrobiologiia, 1998 Nov-Dec, 67(6), 792 - 8 {Comparative study of the homology of DNA in thermophilic and mesophilic lactococcal strains of various origin}; Averina OV et al.; A comparative study of DNA homology among 12 strains of thermophilic streptococci currently used as ferments in the dairy industry in different regions of the CIS and 16 strains of mesophilic lactococci obtained from different sources was performed by the method of optical reassociation in solution . These strains were found to form three groups with DNA homology generally not exceeding 30-35% between each other . These data indicate that strains commonly classified as Streptococcus thermophilus may belong to different taxa . At the same time, the DNA base composition was similar in all these strains (the G + C content was 38-40 mol %) . Among 16 strains of mesophilic lactococci DNA homology was generally higher than 50%, i.e., all these strains indeed belong to the same species, Lactococcus lactis. Eur J Biochem, 1999 May, 262(1), 218 - 23 Active-site mutations which change the substrate specificity of the Clostridium stercorarium cellulase CelZ implications for synergism; Riedel K et al.; CelZ from the cellulolytic thermophile Clostridium stercorarium has been described as a 'monomeric' cellulase able to effect both the endoglucanolytic hydrolysis of internal glycosidic linkages and the exoglucanolytic degradation from the chain ends in a processive mode of action . The putative catalytic residues of this family 9 cellulase, Asp84 and Glu447 located within the N-terminal domain of the modular protein, were replaced by site-directed mutagenesis . A minimized CelZ derivative (CelZC') comprising the catalytic domain and the adjacent cellulose-binding domain (CBD) family IIIc domain C' was used as target for mutagenesis . Six mutant enzymes and the unmodified CelZC' protein were purified to homogeneity and compared with respect to thermoactivity, substrate specificity, product profile and synergism . CD studies revealed that no major changes to the overall structure of the proteins had occurred . Replacement of either one or both catalytic residues completely eliminated the ability of CelZ to attack insoluble Avicel preparations indicative of the exo-activity, whereas the endo-activity measured via hydrolysis of CM-cellulose was retained upon substitution of the catalytic base Asp84 . Thus, endo-active CelZ mutants defective in the exo-activity were available for co-operativity studies with the C . stercorarium exoglucanase CelY . Synergism was found to be dependent on the endo-activity of CelZ . Mutants Asp84Gly and Asp84Glu were able to enhance the degradation of crystalline cellulose significantly, although no products could be released from this substrate by individual action of the mutants. Appl Biochem Biotechnol, 1998 Nov-Dec, 75(2-3), 249 - 59 Hyperalkaline and thermostable phosphatase in Thermus thermophilus; Pantazaki AA et al.; The phosphatases existing in the extreme thermophilic bacterium Thermus thermophilus have been studied . Utilizing ion exchange, hydrophobic, pseudoaffinity, and affinity chromatography, a number of distinct phosphatase activities were identified . At least four phosphatases, with optimum pH ranging between 5.0 and 11.5, were assayed with p-nitrophenylphosphate, and two with optimum pH between 7.0 and 11.0, with 32P-casein as substrate . The authors have focused on the hyperalkaline phosphatase and have tried to purify and characterize it . This hyperalkaline phosphatase reaches a maximal level at the stationary phase of the growth, and is co-purified with alkaline phosphatase with optimum pH of 10.2 . The enzymes present a relative mol wt of 65 and 58 kDa, respectively, as judged by SDS-PAGE and Sephadex G-150 column, and possess similar properties, indicating that they are isoforms . These enzymes barely function in the presence of tartrate, and are inhibited by EDTA, pyrophosphate, and molybdate . Among the metals tested, Hg2+ appeared as the strongest inhibitor of the hyperalkaline phosphatase . The two enzymes are thermostable and, upon treatment at 90 degrees C for 10 min, 75% of their activity remains . The physiological role and function of these phosphatases need further investigation. J Mol Evol, 1999 Jun, 48(6), 756 - 69 DNA polymerase C of the thermophilic bacterium Thermus aquaticus: classification and phylogenetic analysis of the family C DNA polymerases; Huang YP et al.; Bacterial family C DNA polymerases (DNA pol IIIs), the major chromosomal replicative enzymes, have been provisionally classified based on primary sequences and domain structures into three classes: class I (Escherichia coli DNA pol C-type), class II (Bacillus subtilis DNA pol C-type), and class III (cyanobacterial DNA pol C-type), respectively . We have sequenced the structural gene encoding the DNA pol C catalytic subunit of the thermophilic bacterium Thermus aquaticus . This gene, designated the Taq DNA pol C gene, contains a 3660-bp open reading frame which specifies a polypeptide of molecular weight of 137,388 daltons . Comparative sequence analyses revealed that Taq DNA pol C is a class I family C DNA polymerase . The Taq DNA pol C is most closely related to the Deinococcus radiodurans DNA pol C . Although a phylogenetic tree based on the class I family C DNA pols is still in the provisional stage, some important conclusion can be drawn . First, the high-G+C and the low-G+C Gram-positive bacteria are not monophyletic . Second, the low-G+C Gram-positive bacteria contain multigenes of family C DNA pols (classes I and II) . Third, the cyanobacterial family C DNA pol, classified as class III because it is encoded by a split gene, forms a group with the high-G+C Gram-positive bacteria. FEMS Microbiol Lett, 1999 Apr 15, 173(2), 431 - 7 An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus; Tamakoshi M et al.; A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method . First, the leuB gene was replaced with the pyrE gene . Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid . A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host . When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme . These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes. Proteins, 1999 May 1, 35(2), 163 - 72 Structural and dynamic aspects of beta-glycosidase from mesophilic and thermophilic bacteria by multitryptophanyl emission decay studies; Bismuto E et al.; The tryptophanyl emission decay of beta-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (Sbetagly) has been investigated by frequency domain fluorometry . The data were analyzed in terms of sum of discrete lifetimes as well as in terms of quasi- continuous lifetime distributions of different shape . At neutral pH the emission decay is characterized by two components: a long-lived component, centered at 7.4 ns, and a short one at 2.7 ns, irrespective of the decay scheme used for the interpretation of the experimental results . The effects of an irreversible inhibitor, that is, cyclophellitol, and that of a powerful denaturant such as guanidinium hydrochloride on the dynamics of Sbetagly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay . The addition of cyclophellitol to native Sbetagly reduces the contribution of the short-lived component but does not affect the long-lived one . Increasing concentrations of guanidinium hydrochloride differently affect the contributions of the two emission components . Higher concentrations were required to unfold the molecular regions containing the long-lived indolic fluorophores . These results indicate that the long-lived contribution arises from tryptophanyl residues deeply clustered in the interior of the protein matrix, whereas the short-lived one includes residues located in less rigid and more solvent accessible regions, some of which might be located in functionally important parts of protein . The knowledge of the crystallographic structure of Sbetagly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sbetagly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers . These results permitted to divide the tryptophanyl fluorescence of Sbetagly in the contribution of two emitting groups: one consisting of eight closely clustered tryptophans, that is, Trp 33, 36, 60, 84, 151 174, 425, and 433, responsible for the long-lived emission component and the other one, composed of nine tryptophans nearer to the subunit surface, that is, Trp 12, 156, 192, 287, 288, 316, 361, 376, 455, associable to the short-lived emission component . Finally, the examination of the tryptophanyl emission decay of the mesophilic beta-galactosidase from Escherichia coli (Cbetagal) and the Arrhenius analysis of its dependence on temperature indicated that the tryptophanyl environments of the mesophilic enzyme are rather homogeneous in consequence of a larger protein dynamics. Appl Environ Microbiol, 1999 May, 65(5), 2084 - 91 Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme; Bertoldo C et al.; The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli . The pulA gene from F . pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes . The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide . The pulA gene was subcloned without its signal sequence and overexpressed in E . coli under the control of the trc promoter . This clone, E . coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity . A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein . The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C . The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin . Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs . According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I . The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry. Appl Environ Microbiol, 1999 May, 65(5), 1991 - 7 Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp; Tachibana Y et al.; The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan . The cells were irregular cocci, 0.5 to 1.0 micrometers in diameter . The new isolate grew at temperatures between 60 and 95 degrees C (optimum, 85 degrees C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%) . The G+C content of the genomic DNA was 43.0 mol% . The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus . During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase) . The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . Incubation at 120 degrees C with SDS and 2-mercaptoethanol was required for complete unfolding . The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100 degrees C, respectively . The optimum pH for enzyme activity was pH 5.0 to 5.5 . At pH 5.0, the half-life of the enzyme was 40 min at 110 degrees C . The enzyme formed mainly alpha-cyclodextrin with small amounts of beta- and gamma-cyclodextrins from starch . This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea. Bioinformatics, 1999 Mar, 15(3), 187 - 93 10-11 bp periodicities in complete genomes reflect protein structure and DNA folding; Herzel H et al.; MOTIVATION: Completely sequenced genomes allow for detection and analysis of the relatively weak periodicities of 10-11 basepairs (bp) . Two sources contribute to such signals: correlations in the corresponding protein sequences due to the amphipatic character of alpha-helices and the folding of DNA (nucleosomal patterns, DNA supercoiling) . Since the topological state of genomic DNA is of importance for its replication, recombination and transcription, there is an immediate interest to obtain information about the supercoiled state from sequence periodicities . RESULTS: We show that correlations within proteins affect mainly the oscillations at distances below 35 bp . The long-ranging correlations up to 100 bp reflect primarily DNA folding . For the yeast genome these oscillations are consistent in detail with the chromatin structure . For eubacteria and archaea the periods deviate significantly from the 10.55 bp value for free DNA . These deviations suggest that while a period of 11 bp in bacteria reflects negative supercoiling, the significantly different period of thermophilic archaea close to 10 bp corresponds to positive supercoiling of thermophilic archaeal genomes . AVAILABILITY: Protein sets and C programs for the calculation of correlation functions are available on request from the authors (see http://itb.biologie.hu-berlin.de). J Biochem (Tokyo), 1999 May, 125(5), 858 - 63 Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation; Satoh T et al.; The regions contributing to the thermostability of inorganic pyrophosphatase (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our previous study by random chimeragenesis, one of them being estimated to be Ala144-Lys145 {Satoh, T., Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H., Watanabe, M., and Samejima, T . (1999) Biochemistry 38, 1531-1536} . Therefore, we investigated the contributions of these two residues in Tth by preparing a deletion mutant (del.144-145 mutant) of Tth PPase . We examined its thermostability in terms of the CD and fluorescence spectra, and the thermal change in the enzymatic activity . The thermostability of the enzymatic activity of the del.144-145 mutant was similar to that of the wild type Tth PPase, whereas this mutant was more stable against heating . Furthermore, we compared the thermal aggregation of the wild type with that of the del.144-145 mutant . We found that the thermal aggregation of the mutant was reduced relative to that of the wild type . Moreover, the molecular weight of the mutant after heating at 90 degrees C was higher than that of the unheated one, whereas the wild type aggregated under the same conditions . Therefore, we can conclude that although the Ala144-Lys145 residues in Tth PPase may partly cause thermal aggregation, the deletion of these residues may stabilize the Tth PPase molecule structurally against heating and suppress thermal aggregation. FEBS Lett, 1999 Mar 19, 447(1), 25 - 8 Thermostable aminopeptidase from Pyrococcus horikoshii; Ando S et al.; From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity) . This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined . Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides . The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C . The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity . Instead, this enzyme required the cobalt ion for activity . This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures. J Bacteriol, 1999 May, 181(9), 2834 - 9 Purification and characterization of Thermotoga maritima endonuclease IV, a thermostable apurinic/apyrimidinic endonuclease and 3'-repair diesterase; Haas BJ et al.; An endonuclease IV homolog was identified as the product of a conceptual open reading frame in the genome of the hyperthermophilic bacterium Thermotoga maritima . The T . maritima endonuclease IV gene encodes a 287-amino-acid protein with 32% sequence identity to Escherichia coli endonuclease IV . The gene was cloned, and the expressed protein was purified and shown to have enzymatic activities that are characteristic of the endonuclease IV family of DNA repair enzymes, including apurinic/apyrimidinic endonuclease activity and repair activities on 3'-phosphates, 3'-phosphoglycolates, and 3'-trans-4-hydroxy-2-pentenal-5-phosphates . The T . maritima enzyme exhibits enzyme activity at both low and high temperatures . Circular dichroism spectroscopy indicates that T . maritima endonuclease IV has secondary structure similar to that of E . coli endonuclease IV and that the T . maritima endonuclease IV structure is more stable than E . coli endonuclease IV by almost 20 degrees C, beginning to rapidly denature only at temperatures approaching 90 degrees C . The presence of this enzyme, which is part of the DNA base excision repair pathway, suggests that thermophiles use a mechanism similar to that used by mesophiles to deal with the large number of abasic sites that arise in their chromosomes due to the increased rates of DNA damage at elevated temperatures. Biochim Biophys Acta, 1999 Apr 19, 1427(2), 193 - 204 Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover; Ciulla RA et al.; In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions . The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl . However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased . Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked . In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration . Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine . This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte . Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover . These results are discussed in terms of the osmoadaptation strategies of M . thermolithotrophicus. Biochim Biophys Acta, 1999 Apr 21, 1411(1), 147 - 58 Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus; Sakamoto J et al.; Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain . We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J . Sakamoto et al., FEMS Microbiol . Lett . 143 (1996) 151-158) . Compared with proteobacterial counterparts, B . stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity . Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity . In order to clarify the molecular basis of the differences of cytochromes bd between B . stearothermophilus and proteobacteria, the genes encoding for the B . stearothermophilus bd was cloned based on its partial peptide sequences . The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E . coli enzyme . The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da . Homology search indicated that the B . stearothermophilus cbdAB has the highest sequence similarity to ythAB in B . subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome . Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E . coli bd and a more widely distributed type including the B . stearothermophilus enzyme, suggesting that the latter type is evolutionarily older. FEBS Lett, 1999 Mar 26, 447(2-3), 297 - 302 Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C-terminal modulator element; Fujiwara T et al.; Ribosome recycling factor, referred to as RRF, is essential for bacterial growth because of its activity of decomposition of the post-termination complex of the ribosome after release of polypeptides . In this study, we isolated a conditionally lethal amber mutation, named frr-3, in the Escherichia coli RRF gene at amino acid position 161, showing that the truncation of the C-terminal 25 amino acids of RRF is lethal to E . coli . An RRF gene cloned from Thermus thermophilus, whose protein is 44% identical and 68% similar to E . coli RRF, failed to complement the frr-3(Am) allele . However, truncation of the C-terminal five amino acids conferred intergeneric complementation activity on T . thermophilus RRF, demonstrating the modulator activity of the C-terminal tail . Rapid purification of T . thermophilus RRF was achieved by T7-RNA polymerase-driven overexpression for crystallography. J Appl Microbiol, 1999 Apr, 86(4), 622 - 34 Dust-borne bacteria in animal sheds, schools and children's day care centres; Andersson AM et al.; A total of 316 bacterial strains, including psychrophiles, mesophiles and thermophiles, were isolated and identified from indoor dusts in schools, children's day care centres and animal sheds . Several species which had not previously been reported from indoor environments were found: Sphingomonas, Brevibacterium, Nocardiopsis, Deinococcus and Rhodococcus/Gordona . A new psychrophilic actinomycete genus was also found in animal sheds, representing a new undescribed peptidoglycan type and an unusual whole-cell fatty acid composition . The indoor dusts of animal sheds contained mainly the Gram-negative genera Pseudomonas, Pantoea, Flavobacterium and Xanthomonas early in the indoor feeding season, but changed to a composition dominated by Bacillus, Micrococcus and mesophilic and thermophilic actinomycetes towards the end of the season . The dust contained, and air-borne bacterial flora in schools and day care centres were dominated by, Gram-positive bacilli and actinomycetes, notably Bacillus cereus, Brevibacillus brevis, B . licheniformis, B . subtilis and species of Arthrobacter, Corynebacterium, Rhodococcus/Gordona, Nocardiopsis sp., Deinococcus, Staphylococcus and Micrococcus . Indoor air and dust contained Klebsiella oxytoca, Acinetobacter calcoaceticus, Ac . lwoffi, Bacillus cereus and Nocardiopsis dassonvillei with the status of hazard group II . Indoor dusts of animal sheds contained eight different 3-hydroxy fatty acids, the 2-hydroxy fatty acid 14:0 and two 10-methyl fatty acids, whereas in dusts from schools and day care centres, these were below the detection level (< 3.5 ng mg-1) . The 3-and 2-hydroxy fatty acids could be assigned to one or more of the dust-contained cultivable strains, but 10-methyl C16:0 was not present in any of the strains isolated . The dusts from schools and children's day care centres contained 0.2-0.3 ng of endotoxin mg-1 and 0.5-1.4 ng of beta-D-glucan mg-1, whereas the dusts from animal sheds contained more 0.3-41 ng mg-1 and 8-35 ng mg-1, respectively. Biochim Biophys Acta, 1999 Apr 12, 1431(1), 249 - 60 Molecular characterisation of a novel thermophilic nitrile hydratase; Cramp RA et al.; The thermophilic soil isolate, Bacillus pallidus Dac521, expresses a constitutive nitrile hydratase . The purified enzyme was found to be a 110 kDa tetramer composed of two alpha and two beta subunits with molecular masses of 27 kDa and 29 kDa, respectively . The enzyme electrophoresed as a single protein band on native PAGE but two protein bands with isoelectric points of 4.7 and 5.5 on isoelectric focusing suggested the presence of isozymes . The purified enzyme was moderately thermostable up to 55 degrees C and the enzyme activity was stable over a broad pH range . Comparisons of the N-terminal amino acid sequences of the nitrile hydratase subunits with those of other nitrile hydratases showed up to 90% identity for the beta subunit sequence but no significant identity for the alpha subunit . The enzyme hydrolysed a narrow range of aliphatic substrates and did not hydrolyse any of the cyclic, hydroxy-, di- or aromatic nitriles tested . The activity was irreversibly inhibited by the aromatic nitrile, benzonitrile . The kinetic constants for acetonitrile, acrylonitrile and propionitrile compared favourably with those of mesophilic nitrile hydratases. Biochim Biophys Acta, 1999 Apr 12, 1431(1), 87 - 96 Purification, properties and enhanced expression under nitrogen starvation of the NADP+-isocitrate dehydrogenase from the cyanobacterium Phormidium laminosum; Pardo MA et al.; Nitrogen starvation enhances up to 8-fold the cellular level of the NADP+-dependent isocitrate dehydrogenase activity (isocitrate:NADP+ oxidoreductase (decarboxylating), IDH, EC 1.1.1.42) in the thermophilic filamentous non-N2-fixing cyanobacterium Phormidium laminosum . The enzyme was purified 650-fold to electrophoretic homogeneity from nitrogen-starved cells with an activity yield of 25% and a specific activity of 500 U (mg protein)-1 . The native enzyme showed a pI of 5.9 and it was a dimer of 107 kDa consisting of two identical subunits of 53 kDa . The activity required the presence of a divalent metal cation as an essential activator, Mn2+ or Mg2+ being the most effective . The optimum temperature for activity was 55 degrees C and the Ea for catalysis was 39.7 kJ mol-1 . An optimum pH for activity of 8.5 was found and the calculated pKE1, pKE2 and pKES1 of enzyme ionisation groups were 6.0, 8.9 and 6.3, respectively . Km values of 22, 50 and 24 microM were calculated for d,l-isocitrate, NADP and Mn2+, respectively, in the Mn2+-dependent reaction and 70, 32 and 159 microM for d,l-isocitrate, NADP and Mg2+, respectively, in the Mg2+-dependent reaction . The decarboxylating activity was inhibited by ATP, ADP and by its reaction products 2-oxoglutarate and NADPH2 . Polyclonal antibodies raised against the pure IDH were used to assess the presence of the enzyme in cells subjected to nitrogen starvation. Mol Cell Probes, 1999 Apr, 13(2), 127 - 31 Rapid detection of Listeria monocytogenes by a PCR assay specific for an aminopeptidase; Winters DK et al.; Specific and rapid detection of Listeria monocytogenes is very important with regard to food safety since all other species of Listeria appear to be non-pathogenic to humans . Conventional microbiological detection methods are very time consuming . The polymerase chain reaction (PCR) is one of the most promising techniques for rapid detection of micro-organisms in food products . We have developed a PCR assay, specific for L . monocytogenes, based on the gene encoding an aminopeptidase, which previously has not been described for this species . The L . monocytogenes aminopeptidase shares strong sequence similarity with aminopeptidase C from Streptococcus thermophilous, Lactobacillus lactis, Lactobacillus helveticus, and with a cysteine proteinase from Saccharomyces cerevisiae . Polymerase chain reaction primers were synthesized based on the DNA sequence of the aminopeptidase gene . A 90 bp product was apparent with all L . monocytogenes strains tested but not with other species of Listeria or other bacterial genera . The PCR assay, which is performed directly from whole bacterial cells, does not involve DNA purification and can be conducted in 4 h . It provided positive identification of L . monocytogenes in mixed culture . Mol Cell Biol, 1999 May, 19(5), 3624 - 34 A nonessential HP1-like protein affects starvation-induced assembly of condensed chromatin and gene expression in macronuclei of Tetrahymena thermophila; Huang H et al.; Heterochromatin represents a specialized chromatin environment vital to both the repression and expression of certain eukaryotic genes . One of the best-studied heterochromatin-associated proteins is Drosophila HP1 . In this report, we have disrupted all somatic copies of the Tetrahymena HHP1 gene, which encodes an HP1-like protein, Hhp1p, in macronuclei (H . Huang, E . A . Wiley, R . C . Lending, and C . D . Allis, Proc . Natl . Acad . Sci . USA 95:13624-13629, 1998) . Unlike the Drosophila HP1 gene, HHP1 is not essential in Tetrahymena spp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p (DeltaHHP1) . However, during a shift to nongrowth conditions, the survival rate of DeltaHHP1 cells is reduced compared to that of wild-type cells . Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p . Activation of two starvation-induced genes (ngoA and CyP) is significantly reduced in DeltaHHP1 cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells . These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response. J Biol Chem, 1999 Apr 23, 274(17), 11761 - 7 Structural basis for cold adaptation . Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum; Kim SY et al.; Aquaspillium arcticum is a psychrophilic bacterium that was isolated from arctic sediment and grows optimally at 4 degrees C . We have cloned, purified, and characterized malate dehydrogenase from A . arcticum (Aa MDH) . We also have determined the crystal structures of apo-Aa MDH, Aa MDH.NADH binary complex, and Aa MDH.NAD.oxaloacetate ternary complex at 1.9-, 2.1-, and 2.5-A resolutions, respectively . The Aa MDH sequence is most closely related to the sequence of a thermophilic MDH from Thermus flavus (Tf MDH), showing 61% sequence identity and over 90% sequence similarity . Stability studies show that Aa MDH has a half-life of 10 min at 55 degrees C, whereas Tf MDH is fully active at 90 degrees C for 1 h . Aa MDH shows 2-3-fold higher catalytic efficiency compared with a mesophilic or a thermophilic MDH at the temperature range 4-10 degrees C . Structural comparison of Aa MDH and Tf MDH suggests that the increased relative flexibility of active site residues, favorable surface charge distribution for substrate and cofactor, and the reduced intersubunit ion pair interactions may be the major factors for the efficient catalytic activity of Aa MDH at low temperatures. Microbiology, 1999 Jan, 145 ( Pt 1), 127 - 34 Structural and functional analysis of pCI65st, a 6.5 kb plasmid from Streptococcus thermophilus NDI-6; O'Sullivan T et al.; The 6.5 kb cryptic plasmid pCI65st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and sequenced . Five putative ORFs were identified . ORF1 could encode a 315 aa polypeptide almost identical to the RepA protein of previously sequenced S . thermophilus plasmids, indicating that pCI65st is one of the pC194 group of small gram-positive rolling-circle plasmids . ORFs 2 and 4 were virtually identical and could specify proteins of approximately 150 aa with significant similarity to the small heat-shock proteins described from a variety of gram-positive bacteria . ORF3 could encode a 415 aa protein similar to enolase, an enzyme involved in glycolysis and gluconeogenesis . ORF5 could encode a 412 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modification systems . Variants of strain NDI-6 which lacked pCI65st were readily isolated after subculture of the parent strain at 32 degrees C . The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 degrees C to 62 degrees C than its plasmid-free variant and expressed proteins which corresponded with the predicted translation products from ORF2 and ORF4 . In addition, plasmid-free mutants were lysed in broth by bacteriophages to which the parent culture was resistant. Biochem Mol Biol Int, 1999 Feb, 47(2), 283 - 92 Lysosomal glycerophosphocholine phosphodiesterase in Tetrahymena; Florin-Christensen J et al.; The purification and characterization of a novel phosphodiesterase (PDE) is presented . The activity was detected in the extracellular medium of Tetrahymena thermophila cultures, by the release of p-nitrophenol from p-nitrophenylphosphocholine (PNPPC) with an acidic pH optimum . In cell homogenates, it is sedimentable, shows a latency similar to that of acid phosphatase and is co-secreted with this enzyme, indicating that it is a lysosomal hydrolase . PNPPC-PDE was purified to homogeneity from the extracellular medium, yielding a single band of 58 kD on SDS polyacrylamide gel electrophoresis . It catalyzed the release of glycerol from glycerophosphocholine (GPC) and GPC competitively inhibits degradation of PNPPC . We present further evidence indicating that the natural substrate for PNPPC-PDE is GPC . Thus, Tetrahymena becomes the first eukaryote in which a lysosomal GPC-PDE is observed . This finding provides a new pathway for the complete breakdown of phosphatidylcholine in a lysosomal medium. Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4301 - 6 Location of translational initiation factor IF3 on the small ribosomal subunit; McCutcheon JP et al.; The location of translational initiation factor IF3 bound to the 30S subunit of the Thermus thermophilus ribosome has been determined by cryoelectron microscopy . Both the 30S.IF3 complex and control 30S subunit structures were determined to 27-A resolution . The difference map calculated from the two reconstructions reveals three prominent lobes of positive density . The previously solved crystal structure of IF3 fits very well into two of these lobes, whereas the third lobe probably arises from conformational changes induced in the 30S subunit as a result of IF3 binding . Our placement of IF3 on the 30S subunit allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation. J Cell Sci, 1999 Apr, 112 ( Pt 7), 1003 - 11 The conjusome: a novel structure in Tetrahymena found only during sexual reorganization; Janetopoulos C et al.; A unique structure, the conjusome, has been identified and initially characterized in Tetrahymena thermophila . The conjusome appears only during a specific phase of conjugation . Immunofluorescence microscopy reveals that the conjusome is strongly labeled by antibodies to the protein Pdd1p . Pdd1p is a chromodomain protein and participates in the formation of chromatin-containing structures in developing macronuclear anlagen . Recent studies suggest that Pdd1p is physically associated with the elimination of specific germ-line sequences from developing macronuclei (anlagen) and may play a role in heterochromatin assembly . The conjusome contains Pdd1p, but it is devoid of any detectable DNA . The conjusome appears before DNA elimination begins in the developing anlagen and after Pdd1p is found in the parental macronucleus . Transmission electron microscopic observations reveal that the conjusome is not a membrane-bounded structure . The conjusome ranges in size from about 1 microm to sizes approaching 7 microm, depending on its maturity . It is composed of a coarse reticulum of a fibrous, electron dense material, interspersed with apparent background cytoplasm . Our initial characterization does suggest a number of possible functions for what may be a new, transient organelle. J Mol Evol, 1999 May, 48(5), 528 - 41 RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria; Klenk HP et al.; A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, containing the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA-directed RNA polymerase subunits beta and beta' (rpoBC), alanyl-tRNA synthetase (alaS), and subunit A of proteinase Clp (clpA) . Enzymatic activity and extreme thermostability of purified A . pyrophilus RNA polymerase were verified . Transcription initiation on a DNA construct harboring the T7 A1 promoter was demonstrated by elongation of a 32P-labeled trinucleotide . Phylogenetic analyses of the two largest subunits of bacterial RNA polymerases (beta and beta') showed overall consistency with the 16S rRNA-based phylogeny, except for the positions of the hyperthermophiles A . pyrophilus and Thermotoga maritima and for the location of the root of the domain Bacteria . In the phylogenies for both RNA polymerase subunits beta and beta', A . pyrophilus was placed within the Gram-negative bacteria below the epsilon subdivision of the Proteobacteria . No support was found for the 16S rRNA-based hypothesis that A . pyrophilus might be the deepest branch of the Bacteria, but the cell wall-less mycoplasmas were found with a high confidence at the root of the Bacteria phylogenies . This raised doubts not only about whether the original Bacteria were indeed like the hyperthermophiles, but also concerning the value of single-gene phylogenies for hypotheses about the evolution of organisms. J Appl Microbiol, 1999 Mar, 86(3), 531 - 6 Intermittent shedding of thermophilic campylobacters by sheep at pasture; Jones K et al.; The rates at which sheep on different types of pasture shed campylobacters in their faeces were measured over 12 months . Overall, shedding of campylobacters at pasture was between a third and a half of the carriage rate (92%) of the intestines of sheep at slaughter . Shedding was highest during saltmarsh grazing, followed by upland fell and farm grazing . The rate of shedding varied at different times of the year, with the highest rates (100%) coinciding with lambing, weaning, and movement onto new pasture . The lowest rates (0%) occurred when sheep were fed on hay and silage . On the farm, low rates occurred during the whole of gestation, both when the sheep were indoors and outdoors . Campylobacter jejuni was the main species isolated and survived for up to 4 d in sheep faeces . Lambs became colonized by Campylobacter within 1-5 d of being born . Ewes, which were not shedding campylobacters prior to lambing, started to shed after lambing, and ewes which were shedding low numbers of Campylobacter before lambing, increased the numbers of bacteria being shed after lambing. Gene, 1999 Apr 1, 230(1), 7 - 14 Characterization, cloning, and evolutionary history of the chloroplast and cytosolic class I aldolases of the red alga Galdieria sulphuraria; Gross W et al.; Two fructose-1,6-bisphosphate aldolases from the acido- and thermophilic red alga Galdieria sulphuraria were purified to apparent homogeneity and N-terminally microsequenced . Both aldolases had similar biochemical properties such as Km (FBP) (5.6-5.8 microM) and molecular masses of the native enzymes (165kDa) as determined by size exclusion chromatography . The subunit size of the purified aldolases, as determined by SDS-PAGE, was 42kDa for both aldolases . The isoenzymes were not inhibited by EDTA or affected by cysteine or potassium ions, implying that they belong to the class I group of aldolases, while other red algae are known to have one class I and one class II aldolase inhibited by EDTA . cDNA clones of the cytosolic and plastidic aldolases were isolated and sequenced . The gene for the cytosolic isoenzyme contained a 303bp untranslated leader sequence, while the gene for the plastidic isoenzyme exhibited a transit sequence of 56 amino-acid residues . Both isoenzymes showed about 48% homology in the deduced amino-acid sequences . A gene tree relates both aldolases to the basis of early eukaryotic class I aldolases . The phylogenetic relationship to other aldolases, particularly to cyanobacterial class II aldolases, is discussed. Bioelectromagnetics, 1999, 20(3), 172 - 6 Different effects of microwave energy and conventional heat on the activity of a thermophilic beta-galactosidase from Bacillus acidocaldarius; La Cara F et al.; The effect of microwave (f = 10.4 GHz) irradiation on a thermostable enzyme was experimentally tested, showing that irreversible inactivation is obtained . Enzymatic solutions (500 microliters, with concentrations between 10-100 micrograms/ml) were exposed at 70 degrees C, at SAR levels of 1.1 and 1.7 W/g for 15, 30, 45, or 60 min, and their activity was compared to that of a sample heated in a water bath at the same temperature . The residual activity of the exposed samples depends on enzyme concentration, microwave power level, and exposure time; activity was reduced to 10% in 10 micrograms/ml solutions treated at 1.7 W/g for 60 min . Microwave effects disappeared at concentrations above 50 micrograms/ml . These results were not found following water bath heating at the same temperature and durations. Biosci Biotechnol Biochem, 1999 Feb, 63(2), 261 - 70 Gene cloning and overexpression of a geranylgeranyl diphosphate synthase of an extremely thermophilic bacterium, Thermus thermophilus; Ohto C et al.; A geranylgeranyl diphosphate (GGPP) synthase gene of an extremely thermophilic bacterium, Thermus thermophilus, was cloned and sequenced . T . thermophilus GGPP synthase, overexpressed in Escherichia coli cells as a glutathione S-transferase fusion protein, was purified and characterized . The fusion protein, retaining thermostability, formed a homodimer, and showed higher specific activity than did a partially purified thermostable enzyme previously reported . Optimal reaction conditions and kinetic parameters were also examined . The deduced amino acid sequence indicated that T . thermophilus GGPP synthase was excluded from the group of bacterial type GGPP synthases and lacked the insertion amino acid residues in the first aspartate-rich motif as do archaeal and eukaryotic short-chain prenyltransferases. Biodegradation, 1998, 9(5), 337 - 41 Degradation of pinene by Bacillus pallidus BR425; Savithiry N et al.; An aerobic thermophile has been isolated from an alpha-pinene enrichment culture . The isolate, which was designated BR425, has been tentatively identified as Bacillus pallidus using 16S ribosomal RNA gene sequencing and organism morphology . Monophasic and biphasic incubations of BR425 cells with alpha-pinene, beta-pinene, and limonene yielded a number of oxidized monoterpene metabolites with carveol as a common metabolite . A pinene degradation pathway with carveol and carvone as central metabolic intermediates is suggested. Cell Biochem Funct, 1999 Mar, 17(1), 29 - 33 Colonization by diatoms and antirheumatic activity of thermal mud; Tolomio C et al.; We have identified diatoms among other thermophilic microorganisms as the main agents for the colonization of thermal mud resulting in a 'maturation' which renders the mud suitable to be used for mud-pack treatment of osteoarthrosis patients . The main effects of the diatom growth are the progressive enrichment of mud extracts in chlorophyll a parallel to the building up of a sulfoglycolipid endowed with an anti-inflammatory action . The sulfoglycolipid was also produced by diatoms isolated from the mud and cultivated in vitro. J Microbiol Methods, 1999 Mar, 35(2), 177 - 82 Preservation of some extremely thermophilic chemolithoautotrophic bacteria by deep-freezing and liquid-drying methods; Malik KA; Long-term preservation methods for extreme thermophilic chemolithoautotrophic bacteria representing various species are described . The cultures were cryopreserved in liquid nitrogen under anaerobic conditions using 5% dimethylsulfoxide as a cryoprotectant . For easy storage and transport, the cultures were successfully liquid-dried, directly from the liquid phase without involving freezing under semiaerobic conditions using effective protective agents such as ethylenediamine and meso-inositol . The tested cultures showed good stability and survival rates after drying, after cryopreservation and on long-term storage . All tested strains were successfully preserved and reactivated within relatively short time . The viability, stability and ability of chemolithoautotrophic growth was not affected . Cryopreservation, liquid-drying and reactivation under microaerobic conditions proved very effective for these oxygen sensitive cultures. J Microbiol Methods, 1999 Mar, 35(2), 151 - 6 High purity 14CH4 generation using the thermophilic acetotrophic methanogen Methanothrix sp . strain CALS-1; Miller DN et al.; Methane-oxidizing activity in natural samples is typically measured by amending 14CH4 to the sample and then following the accumulation of 14CO2 . Current biological techniques to synthesize 14CH4 yield significant quantities of 14CO that when oxidized to 14CO2 would artificially inflate the measured methane-oxidizing activity of a sample . We present here a new method to biologically produce highly-pure 14CH4 using Methanothrix sp . Strain CALS-1 which produces very little CO . Using this method, 14CH4 was produced at nearly 100% efficiency and at a high specific activity (2.2 GBq.mmol-1) equal to the parent compound, {2-14C} sodium acetate . Furthermore, only trace quantities of H2 and CO were produced with only one molecule of CO produced for every 17,000 molecules of CH4 . When compared to the standard CH4 generation method, this technique produced 97% purer CH4. Biochemistry (Mosc), 1999 Feb, 64(2), 181 - 8 Mossbauer spectroscopic study of functional thermal destruction of iron--sulfur proteins in membranes of thermophilic cyanobacteria synechococcus elongatus Kaurov YN, Novakova AA, Davletshina LN, Aleksandrov AY, Khval'kovskaya EA, Semin BK, Belevich NP, Ivanov II II, Rubin AB. A mathematical model of the Mossbauer spectrum (80K) of native membranes of Synechococcus elongatus was constructed on the basis of values of the quadruple splitting (Delta) and the isomeric shift (delta) of the iron-containing components of the photosynthetic apparatus obtained from the literature . Thermally induced changes in the intensity of the spectral components of membranes and isolated preparations of photosystem (PS) I were studied using this model . It was shown that exposure of membranes to 70-80 degrees C causes a decrease in the intensity of the components related to the FX, FA, and FB centers and surface-located ferredoxins of PS I, an increase in the intensity of the doublets of oxidized iron clusters that are nonspecifically absorbed by the membranes, and formation of a new doublet . Spectral parameters of this doublet (Delta = 3.10 mm/sec and delta = 1.40 mm/sec) are typical of inorganic hydrated forms of reduced iron . Heating of PS I preparations also causes a decrease in the intensity of doublets of the FX, FA, and FB centers and an increase in the intensity of doublets of nonspecifically bound oxidized iron . However, this does not cause formation of inorganic reduced iron . Comparison between the intensities of the Mossbauer spectral components in intact and heated samples suggests that the main source of reduced iron in membranes is surface-located ferredoxins . Nonspecifically bound oxidized iron is formed at the expense of the FX, FA, and FB centers . Disappearance of spectral components associated with ferredoxins and accumulation of reduced iron in membranes occur within the temperature range critical for inhibition of electron transport through PS I to oxygen . These findings suggest that the thermally induced processes of accumulation of reduced iron and inhibition of electron transport in PS I in membranes of thermophilic cyanobacteria are interrelated and caused mainly by degradation of the Fe--S centers of ferredoxins . The possible role of reduced iron accumulation in the degradation of the photosynthetic apparatus induced by heat and other extreme physical and chemical factors is discussed. J Eukaryot Microbiol, 1999 Jan-Feb, 46(1), 6 - 11 Interface-mediated death of unconditioned Tetrahymena cells: effect of the medium composition; Hagemeister JJ et al.; We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic . The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68 . Here, we report that cell death can also be caused by the medium . The specific effects of several medium constituents were tested in the presence and absence of an interface . Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca2+ (68 microM) and Mg2+ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death . The effect of the amino acids and the salt ions Ca2+ and Mg2+ can be abolished by the addition of insulin (10(-6) M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions . On the basis of our findings, a new recipe for a chemically-defined medium has been formulated . Single cells can grow in this medium in the presence of medium-air interface without any supplements. FEMS Microbiol Lett, 1999 Mar 15, 172(2), 179 - 86 Cloning and characterization in Escherichia coli of the gene encoding the principal sigma factor of an extreme thermophile, Thermus thermophilus; Nishiyama M et al.; The nucleotide sequence of the upstream region of the aspartate kinase genes of Thermus thermophilus HB27 revealed the presence of two open reading frames in the orientation opposite to that of the aspartate kinase genes . The upstream open reading frame termed ORF375 encodes a protein composed of 375 amino acid residues, possessing amino acid sequence motifs for methylases . Another open reading frame designated as sigA encodes a protein of 423 amino acid residues which shows significant identity in amino acid sequence to the principal sigma factor, a component of the DNA-dependent RNA polymerase holoenzyme . The close proximity of the open reading frames suggested that the two genes are transcribed in a polycistronic manner . By the use of an Escherichia coli expression system, SigA was produced in a soluble form . An in vitro transcription assay of purified SigA reconstituted with the core RNA polymerase of E . coli showed that Thermus SigA functioned as a sigma factor to initiate specific transcription. J Biol Chem, 1999 Apr 9, 274(15), 9976 - 83 Molecular cloning and expression of a stress-responsive mitogen-activated protein kinase-related kinase from Tetrahymena cells; Nakashima S et al.; To identify genes responsive to cold stress, we employed the differential display mRNA analysis technique to isolate a novel gene from Tetrahymena thermophila which encodes a protein kinase of 430 amino acids . A homolog of this kinase with 90% amino acid sequence identity was also found in T . pyriformis . Both kinases contain 11 subdomains typical of protein kinases . Sequence analysis revealed that the predicted amino acid sequences resemble those of mitogen-activated protein kinase (MAPK), especially p38 and stress-activated protein kinase which are known to be involved in various stress responses . However, it should be noted that the tyrosine residue in the normally conserved MAPK phosphorylation site (Thr-X-Tyr) is replaced by histidine (Thr226-Gly-His228) in this MAPK-related kinase (MRK) . The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated . However, the mutated recombinant protein in which Thr226 was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr226 residue is essential for kinase activity . The MRK mRNA transcript in T . thermophila increased markedly upon temperature downshift from 35 to 15 degrees C (0.8 degrees C/min) . Interestingly, osmotic shock either by sorbitol (100-200 mM) or NaCl (25-100 mM) also induced mRNA expression of the MRK in T . pyriformis . In addition, the activity of the kinase as determined by an immune complex kinase assay using MBP as a substrate was also induced by osmotic stress . This is the first demonstration of a MAPK-related kinase in the unicellular eukaryotic protozoan Tetrahymena that is induced by physical stresses such as cold temperature and osmolarity . The present results suggest that this MRK may function in the stress-signaling pathway in Tetrahymena cells. Appl Environ Microbiol, 1999 Apr, 65(4), 1644 - 51 Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain; Kim TJ et al.; A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium, Thermus strain IM6501 . The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119 . The optimal temperature of ThMA was 60 degrees C, which was higher than those of other maltogenic amylases reported so far . Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA . ThMA harbored both hydrolysis and transglycosylation activities . It hydrolyzed beta-cyclodextrin and starch mainly to maltose and pullulan to panose . ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc . Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4 . The transglycosylation of sugar to methyl-alpha-D-glucopyranoside by forming an alpha-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA . Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product. Eur J Biochem, 1999 Mar, 260(3), 752 - 60 Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I; Kristjansson MM et al.; An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases . The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein . In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively . The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I . The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I . One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity . Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected . The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant . The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I . The same order of reactivity was also observed for the inactivation of the enzymes by H2O2-oxidation, as a function of temperature . The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures. Biosens Bioelectron, 1999 Feb, 14(2), 171 - 8 Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis; Pasco N et al.; Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313-318 K in a flow injection analysis (FIA) system . The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions . The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD (n = 10) for 5 mM L-glutamate was 7.6% . The response to some potential interferents has been quantified . Addition of finely ground hexaammineruthenium (III) trichloride ({Ru(NH3)6}Cl3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current . The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO. Biotechnol Bioeng, 1998 Dec 20, 60(6), 664 - 9 A sensitive photosystem II-based biosensor for detection of a class of herbicides; Koblizek M et al.; We have developed a biosensor for the detection of residual triazine-, urea- and phenolic-type herbicides, using isolated photosystem II (PSII) particles from the thermophilic cyanobacterium, Synechococcus elongatus, as biosensing elements . The herbicide detection was based on the fact that, in the presence of artificial electron acceptors, the light-induced electron transfer through isolated PSII particles is accompanied by the release of oxygen, which is inhibited by the herbicide in a concentration-dependent manner . The PSII particles were immobilized between dialysis membrane and the Teflon membrane of the Clark oxygen electrode mounted in a flow cell that was illuminated . Inclusion of the antibiotic chloramphenicol in the reaction mixtures prolonged, by 50%, the lifetime of the biosensor . The use of highly active PSII particles in combination with the flow system resulted in a reusable herbicide biosensor with good stability (50% of initial activity was still remaining after 35-h use at 25 degrees C) and high sensitivity (detection limit for diuron was 5 x 10(-10) M) . Biotechnol Bioeng, 1998 Jul 5, 59(1), 108 - 15 Advantages in using immobilized thermophilic beta-glycosidase in nonisothermal bioreactors; Febbraio F et al.; Catalytic membranes, obtained by immobilizing thermophilic beta-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction . Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity . The results showed that the thermophilic enzyme behaved as the mesophilic beta-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference . The efficiency of the system proposed was determined by calculating two constants, alpha and beta, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1 degrees C or a temperature gradient of 1 degrees C cm-1 were applied across the catalytic membrane . The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times . The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed . Biotechnol Bioeng, 1998 Jun 20, 58(6), 663 - 7 Effect of yeast extract supplementation in leach solution on bioleaching rate of pyrite by acidophilic thermophile acidianus brierleyi Konishi Y, Yoshida S, Asai S. The bioleaching rate of pyrite (FeS2) by the acidophilic thermophile Acidianus brierleyi was studied at 65 degrees C and pH 1.5 with leach solutions supplemented with yeast extract . In the absence of yeast extract supplementation, A . brierleyi could grow autotrophically on pyrite, and the leaching percentage of pyrite particles (25-44 mum) reached 25% for 7 d . The bacterial growth and consequent pyrite oxidation were enhanced by the addition of yeast extract between 0.005 and 0.25% w/v: the pyrite particles were completely solubilized within 6 d . The bioleaching rate was enhanced by a factor of 1.5 when the yeast extract concentration was changed from 0.005 to 0.05% w/v . However, there was only a slight effect on the leaching rate at the yeast extract concentrations of 0.05 to 0 . 25% w/v, suggesting that the organic supplement level was in large excess in the pyrite bioleaching . Biotechnol Bioeng, 1998 Mar 5, 57(5), 624 - 9 Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli; Ikeda M et al.; A genomic library of the hyperthermophilic archaeon Pyrococcus furiosus was constructed in Escherichia coli using pBluescript II SK(+) as a cloning vector . One positive clone exhibiting thermophilic ester-hydrolyzing activity was directly detected by an in situ plate assay using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-acetate . The plasmid isolated from the clone contained a 3.8 kb HindIII fragment from P . furiosus . Expression of active thermostable esterase in E . coli was independent of isopropyl-beta-D-thiogalactopyranoside, suggesting that the archaeal esterase gene was heterologously controlled by its own promoter sequence, not by the vector-located lac promoter . Assays of esterase activity in heat-treated extract of the recombinant E . coli showed the highest temperature optimum (100 degrees C) and thermostability (a half-life of 50 min at 126 degrees C) among esterases reported to date . Biotechnol Bioeng, 1998 Feb 5, 57(3), 342 - 55 Methanogenic population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids; Griffin ME et al.; An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions . The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S . production rates . The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days . Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters . Chemical analyses were performed to evaluate digester performance . The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum . After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals . In contrast, the mesophilic digester did not respond favorably to the start-up method . The concentrations of volatile fatty acids increased dramatically and pH control was difficult . After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high . Methanogenic population dynamics correlated well with performance measures . Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed . Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up . The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively) . Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester . Epidemiol Infect, 1999 Feb, 122(1), 7 - 13 Presence of Campylobacter and Salmonella in sand from bathing beaches; Bolton FJ et al.; The purpose of this study was to determine the presence of thermophilic Campylobacter spp . and Salmonella spp . in sand from non-EEC standard and EEC standard designated beaches in different locations in the UK and to assess if potentially pathogenic strains were present . Campylobacter spp . were detected in 82/182 (45%) of sand samples and Salmonella spp . in 10/182 (6%) . Campylobacter spp . were isolated from 46/92 (50%) of samples from non-EEC standard beaches and 36/90 (40%) from EEC standard beaches . The prevalence of Campylobacter spp . was greater in wet sand from both types of beaches but, surprisingly, more than 30% of samples from dry sand also contained these organisms . The major pathogenic species C . jejuni and C . coli were more prevalent in sand from non-EEC standard beaches . In contrast, C . lari and urease positive thermophilic campylobacters, which are associated with seagulls and other migratory birds, were more prevalent in sand from EEC standard beaches . Campylobacter isolates were further characterized by biotyping and serotyping, which confirmed that strains known to be of types associated with human infections were frequently found in sand on bathing beaches. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3600 - 5 Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius; Hopfner KP et al.; Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions . We describe here the 2.5 A resolution crystal structure of a DNA polymerase from the Archaea Thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function . Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA-protein interface . In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site . A physiological role of this "closed" conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site . This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3578 - 83 Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species; Haney PJ et al.; The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile . In this paper, sequences of 115 proteins from M . jannaschii are compared with their homologs from mesophilic Methanococcus species . Although the growth temperatures of the mesophiles are about 50 degrees C below that of M . jannaschii, their genomic G+C contents are nearly identical . The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln) . These are recurring themes, with all trends applying to 83-92% of the proteins for which complete sequences were available . Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias . We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement. Res Microbiol, 1999 Jan-Feb, 150(1), 21 - 32 The role of the codon first letter in the relationship between genomic GC content and protein amino acid composition; Wilquet V et al.; Analysis of the statistical distribution of amino acid compositions within 22 protein families shows that a GC bias generally affects proteins with a variety of functions from the extreme thermophile Thermus . This results in evident enrichment in amino acids of the group L, V, A, P, R and G and underrepresentation of amino acids of the group I, M, F, S, T, C and W . The strong amino acid composition biases noted in Thermus proteins are not related to thermoadaptation; they were also found in mesophilic homologues encoded by GC-rich genes . The results of a comparative analysis on large samples of translated sequences from 30 organisms, representing the three major kingdoms of life and including extremophiles, indicate a universal correlation between the usage of particular amino acids and the genomic GC content . It is concluded that the codon first letter plays a dominant role in translating the genomic GC signature into protein amino acid composition and sequences. RNA, 1999 Mar, 5(3), 434 - 45 An engineered class I transfer RNA with a class II tertiary fold; Nissan TA et al.; Structure-based engineering of the tertiary fold of Escherichia coli tRNA(Gln)2 has enabled conversion of this transfer RNA to a class II structure while retaining recognition properties of a class I glutamine tRNA . The new tRNA possesses the 20-nt variable stem-loop of Thermus thermophilus tRNA(Ser) . Enlargement of the D-loop appears essential to maintaining a stable tertiary structure in this species, while rearrangement of a base triple in the augmented D-stem is critical for efficient glutaminylation . These data provide new insight into structural determinants distinguishing the class I and class II tRNA folds, and demonstrate a marked sensitivity of glutaminyl-tRNA synthetase to alteration of tRNA tertiary structure. Eur J Biochem, 1999 Feb, 259(3), 592 - 601 Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA; Kato R et al.; Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures . ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C . The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively . Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2 . At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity . The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA) . Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C . At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability . At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA . The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA . The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD . These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA . The specific structure of ssDNA was altered by heat or binding of ecRecA . After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature. J Mol Biol, 1999 Apr 2, 287(3), 555 - 68 Crystal structures of phenylalanyl-tRNA synthetase complexed with phenylalanine and a phenylalanyl-adenylate analogue; Reshetnikova L et al.; The crystal structures of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) complexed with phenylalanine and phenylalaninyl-adenylate (PheOH-AMP), the synthetic analogue of phenylalanyl-adenylate, have been determined at 2.7A and 2.5A resolution, respectively . Both Phe and PheOH-AMP are engulfed in the active site cleft of the catalytic alpha-subunit of PheRS, and neither makes contact with the PheRS beta-subunit . The conformations and binding of Phe are almost identical in both complexes . The recognition of Phe by PheRS is achieved through a mixture of multiple van der Waals interactions and hydrogen bonds . The side-chain of the Phe substrate is sandwiched between the hydrophobic side-chains of Phealpha258 and Phealpha260 on one side, and the main-chain atoms of the two adjacent beta-strands on the other . The side-chains of Valalpha261 and Alaalpha314 form the back wall of the amino acid binding pocket . In addition, PheRS residues (Trpalpha149, Seralpha180, Hisalpha178, Argalpha204, Glnalpha218, and Glualpha220) form a total of seven hydrogen bonds with the main-chain atoms of Phe . The conformation of PheOH-AMP and the network of interactions of its AMP moiety with PheRS are reminiscent of the other class II synthetases . The structural similarity between PheRS and histidyl-tRNA synthetase extends to the amino acid binding site, which is normally unique for each enzyme . The complex structures suggest that the PheRS beta-subunit may affect the first step of the reaction (formation of phenylalanyl-adenylate) through the metal-mediated conserved alpha/beta-subunit interface . The modeling of tyrosine in the active site of PheRS revealed no apparent close contacts between tyrosine and the PheRS residues . This result implies that the proofreading mechanism against activated tyrosine, rather than direct recognition, may play the major role in the PheRS specificity . J Mol Biol, 1999 Apr 2, 287(3), 511 - 25 The functional cycle and regulation of the Thermus thermophilus DnaK chaperone system; Klostermeier D et al.; The Escherichia coli DnaK (DnaKEco) chaperone cycle is tightly regulated by the cochaperones DnaJ, which stimulates ATP hydrolysis, and GrpE, which acts as a nucleotide exchange factor . The Thermus thermophilus DnaK (DnaKTth) system additionally comprises the DnaK-DnaJ assembly factor (DafATth) that is mediating formation of a 300 kDa DnaKTth . DnaJTth.DafATth complex.A model peptide derived from the tumor suppressor protein p53 was used to dissect the regulation of the individual kinetic key steps of the DnaKTth nucleotide/chaperone cycle . As with DnaKEco the DnaKTth.ATP complex binds substrates with reduced affinity and large exchange rates compared to the DnaKTth.ADP.Pi state . In contrast to DnaKEco, ADP-Pi release is slow compared to the rate of hydrolysis, reversing the balance of the two functional nucleotide states . Whereas GrpETth stimulates nucleotide release from DnaKTth, DnaJTth does not accelerate ATP hydrolysis under various experimental conditions . However, it exerts influence on the interaction of DnaKTth with substrates: in the presence of DafATth, DnaJTth inhibits substrate binding, and substrate already bound to DnaKTth is displaced by DnaJTth and DafATth, indicating competitive binding of DnaJTth/DafATth and substrate . It thus appears that the DnaKTth . DnaJTth.DafATth complex as isolated from T . thermophilus does not represent the active species in the DnaKTth chaperone cycle . Isothermal titration calorimetry showed that the ternary complex of DnaKTth, DnaJTth and DafATth is assembling with high affinity, whereas binary complexes of DnaKTth and DnaJTth or DafATth were not detectable, indicating highly synergistic formation of the 300 kDa DnaKTth . DnaJTth.DafATth complex.Based on these results, a model describing the DnaKTth chaperone cycle and its regulation by cochaperones is proposed where DnaKTth . DnaJTth.DafATth constitutes the resting state, and a DnaKTth . substrate.DnaJTth complex is the active chaperone species . The novel factor DafATth that mediates interaction of DnaKTth with DnaJTth would thus serve as a "template" to stabilise the ternary DnaKTth.DafATth.DnaJTth complex until it is replaced by substrate proteins under heat shock conditions . Biochemistry, 1999 Mar 23, 38(12), 3831 - 6 A thermodynamic comparison of mesophilic and thermophilic ribonucleases H; Hollien J et al.; The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues . To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus . The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins . Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T . thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature . In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1382 - 6 Crystals of Thermus thermophilus tRNAAsp complexed with its cognate aspartyl-tRNA synthetase have a solvent content of 75% . Comparison with other aminoacylation systems; Briand C et al.; Thermus thermophilus tRNAAsp, purified from a non-recombinant source, has been crystallized in a complex with its cognate dimeric (alpha2) aspartyl-tRNA synthetase . Crystals diffract to 2.9 A resolution and belong to space group P63 with cell parameters a = b = 258, c = 90.9 A . The crystals contain one aspartyl-tRNA synthetase dimer and two tRNA molecules in the asymmetric unit, corresponding to a Vm of 4.85 A3 Da-1 and 75% solvent content . When compared with those obtained for globular proteins these values are high, but fall within the range observed for other aminoacyl-tRNA synthetases, either free or complexed with their tRNAs . A comparative survey is presented here. Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 704 - 5 Crystallization and preliminary X-ray diffraction studies of a DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8; Shibata A et al.; A DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8 was crystallized by the vapor-diffusion method using lithium sulfate as the precipitant and beta-octylglucoside as an additive . The crystals belong to the trigonal space group P3121 or P3221, with unit-cell dimensions of a = b = 136.0 and c = 108.1 A . The crystal is most likely to contain one UvrB protein in an asymmetric unit with the Vm value of 3.8 A3 Da-1 . The crystals diffracted X-rays beyond 2.9 A resolution . Although the crystals were sensitive to X-ray irradiation at room temperature, the frozen crystals at 100 K showed no apparent decay during the intensity measurement. Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 345 - 6 Epub 1999 Jan 01. Medium temperature, 310 K, provides single crystals of orotate phosphoribosyltransferase from Thermus thermophilus; Hamana H et al.; Crystallization of orotate phosphoribosyltransferase from a thermophilic organism, Thermus thermophilus, was achieved using the hanging-drop vapour-diffusion method coupled with a macroseeding starter . Small needle-like microcrystals were grown in a fresh protein solution in the presence of 2-methyl-2,4-pentanediol at 298 K or below . Although these normal temperature conditions caused stacking crystallization, an increase of temperature to 310 K permitted crystal growth . This was because of increased enzyme solubility at the higher temperature . The crystal was found to belong to the monoclinic space group P21with unit-cell parameters a = 44.4, b = 59.6, c = 67.8 A and beta = 98.3 degrees. J Food Prot, 1999 Mar, 62(3), 287 - 91 Effects of lactic acid bacteria ingestion of basal cytokine mRNA and immunoglobulin levels in the mouse; Tejada-Simon MV et al.; An increasing number of functional foods and pharmaceutical preparations containing lactic acid bacteria are being promoted with health claims based on the potential probiotic characteristics and on their capacity for stimulating the host immune system . However, the specific immune effects of oral administration of these microbes remain undefined . In this study, we tested the hypothesis that basal gastrointestinal immune status in mice is affected by orally administered lactic acid bacteria . The specific objective of this research was to evaluate the effects of repeated oral exposure to viable and nonviable lactic acid bacteria (Lactobacillus acidophilus, L . bulgaricus, L . casei, and Streptococcus thermophilus) in mice on basal cytokine mRNA expression in mucosal (Peyer's patches), systemic (spleen), and lymphoid tissue and on immunoglobulin levels . The results indicated that oral exposure to 10(9) CFU/day for up to 14 days did not significantly affect basal interferon-gamma, tumor necrosis factor-alpha, or interleukin-6 mRNA expression or total serum and intestinal immunoglobulins. Extremophiles, 1999 Jan, 3(1), 63 - 70 Dissimilatory sulfite reductase from Archaeoglobus profundus and Desulfotomaculum thermocisternum: phylogenetic and structural implications from gene sequences; Larsen O et al.; The genes encoding the alpha- and beta-subunits of dissimilatory sulfite reductase, dsrAB, from the hyperthermophilic archaeon Archaeoglobus profundus and the thermophilic gram-positive bacterium Desulfotomaculum thermocisternum were cloned and sequenced . The dsrAB genes are contiguous, and most probably comprise an operon also including a dsrD homolog, a conserved gene of unknown function located downstream of dsrAB in all four sulfate reducers so far sequenced . Sequence comparison confirms that dissimilatory sulfite reductase, Dsr, is a highly conserved enzyme . A phylogenetic analysis using the available Dsr sequences, including Dsr-like proteins from nonsulfate reducers, suggests a paralogous origin of the alpha- and beta-subunits . Furthermore, the Dsr from sulfate reducers forms a separate cluster, with Dsr from the bacterial sulfate reducers Desulfotomaculum thermocisternum and Desulfovibrio vulgaris branching together, next to Dsr from Archaeoglobus profundus and Archaeoglobus fulgidus . Based on an alignment with the assimilatory sulfite reductase from Escherichia coli, the amino acid residues involved in binding of sulfite, siroheme, and {Fe4S4}-clusters have been tentatively identified, which is consistent with the binding of two sirohemes and four {Fe4S4}-clusters per alpha2beta2 structure . The evolution of Dsr and the structural basis for the binding of substrate and cofactors are discussed. Proteins, 1999 Mar 1, 34(4), 435 - 42 Characterization and functional properties of the extracellular coelomic hemoglobins from the deep-sea, hydrothermal vent scaleworm Branchipolynoe symmytilida; Hourdez S et al.; Polychaete species belonging to the genus Branchipolynoe are commensal with mussels from deep-sea hydrothermal vents and cold-seeps . Possessing hemoglobins (Hbs), the species B . symmytilida, which is found in the mussel Bathymodiolus thermophilus on the East Pacific Rise, is exceptional in a family normally devoid of respiratory pigments . In a previous paper we described two major coelomic extracellular hemoglobins with unique quaternary structures . Aiming to discern respiratory adaptations to the highly variable hydrothermal environment, this paper characterizes the functional properties of these Hbs and the coelomic fluid . The two major hemoglobins (C1 and C2) exhibit spectrophotometric characteristics of both intra- and extracellular hemoglobins . However, their amino acid content is very different from other known hemoglobins and is characterized by a high proportion of alanine and glycine (up to 40% cumulated in C1) . C1 and C2 differ markedly by their cysteine content (0.8% and 13% respectively) . The coelomic fluid exhibits a strong buffer capacity due to the high hemoglobin content (3 mM heme) . In vitro, CO2 accumulation (up to 10-12 mM CO2 for PCO2 = 7.5 Torr) occurs with limited pH changes and is only partly accounted for by carbamino-Hb formation . The two hemoglobins exhibit high oxygen-affinities (P50 0.4 Torr for C1 and 0.9 Torr for C2, at 10 degrees C, pH 8) and a normal Bohr effect (phi values ranging from -0.54 and -0.37 at 10 degrees C, to -0.24 and -0.28 at 30 degrees C, for C1 and C2, respectively) . Cooperativity values range from 0.8 to 1.9 for C1 and from 0.8 to 1.7 for C2 . The temperature sensitivity of O2 affinity reflect deltaH values that decrease from -30 to -60 kJ x mol(-1) with increasing pH . C2 exhibits a slight specific effect of CO2 on oxygenation properties. FEMS Microbiol Lett, 1999 Mar 1, 172(1), 85 - 90 Phylogenetic analysis of transformable strains of thermophilic Bacillus species; Studholme DJ et al.; Few strains of thermophilic Bacillus spp are readily transformable with plasmid DNA . Given the considerable phylogenetic and phenotypic diversity amongst thermophilic bacilli, we have examined whether transformability is a trait associated with a particular phylogenetic group, by sequencing the 16S ribosomal RNA genes from transformable strains NUB3621, K1041, and NRRL1174 . Although all of these strains were described in the literature as B . stearothermophilus, only NRRL1174 is closely related to the type strain of this species . Based on its 16S rDNA sequence and physiological data K1041 appeared to belong to the species B . thermodenitrificans, while NUB3621 showed a slightly closer relationship to B . thermoglucosidasius than to B . stearothermophilus . Therefore we conclude that the trait of transformability, though possibly strain-specific, is not limited to a single species of thermophilic Bacillus. Curr Genet, 1999 Mar, 35(2), 88 - 102 Systematic mutagenesis of the fission yeast Srp54 protein; Martinez-Force E et al.; The signal recognition particle (SRP) is a ribonucleoprotein required for targeting a subset of nascent pre-secretory proteins to the endoplasmic reticulum membrane . Of the six SRP polypeptides, the most highly conserved is Srp54p, a modular protein consisting of an amino-terminal (N) domain of unknown function, a central GTPase (G) domain, and a carboxyl-terminal (M) domain implicated in the recognition of both signal sequences and SRP RNA . To identify regions of Srp54p that interact with other SRP subunits or regulatory proteins, we carried out systematic mutagenesis of the fission yeast homolog, principally using a "clustered charged-to-alanine" strategy . Of the 35 alleles examined, 13 are unable to support growth, two confer cold-sensitivity, five confer heat-sensitivity, and 15 produce no discernible phenotype . The lethal and conditional mutations map throughout the protein to several conserved regions, confirming that these motifs play critical roles in Srp54p function . The effects of the amino-acid substitutions are analyzed with reference to the recently determined tertiary structures of the N/G domain and the intact protein from a thermophilic bacterium. J Mol Evol, 1999 Apr, 48(4), 408 - 20 Archaeabacterial seryl-tRNA synthetases: adaptation to extreme environments and evolutionary analysis; Taupin CM et al.; The aminoacyl-tRNA synthetases are ubiquitous enzymes which catalyze a crucial step of the cell life, the specific attachment of amino acids to their cognate tRNA . The amino acid sequences of three archaeal seryl-tRNA synthetases (SerRS) from Haloarcula marismortui and Methanococcus jannaschii, both belonging to the group of Euryarchaeota, and from Sulfolobus solfataricus, of the group of Crenarchaeota, were aligned with other eubacterial and eukaryal available SerRS sequences . In an attempt to identify some features of adaptation to extreme environments of these organisms, amino acid composition and amino acid substitutions between mesophilic and thermophilic SerRS were analyzed . In addition, universal phylogenetic trees of SerRS including the three known archaeal sequences, rooted by the threonyl-tRNA synthetases were inferred . Amino acid analyses of the SerRS revealed two ways of adaptation to thermophilic environments between the Eubacteria and the Archaea; most of the usually described amino acid substitutions were nonsignificant in the case of archaeal thermophilic SerRS and most amino acid composition biases seemed to be linked to the genome G+C content pressure . The phylogenetic analysis of the SerRS showed the Archaea to be paraphyletic, H . marismortui emerging with the Gram-positive Bacteria, M . jannaschii being near the root of the tree, and S . solfataricus branching with Eucarya. Biochim Biophys Acta, 1999 Jan 27, 1410(1), 7 - 18 Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I; Jin P et al.; PsaD is a peripheral protein on the reducing side of photosystem I (PS I) . We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus . The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF . The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M . laminosus . The soluble PsaD could assemble into the PsaD-less PS I . As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7x10(6) M-1, and the enthalpy change of -93.6 kJ mol-1 . This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein . To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped . The Cys66, Lys21, Arg118 and Arg119 residues were exposed on the surface of soluble PsaD whereas the Lys129 and Lys131 residues were not exposed on the surface . Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of alpha-helical conformation. Nucleic Acids Res, 1999 Apr 1, 27(7), 1690 - 7 Identification, cloning and expression of p25, an AT-rich DNA-binding protein from the extreme thermophile, Thermus aquaticus YT-1; Du X et al.; Although the G+C content of Thermus aquaticus YT-1 chromosomal DNA is 67.4%, regions with lower G+C content have also been observed . AT-rich DNA-binding proteins may contribute to the thermostability and biological functions of these DNA regions at Thermus growth temperatures . Using double-stranded DNA (dsDNA)-cellulose chromatography, a T.aquaticus YT-1 protein, designated as p25, was identified to bind preferentially to AT-rich DNA . The gene encoding p25 was cloned and sequenced after immunoscreening T.aquaticus YT-1 expression libraries . The deduced primary structure of p25 is 211 amino acids in length with a molecular weight of 23 225 Da . Native p25 was purified and characterized as a homodimer with modification possibly at lysine and arginine residues . Its preferential and temperature-dependent binding to AT-rich DNA was confirmed with mobility-shift DNA-binding assays . The protein was demonstrated to bind preferentially to dsDNA instead of single-stranded DNA . The binding of p25 to dsDNA also improved the thermotolerence of this protein . Overexpression study of fusion p25 suggested that the N-terminus of the protein might form the DNA-binding domain or be closely involved in DNA-binding activity. J Bacteriol, 1999 Mar, 181(6), 1861 - 7 Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima; Bock AK et al.; Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity . PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure . The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases . The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5 . The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation) . In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%) . The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h . AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure . The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila . The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0 . The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation) . AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%) . Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective . The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts . In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h . The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions. J Bacteriol, 1999 Mar, 181(6), 1713 - 8 Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via alpha-aminoadipic acid not via diaminopimelic acid; Kobashi N et al.; An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed . The mutant strain did not grow in a minimal medium, suggesting that T . thermophilus contains a single aspartate kinase . Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth . To further elucidate the lysine biosynthetic pathway in T . thermophilus, lysine auxotrophic mutants of T . thermophilus were obtained by chemical mutagenesis . For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi . A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned . Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment . Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid . All of these results indicated that in T . thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate. Mol Biol Cell, 1999 Mar, 10(3), 771 - 84 Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila; Lee S et al.; In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function . The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes . Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced . Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2 . The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis . In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons . We conclude that the two dyneins perform separate tasks in Tetrahymena. J Pediatr Gastroenterol Nutr, 1999 Mar, 28(3), 252 - 6 Reverse transcription-polymerase chain reaction in the diagnosis of Helicobacter pylori infection in Finnish children; Oksanen K et al.; BACKGROUND: The purpose of this study was to design a simplified polymerase chain reaction (PCR) technique for the detection of Helicobacter pylori and to compare it with conventional diagnostic methods-culture and histology of gastric biopsy specimens . In addition, the capability of this technique to detect H . pylori in the gastric mucosal biopsies of originally H . pylori-negative children with gastritis or recurrent abdominal pain was investigated . METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) using polymerase from Thermus thermophilus was applied to detect H . pylori 16S rRNA . Twenty-five children H . pylori-positive by culture and/or histology were used as positive control subjects . Sixteen healthy H . pylori-negative children served as negative control subjects . Biopsy specimens from gastric antrum and corpus from 81 children were examined by RT-PCR . Altogether, 30 had histologic gastritis and 51 had nonspecific abdominal pain only, with no disease in histologic specimens . Histology and culture of H . pylori were negative in both patient groups . RESULTS: Reverse transcription-polymerase chain reaction detected 24 of 25 tissue-positive and 0 of 16 tissue-negative cases, indicating 96% sensitivity and 100% specificity for the test . None of the culturally and histologically H . pylori-negative samples showed H . pylori colonization when analyzed by RT-PCR . CONCLUSIONS: RT-PCR using Thermus thermophilus polymerase is a fast and simple means of detecting H . pylori in gastric biopsy specimens . It is at least as specific and sensitive as conventional methods . In pediatric patients it may be necessary to take more than two biopsy specimens to increase sensitivity in cases of local or patchy colonization. Biochem Biophys Res Commun, 1999 Mar 5, 256(1), 20 - 6 Isolation and partial characterization of an extracellular low-molecular mass component with high phenoloxidase activity from Thermoascus aurantiacus; Machuca A et al.; An extracellular low-molecular mass component (LMMC) with catalytic properties was isolated from liquid cultures containing wheat bran of ascomycete thermophilic Thermoascus aurantiacus . The partially purified LMMC showed very high activity with typical phenoloxidase substrates in the absence of hydrogen peroxide at acidic pH (2.8) . However, in this pH range, the phenoloxidase (PO) activity was quickly lost . The LMMC showed a high optimum temperature (80 degrees C) and an elevated thermostability . The molecular mass of the component estimated by gel filtration chromatography was 530 Da . IR and 1H- and 13C-NMR spectra indicated the presence of hydroxamic acid moiety . Qualitative determination of metal ions by several techniques revealed the presence of mainly iron associated with this structure . Iron may be the responsible for the ability for catalyze oxidation reactions, such as o-dianisidine oxidation, by the LMMC . These results suggested the existence of a hydroxamate-type metal-binding component, most likely hydroxamate siderophore . In addition, the chrome azurol S (CAS) universal assay for noncomplexed siderophores detection revealed the production of these compounds by T.aurantiacus in solid and liquid media . Protein Eng, 1999 Jan, 12(1), 47 - 53 Directed evolution converts subtilisin E into a functional equivalent of thermitase; Zhao H et al.; We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris . Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83 degrees C (3.5 min) and temperature optimum for activity (Topt, 76 degrees C) are identical with those of thermitase . The Topt of the evolved enzyme is 17 degrees C higher and its half-life at 65 degrees C is >200 times that of wild-type subtilisin E . In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90 degrees C . Thermitase differs from subtilisin E at 157 amino acid positions . However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable . The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme . Only two (N218S, N181D) are found in thermitase . Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity. J Mol Biol, 1999 Mar 12, 286(5), 1449 - 59 Glycyl-tRNA synthetase uses a negatively charged pit for specific recognition and activation of glycine; Arnez JG et al.; The crystal structures of glycyl-tRNA synthetase (GlyRS) from Thermus thermophilus, a homodimeric class II enzyme, were determined in the enzyme-substrate and enzyme-product states corresponding to the first step of aminoacylation . GlyRS was cocrystallized with glycine and ATP, which were transformed by the enzyme into glycyl-adenylate and thus gave the enzyme-product complex . To trap the enzyme-substrate complex, the enzyme was combined with the glycine analog ethanolamine and ATP . The ligands are bound in fixed orientations in the substrate-binding pocket of the N-terminal active site domain, which contains the classical class II aminoacyl-tRNA synthetase (aaRS) fold . Since glycine does not possess a side-chain, much of the specificity of the enzyme is directed toward excluding any additional atoms beyond the alpha-carbon atom . Several carboxylate residues of GlyRS line the glycine binding pocket; two of them interact directly with the alpha-ammonium group . In addition, the enzyme utilizes the acidic character of the pro-L alpha-hydrogen atom by contacting it via a glutamate carboxylic oxygen atom . A guanidino eta-nitrogen atom of the class II aaRS-conserved motif 2 arginine interacts with the substrate carbonyl oxygen atom . These features serve to attract the small amino acid substrate into the active site and to position it in the correct orientation . GlyRS uses class II-conserved residues to interact with the ATP and the adenosine-phosphate moiety of glycyl-adenylate . On the basis of this similarity, we propose that GlyRS utilizes the same general mechanism as that employed by other class II aminoacyl-tRNA synthetases . Biol Chem, 1999 Jan, 380(1), 55 - 62 The recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri: in vitro analysis of its chaperone activity; Minuth T et al.; The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known . It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity . Since its synthesis is not increased upon heat shock, we set out to test its chaperone function . In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E . coili BL21 (DE3) cells . Purification yielded soluble, high-molecular-mass double-ring complexes, indistinguishable from the natural thermosome . In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates . The results demonstrate that the recombinant M . kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation . However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 degrees C. J Appl Microbiol, 1999 Feb, 86(2), 353 - 8 Note: evaluation of selective media for the enumeration of Bifidobacterium sp . in milk; Payne JF et al.; Pure cultures of three species of bifidobacteria (Bifidobacterium longum, Bif . adolescentis and Bif . bifidum), Lactobacillus acidophilus and a mixed culture of Lact . delbrueckii subsp . bulgaricus and Streptococcus salivarius subsp . thermophilus were each enumerated on two differential media and six selective media for the enumeration of bifidobacteria . The appearance of the colonies on the differential media was as expected but when mixed cultures were present, it proved extremely difficult to distinguish one species from another . Of the selective media, AMC, RMS, NPNL and BL-OG performed well in that they gave good recoveries of bifidobacteria and were inhibitory to the growth of Lact . delbrueckii subsp . bulgaricus, Strep . salivarius subsp . thermophilus and Lact . acidophilus . However, of these four media, AMC was most convenient as it is based on a commercially available medium, whereas the others must be made up from individual constituents . The AMC agar is thus a good choice for the routine enumeration of bifidobacteria from mixed cultures. J Appl Microbiol, 1999 Feb, 86(2), 275 - 83 Electrotransformation of industrial strains of Streptococcus thermophilus; O'Sullivan TF et al.; A standard electroporation procedure was utilized to introduce a range of Gram-positive plasmid vectors into nine industrial strains of Streptococcus thermophilus . All the strains were transformable with at least two of the plasmids assessed, but electrotransformation frequencies depended on both the strain and the nature of transforming DNA . In general, small rolling circle (RC) plasmids could be electroporated at high frequency into a wide range of strains with efficiencies of 10(2)-10(5) transformants microgram-1 of transforming DNA . The presence of these plasmids did not influence doubling times during growth in broth, and they were generally extremely stable in slow milk acidifying strains, with 85-100% of transformants retaining the selective markers over 105 generations . Vectors were less stable in fast-growing cultures . Of the three theta-type plasmids assessed, only one, pIL253, could be electroporated at low frequency into some slow growing strains . The presence of this plasmid caused a 40% increase in doubling time and it was lost from cells at a rate of 3% per generation . Attempts to alter the proteolytic status of slow acidifying strains of Strep . thermophilus by the introduction of heterologous proteinase genes are also described. J Appl Microbiol, 1999 Feb, 86(2), 226 - 30 A method for the preparation of Tetrahymena thermophila phospholipase A1 suitable for large-scale production; Guberman A et al.; A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented . Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids . The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation . Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis . In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416-fold increase in specific activity . This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources . This practical method for the purification of a microbial PLA1 opens the way to large-scale production of these types of enzyme, which are not as yet commercially available. Appl Environ Microbiol, 1999 Mar, 65(3), 1304 - 7 Kinetics of sulfate and hydrogen uptake by the thermophilic sulfate-reducing bacteria thermodesulfobacterium sp . Strain JSP and thermodesulfovibrio sp . Strain R1Ha3 Sonne-Hansen J, Westermann P, Ahring BK. Half-saturation constants (Km), maximum uptake rates (Vmax), and threshold concentrations for sulfate and hydrogen were determined for two thermophilic sulfate-reducing bacteria (SRB) in an incubation system without headspace . Km values determined for the thermophilic SRB were similar to the constants described for mesophilic SRB isolated from environments with low sulfate concentrations. FEBS Lett, 1999 Feb 12, 444(2-3), 222 - 6 Synechocystis sp . slr0787 protein is a novel bifunctional enzyme endowed with both nicotinamide mononucleotide adenylyltransferase and 'Nudix' hydrolase activities; Raffaelli N et al.; Synechocystis sp . slr0787 open reading frame encodes a 339 residue polypeptide with a predicted molecular mass of 38.5 kDa . Its deduced amino acid sequence shows extensive homology with known separate sequences of proteins from the thermophilic archaeon Methanococcus jannaschii . The N-terminal domain is highly homologous to the archaeal NMN adenylyltransferase, which catalyzes NAD synthesis from NMN and ATP . The C-terminal domain shares homology with the archaeal ADP-ribose pyrophosphatase, a member of the 'Nudix' hydrolase family . The slr0787 gene has been cloned into a T7-based vector for expression in Escherichia coli cells . The recombinant protein has been purified to homogeneity and demonstrated to possess both NMN adenylyltransferase and ADP-ribose pyrophosphatase activities . Both activities have been characterized and compared to their archaeal counterparts. Int J Food Microbiol, 1999 Jan 12, 46(1), 81 - 5 Origin and identification of bifidobacteria strains isolated from meat and meat products; Gavini F et al.; Forty-seven strains of bifidobacteria isolated from meat and meat products have been identified following phenotypic numerical analysis and DNA-DNA hybridization . Twenty-three strains were identified to the species B . thermophilum and 14 to B . pseudolongum subsp . pseudolongum . All others were also of animal origin, except for two strains -- B . longum and B . pseudocatenulatum -- that were of human origin . These strains were isolated from artificially contaminated meat by manual handling. J Biochem (Tokyo), 1999 Mar, 125(3), 487 - 94 Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase; Kikuchi N et al.; Based on partial amino acid sequences of p50 purified from a high-salt buffer extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted . The sequence contained helicase motifs, and showed homology with RuvB DNA helicase of Thermus thermophilus and an open reading frame for an unknown 50.5 k protein of Saccharomyces cerevisiae . p50 was expressed as a GST-fusion protein and antiserum against the protein was generated . p50 was localized to the nuclear matrix by cell fractionation and immunoblotting . p50 bound to ATP-Sepharose beads . Ultracentrifugation and gel filtration analyses showed that p50 in rat liver and Xenopus egg mitotic extracts exists as large complexes corresponding to 697 k and 447 k, respectively . A 50 k protein reactive with p50 antibodies was detected not only in rat liver nuclei, but also in a Xenopus egg cytoplasm fraction and a S . cerevisiae extract . This suggests that this putative DNA helicase is present in a wide variety of species ranging from yeast to mammals. J Biochem (Tokyo), 1999 Mar, 125(3), 454 - 9 Overexpression of the alanine carrier protein gene from thermophilic bacterium PS3 in Escherichia coli; Kanamori M et al.; The alanine transporter (alanine carrier protein, ACP) gene of thermophilic bacterium PS3 was previously cloned and expressed in a functionally active form in Escherichia coli cells . To achieve controlled overproduction of the ACP protein, we designed a plasmid encoding a fusion protein comprising ACP joined to the carboxyl terminus of the maltose binding protein (MBP-ACP) . Upon transduction of the plasmid into E . coli RM1 cells defective in alanine/glycine transport, the transport activity was expressed even before induction with 1-thio-beta-D-galacto-pyranoside (IPTG), and increased slightly on induction with IPTG at low concentrations . However, overexpression of the MBP-ACP gene, induced by higher concentrations of IPTG, resulted in death of the host cells . Hence we screened other host cells and found that the MBP-ACP fusion protein was produced in a large quantity in E . coli TB1 cells 3 h after IPTG induction . The MBP-ACP fusion protein was accumulated in cytoplasmic membranes in an amount reaching more than 20% of the total membrane protein . The affinity-purified MBP-ACP exhibited very low transport activity when reconstituted into proteoliposomes. Appl Environ Microbiol, 1999 Mar, 65(3), 1280 - 8 Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules; Sekiguchi Y et al.; 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater . The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes . In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells . Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta . For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y . Sekiguchi, Y . Kamagata, K . Syutsubo, A . Ohashi, H . Harada, and K . Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species . The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections . The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections . These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges. Appl Environ Microbiol, 1999 Mar, 65(3), 1214 - 21 Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate; Kieft TL et al.; A thermophilic bacterium that can use O2, NO3-, Fe(III), and S0 as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine . This organism, designated SA-01, clustered most closely with members of the genus Thermus, as determined by 16S rRNA gene (rDNA) sequence analysis . The 16S rDNA sequence of SA-01 was >98% similar to that of Thermus strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico . Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors . Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth . Thermus strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced . Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers . Suspensions of Thermus strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI) . Mn(IV)-oxide was reduced in the presence of either lactate or H2 . Both strains were also able to mineralize NTA to CO2 and to couple its oxidation to Fe(III) reduction and growth . The optimum temperature for growth and Fe(III) reduction by Thermus strains SA-01 and NMX2 A.1 is approximately 65 degrees C; their optimum pH is 6.5 to 7.0 . This is the first report of a Thermus sp . being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S. Appl Environ Microbiol, 1999 Mar, 65(3), 910 - 5 Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose; Terada Y et al.; The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced . The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da . The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%) . The amylomaltase gene was overexpressed in E . coli, and the enzyme was purified . This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C . When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme . The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84% . However, the minimum degree of polymerization (DP) of the cycloamylose produced by T . aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17 . The T . aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides . A detailed analysis of the activity of T . aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E . coli amylomaltase and the plant D-enzyme. Virology, 1999 Mar 1, 255(1), 63 - 76 Complete genomic sequence of the lytic bacteriophage DT1 of Streptococcus thermophilus; Tremblay DM et al.; Streptococcus thermophilus lytic bacteriophage DT1, isolated from a mozzarella whey, was characterized at the microbiological and molecular levels . Phage DT1 had an isometric head of 60 nm and a noncontractile tail of 260 x 8 nm, two major structural proteins of 26 and 32 kDa, and a linear double-stranded DNA genome with cohesive ends at its extremities . The host range of phage DT1 was limited to 5 of the 21 S . thermophilus strains tested . Using S . thermophilus SMQ-301 as a host, phage DT1 had a burst size of 276 +/- 36 and a latent period of 25 min . The genome of phage DT1 contained 34,820 bp with a GC content of 39.1% . Forty-six open reading frames (ORFs) of more than 40 codons were found and putative functions were assigned to 20 ORFs, mostly in the late region of phage DT1 . Comparative genomic analysis of DT1 with the completely sequenced S . thermophilus temperate phage O1205 revealed two large homologous regions interspersed by two heterologous segments . The homologous regions consisted of the early replication genes, the late morphogenesis genes, and the lysis cassette . The divergent segments contained the DNA packaging machinery, the major structural proteins, and remnants of a lysogeny module . Biochem Biophys Res Commun, 1999 Feb 24, 255(3), 765 - 73 Human phenylalanyl-tRNA synthetase: cloning, characterization of the deduced amino acid sequences in terms of the structural domains and coordinately regulated expression of the alpha and beta subunits in chronic myeloid leukemia cells; Rodova M et al.; Unlike the catalytic alpha-subunit, the beta-subunit of heterodimeric (alphabeta)2 phenylalanyl-tRNA synthetase (PheRS) has no invariant functional amino acids directly involved in the aminoacylation process as it is evident from the crystal structure of the T . thermophilus enzyme complexed with tRNAPhe . Having no catalytic function, the prokaryotic beta-subunit comprises OB-, RNP-, SH3-, and DNA-binding-like domains involved in a variety of biological functions in other proteins . It was shown that the mRNA of the human alpha-subunit overexpressed in the tumorigenic versus the nontumorigenic variant of the same acute-phase chronic myeloid leukemia cell line (CML) . We cloned, sequenced, and expressed human PheRS . The layout of the human sequence indicates that the general tRNA binding mode and anticodon recognition differ between prokaryotes and eukaryotes for the phenylalanine system . Northern blot hybridization analysis from malignant and normal human tissues enabled us to assess the relative expression levels of the alpha- and beta-subunits independently, in view of the additional cellular role proposed for the beta-subunit in tumorigenic events . The levels of mRNA corresponding to the alpha- and beta-subunits were remarkably similar in all cell types and tissues examined, thus indicating the implication of the entire (alphabeta)2 heterodimer in tumorigenic events . Arch Biochem Biophys, 1999 Mar 1, 363(1), 135 - 47 The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization; Hansen T et al.; Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities . S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ >> Mn2+ >> Ni2+, Ca2+ . CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E . coli (E-PPase) and Thermus thermophilus (T-PPase) . The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model . S-PPase is extremely heat resistant . Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations . More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization . The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase . The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate . Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers . The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases . The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase . J Bacteriol, 1999 Mar, 181(5), 1643 - 51 Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum; Cann IK et al.; The manA gene of Thermoanaerobacterium polysaccharolyticum was cloned in Escherichia coli . The open reading frame of manA is composed of 3,291 bases and codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da . The start codon is preceded by a strong putative ribosome binding site (TAAGGCGGTG) and a putative -35 (TTCGC) and -10 (TAAAAT) promoter sequence . The ManA of T . polysaccharolyticum is a modular protein . Sequence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, and SLH-3) . The CBD domains show no sequence homology to any cellulose binding domain yet reported, hence suggesting a novel CBD . The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion resulted in both failure to bind to cellulose and an apparent loss of carboxymethyl cellulase and mannanase activities . At the C-terminal region of the gene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xylanases and cellulases of thermophilic members of the Bacillus-Clostridium cluster . The ManA of T . polysaccharolyticum, besides being an extremely active enzyme, is the only mannanase gene cloned which shows this domain structure. Biochemistry, 1999 Feb 23, 38(8), 2570 - 6 Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme; Beadle BM et al.; Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not . To test these models, we reversibly melted two analogous protein domains in a two-state manner . E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca . CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi . When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0 . 32 kcal/(mol*K) . The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30 . This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts . This was further explored by urea denaturation . Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol . Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C . We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility . These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30. Biochemistry, 1999 Feb 23, 38(8), 2413 - 24 Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate; Nakai T et al.; The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively . The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain . AspATs from many species were classified into aminotransferase subgroups Ia and Ib . The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved . The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them . (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site . However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate . (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate . The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT. Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 7 - 17 Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp . nov; Kim B et al.; A polyphasic taxonomic study was undertaken to clarify relationships within and between representative thermophilic alkalitolerant streptomycetes isolated from soil and appropriate marker strains . The resultant data, notably those from DNA-DNA relatedness studies, support the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris . However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and S . thermovulgaris (Henssen 1957) Goodfellow et al . 1987 represent a single species . On the basis of priority, S . thermonitrificans is a later subjective synonym of S . thermovulgaris . Similarly, 10 out of the 11 representative thermophilic alkalitolerant isolates had a combination of properties consistent with their classification as S . thermovulgaris . The remaining thermophilic alkalitolerant isolate, Streptomyces strain TA56, merited species status . The name Streptomyces thermoalcalitolerans sp . nov . is proposed for this strain . A neutrophilic thermophilic isolate, Streptomyces strain NAR85, was identified as S . thermodiastaticus. Biochemistry, 1999 Feb 9, 38(6), 1780 - 8 Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA; Khaitovich P et al.; Functionally active large ribosomal subunits of thermophilic bacterium Thermus aquaticus have been assembled in vitro from ribosomal proteins and either natural or in vitro-transcribed 23S rRNA and 5S rRNA . Sedimentation properties of reconstituted subunits were similar to those of native ribosomal 50S subunits . Subunits reconstituted with in vitro-transcribed rRNAs exhibited high activity in the peptidyl transferase assay and in a poly(U)-dependent cell-free translation system (22 and 30%, respectively, compared to that of native 50S subunits) . Catalytic activity of reconstituted subunits critically depended on the presence of 5S rRNA . rRNA mutations known to affect functions of the native ribosome produced similar effects in reconstituted T . aquaticus 50S subunits . Subunits assembled with in vitro-transcribed T . aquaticus 23S rRNA containing the G2267A mutation (G2252A in Escherichia coli), which interferes with binding of peptidyl-tRNA in the ribosomal P-site, showed drastically reduced peptidyl transferase activity, whereas clindamycin resistance mutation A2084G (A2058G in E . coli) rendered assembled subunits tolerant to clindamycin inhibition . Thus, reconstitution of functional subunits with in vitro-transcribed rRNA makes possible the use of in vitro genetics for mutational analysis of 23S rRNA functions in translation . In addition, the ability to assemble catalytically active 50S subunits from the rRNA transcript lacking any posttranscriptional modifications clearly demonstrates that modified nucleotides in 23S rRNA are dispensable for the principal activities of the ribosome. J Biol Chem, 1999 Feb 26, 274(9), 5701 - 6 Cross-linking of two beta subunits in the closed conformation in F1-ATPase; Tsunoda SP et al.; In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation . In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact . We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond . The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide . The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP. Mol Cell Biol, 1999 Mar, 19(3), 2061 - 8 A novel H2A/H4 nucleosomal histone acetyltransferase in Tetrahymena thermophila; Ohba R et al.; Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A) . Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output . Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei . This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast . A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members . Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion . Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be approximately 80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity . Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena. Biodegradation, 1998, 9(3-4), 225 - 32 Thermophilic anaerobic treatment of sulphur rich forest industry wastewater; Rintala JA et al.; Thermophilic anaerobic treatment of sulphur-rich paper mill wastewater (0.8-3.1 gCOD/1, 340-850 mgSO4/l; COD:SO4 3.4-5.3) was studied in three laboratory-scale, upflow anaerobic sludge blanket (UASB) reactors and in bioassays . The reactors were inoculated with non-adapted thermophilic granular sludge . In the bioassays, no inhibition of the inoculum was detected and about 62% COD removal (sulphide stripped) was obtained . About 70 to 80% of the removed COD was methanised . In the reactors, up to 60-74% COD removal (effluent sulphide stripped) was obtained at loading rates up to 10-30 kgCOD/m3d and hydraulic retention times down to 6 to 2 hours . The effluent total sulphide was up to 150-250 mg/l . Sulphide inhibition could not be confirmed from the reactor performances . The results from bioassays suggested that both the inoculum and sludge from the UASB reactor used acetate mainly for methane production, while sulphide was produced from hydrogen or its precursors. J Biotechnol, 1999 Jan 22, 67(2-3), 85 - 97 Comparison of gene structures and enzymatic properties between two endoglucanases from Humicola grisea; Takashima S et al.; We have cloned two endoglucanase genes (egl3 and egl4) from a thermophilic fungus, Humicola grisea . The coding region of the egl3 gene was interrupted by an intron of 56-bp, and the deduced amino acid sequence of the egl3 gene was 305 amino acids in length and showed 98.4% identity with Humicola insolens EGV . The coding region of the egl4 gene was also interrupted by an intron of 173-bp, which contains 34 TTC repeated sequence units, and the deduced amino acid sequence of the egl4 gene was 227 amino acids in length and showed 61.5% identity with H . grisea EGL3 . The typical hinge and the cellulose-binding domain were observed in the C-terminal region of EGL3, but they were not observed in EGL4 . In the 5' upstream region of both genes, there were a TATA box or its similar sequence, CAAT motifs, and 6-bp sites which are identical or similar to the consensus sequence for binding a catabolite repressor CREA in Aspergillus nidulans . The egl3 and the egl4 genes were expressed in Aspergillus oryzae, and the translation products were purified . The fusion protein, EGL4CBD, which consists of a catalytic domain of EGL4 and the C-terminal region of EGL3, was also constructed and produced by A . oryzae, and purified . These enzymes showed relatively high activity toward carboxymethyl cellulose (CMC) and could not hydrolyze p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-cellobioside . The positive effect of substituting the C-terminal region of EGL4 with that of EGL3 was observed in the hydrolysis of CMC. Bioorg Med Chem Lett, 1999 Jan 4, 9(1), 35 - 8 Chemical and enzymatic synthesis of glycoconjugates . 5: One-pot regioselective synthesis of bioactive galactobiosides using a CLONEZYME thermophilic glycosidase library; Li J et al.; Enzymatic synthesis of galactobiosides using a versatile CLONEZYME thermostable glycosidase library was studied . One-pot transglycosylation reactions were demonstrated to synthesize beta(1-->4), beta(1-->6), and alpha(1-->6) disaccharide sequences with high regioselectivity and moderate to high yields. Arch Biochem Biophys, 1999 Feb 15, 362(2), 346 - 55 Purification and cloning of a thermostable manganese catalase from a thermophilic bacterium; Kagawa M et al.; We have purified a heat-stable catalase from a thermophilic bacterium, Thermus species strain YS 8-13 . The enzyme was purified 160-fold from crude cellular extracts and possessed a specific activity of 8000 units/mg at 65 degrees C . The purified enzyme displayed the highest activity at pH 7 to 10 and temperatures around 85 degrees C . The catalase was determined to be a manganese catalase, based on results from atomic absorption spectra and inhibition experiments using sodium azide . The enzyme was composed of six identical subunits of molecular weight 36,000 . Amino acid sequences determined from the purified protein were used to design oligonucleotide primers, which were in turn used to clone the coding gene . The nucleotide sequence of a 1.4-kb fragment of Thermus sp . YS 8-13 genomic DNA containing a 909-bp open reading frame was determined . The gene encoded a 302-residue polypeptide of deduced molecular weight 33,303 . The deduced amino acid sequence displayed a region-specific homology with the sequences of the manganese catalase from a mesophilic organism, Lactobacillus plantarum . J Mol Biol, 1999 Feb 19, 286(2), 563 - 77 Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization; Park YC et al.; A dense cluster of eight residues was identified at the crossing of two alpha-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus . Its mechanism of evolution was characterized . Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli . The corresponding mutations were constructed in TyrRS(Delta1), a derivative of TyrRS from B . stearothermophilus in which the anticodon binding domain is deleted . Mutations I52L (i.e . Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Delta1) in the pyrophosphate exchange reaction whereas T51P increased it . The kinetic stabilities of TyrRS(Delta1) and its mutant derivatives at 68.5 degreesC were determined from experiments of irreversible thermal precipitation . They were in the order L105V<I52L<T51P<Wild Type</=M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V . Mutations I52L and L105V affected the stability of the dimeric TyrRS(Delta1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectrofluorometry or size exclusion chromatography . I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A away from the subunit interface . L105V destabilized the monomeric intermediate of unfolding . The two mutational pathways, going from the wild-type TyrRS(Delta1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer . One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step . Mutation I52L allowed L105V along the first pathway and compensated it along the second pathway . Thus, the effects of I52L and L105V on stability depended on the structural context . The gain in activity due to T51P was at the expense of a slight destabilization . J Mol Biol, 1999 Feb 19, 286(2), 403 - 15 Identification of three aspartic acid residues essential for catalysis by the RusA holliday junction resolvase; Bolt EL et al.; RusA is a Holliday junction resolvase encoded by the cryptic prophage DLP12 of Escherichia coli K-12 that can be activated to promote homologous recombination and DNA repair in resolution-deficient mutants lacking the RuvABC proteins . Database searches with the 120 amino acid residue RusA sequence identified 11 homologues from diverse species, including one from the extreme thermophile Aquifex aeolicus, which suggests that RusA may be of ancient bacterial ancestry . A multiple alignment of these sequences revealed seven conserved or invariant acidic residues in the C-terminal half of the E . coli protein . By making site-directed mutations at these positions and analysing the ability of the mutant proteins to promote DNA repair in vivo and to resolve junctions in vitro, we identified three aspartic acid residues (D70, D72 and D91) that are essential for catalysis and that provide the first insight into the active-site mechanism of junction resolution by RusA . Substitution of any one of these three residues with asparagine reduces resolution activity >80-fold . The mutant proteins retain the ability to bind junction DNA regardless of the DNA sequence or of the mobility of the crossover . They interfere with the function of the RuvABC proteins in vivo, when expressed from a multicopy plasmid, an effect that is reproducible in vitro and that reflects the fact that the RusA proteins have a higher affinity for junction DNA in the presence of Mg2+ than do the RuvA and RuvC proteins . The D70N protein has a greater affinity for junctions in Mg2+ than does the wild-type, which indicates that the negatively charged carboxyl group of the aspartate residue plays a critical role at the active site of RusA . Electrostatic repulsions between D70, D72 and D91 may help to form a classical Mg2+-binding pocket . Int J Radiat Biol, 1999 Jan, 75(1), 59 - 65 Replication in vitro and cleavage by restriction endonuclease of 5-formyluracil- and 5-hydroxymethyluracil-containing oligonucleotides; Zhang QM et al.; PURPOSE: To investigate the biological consequences of 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU) . MATERIALS AND METHOD: The authors constructed 22-mer oligonucleotides containing a 5-foU or 5-hmU residue at the same sites . The effects of such modifications on the ability to serve as a template for DNA polymerase and on the cleavage by sequence-specific restriction endonuclease were examined . RESULTS: The Klenow fragment of DNA polymerase I and Thermus thermophilus DNA polymerase read through the sites of 5-foU and 5-hmU in the templates . 5-FoU directed the incorporation of dCMP in addition to dAMP opposite the lesion during DNA synthesis . The DNA polymerases incorporated only dAMP opposite the 5-hmU . The substitution of thymine by 5-foU within the recognition site of the restriction endonucleases HincII and SalI inhibited or prevented the cleavage by the enzymes, whereas the enzymes cleaved the 5-hmU-containing oligonucleotides at the same rate as the T-containing oligonucleotides . CONCLUSIONS: These results indicated that the 5-foU-A base pair is less stable than the T-A base pair and that 5-foU can form a base pair with C in addition to A . It was also demonstrated that the oxidation of thymine to 5-hmU does not result in substantial deterioration. Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2375 - 81 Cloning, sequencing, high expression, and crystallization of the thermophile Thermus aquaticus glycerol kinase; Huang HS et al.; Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism in bacteria . Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus . The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814 . The amino acid sequence deduced from T . aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes . After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography . The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22 degrees C . Comparison of the amino acid sequence with that of the B . subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes. J Pediatr Gastroenterol Nutr, 1999 Feb, 28(2), 191 - 8 Intestinal barrier function and cow's milk sensitization in guinea pigs fed milk or fermented milk; Terpend K et al.; BACKGROUND: The respective effect of milk and fermented milks on intestinal barrier capacity and on sensitization to beta-lactoglobulin was studied using a guinea pig model of cow's milk allergy . METHODS: Guinea pigs were fed a control diet or the same diet supplemented with milk, fermented milk (Streptococcus thermophilus and Bifidobacterium breve), or dehydrated fermented milk . Intestinal barrier capacity to macromolecules was assessed in an Ussing chamber, and sensitization to cow's milk proteins was measured by systemic anti-beta-lactoglobulin immunoglobulin G1 titers and by intestinal anaphylaxis, the latter assessed by the beta-lactoglobulin-induced increase in short-circuit current of jejunal fragments (deltaIsc(beta-LG)) . RESULTS: The electrical resistance of jejunum was similar in the four groups (approximately 80 omega/cm2) suggesting the same paracellular permeability . The transport of 14C-beta-lactoglobulin from mucosa to serosa was significantly decreased in the animals fed dehydrated fermented milk (403+/-131 ng / hr x cm2) compared with that in control animals or animals fed milk (767+/-250 ng / hr x cm2 and 749+/-475 ng / hr x cm2, respectively; p < 0.05) . Milk fermentation did not modify native beta-lactoglobulin concentration but anti-beta-lactoglobulin immunoglobulin G1 titers were higher in fermented milk and dehydrated fermented milk (log10 titer = 2.86 and 2.79, respectively) than in guinea pigs fed milk (log10 titer = 2.5; p < 0.007) . However, beta-lactoglobulin-induced intestinal anaphylaxis remained the same in the three groups (deltaIsc(beta-LG), 9.6+/-4.1 microA/cm2, 8.5+/-4.3 microA/cm2, and 8.5+/-3.4 microA/cm2 in milk-fed, fermented milk-fed, and dehydrated fermented milk-fed guinea pigs, respectively) . CONCLUSIONS: The intestinal barrier capacity to milk proteins seems to be reinforced by dehydrated fermented milk, but milk and fermented milks are equally efficient in inducing cow's milk allergy in guinea pigs. J Mol Biol, 1999 Feb 12, 286(1), 189 - 205 Iron superoxide dismutase from the archaeon Sulfolobus solfataricus: analysis of structure and thermostability; Ursby T et al.; The crystal structure of superoxide dismutase (SOD) from the hyper thermophile Sulfolobus solfataricus has been determined at 2.3 A resolution by molecular replacement and refined to a crystallographic R-factor of 16.8 % (Rfree 19.8 %) . The crystals belong to the space group C2 (a=76.3 A, b=124.3 A, c=60.3 A, beta=128.8 degrees) with two identical monomers in the asymmetric unit . The monomer has a molecular weight of 24 kDa and consists of 210 amino acid residues of which 205 are visible in the electron density map . The overall fold of the monomer of S . solfataricus SOD is similar to that of the other known Fe or Mn-SODs . S . solfataricus SOD forms a very compact tetramer of a type similar to that of SOD from the hyperthermophile Aquifex pyrophilus . Both structures show an elevated number of inter-subunit ion-pairs compared with the mesophilic SOD from Mycobacterium tuberculosis and the thermophilic SOD from Thermus thermophilus . However, in contrast to the A . pyrophilus SOD structure, the number of intra-subunit ion-pairs as well as inter- subunit hydrogen bonds is not higher than in the compared mesophilic and thermophilic SOD structures . The electron density also revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site . Its involvement in the specific activity of the enzyme is discussed . J Mol Biol, 1999 Feb 12, 286(1), 121 - 34 Isolation and characterization of a protein with high affinity for DNA: the glutamine synthetase of Thermus thermophilus 111; Mary J et al.; In a search of proteins from the thermophilic bacterium Thermus thermophilus 111 with a high affinity for DNA, the selected protein from this screening appears to be the glutamine synthetase (GS) . The purified product gives one band in SDS-polyacrylamide gel electrophoresis (53,700 Da) . The N-terminal 32 residues have been identified and present an homology of 80% with the glutamine synthetase of Bacillus subtilis and 76% with that of Thermotoga maritima . The protein displays the characteristic dodecameric structure of the eubacteria glutamine synthetase . From a detailed study of the interaction of this protein with DNA by dark-field electron microscopy and agarose gel electrophoresis, it is concluded that double-stranded DNA wraps the protein by a full turn of 150 bp length . An even number of GS molecules bound to a closed relaxed plasmid DNA does not alter its null topology . By using an inverted dimer DNA fragment, which contains twice a curved kinetoplast DNA insert in its central part, it is shown that DNA curvature rules the order in which GS binds to the DNA . DNA ends are also sites of high affinity for the GS . Supercoiling does not favor the binding of GS to the DNA with the exception of the apices that are by essence bent regions . By saturating a DNA molecule with GS one obtains a novel characteristic scalloped configuration in which the DNA undulates from one GS to the next . The DNA is condensed at least three times in these structures . By increasing the ratio of GS to DNA in solution the resulting material migrates as discrete bands relative to the free DNA in an agarose gel . By gel retardation and EM statistical distribution analysis of GS within the complexes, an average affinity constant of 10(7) M-1 was obtained . The potential implications of this novel interaction of the glutamine synthetase with DNA for the regulation of its own gene are briefly discussed . Biochemistry, 1999 Feb 2, 38(5), 1531 - 6 A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability; Satoh T et al.; Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth) . Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E . coli PPase) and T1-149E {residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E . coli PPase}, were constructed by random chimeragenesis . After the genes were overexpressed in the E . coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases . We found that the two chimeras had higher activity than either parent PPase at the optimum temperature . We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity . The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable . This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras . By comparing the three-dimensional structures of Tth and E . coli PPases, we deduced that the following two factors may contribute to differences in thermostability . (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E . coli PPase) in the vicinity of the trimer-trimer interface were different . (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E . coli PPase and also in the T1-135E chimera . Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes. Biochemistry, 1999 Jan 26, 38(4), 1332 - 7 Urea-induced unfolding and conformational stability of 3-isopropylmalate dehydrogenase from the Thermophile thermus thermophilus and its mesophilic counterpart from Escherichia coli; Motono C et al.; To reveal the basis of the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli has been studied . The urea-induced equilibrium unfolding of T . thermophilus and E . coli IPMDHs at 27 degreesC was monitored by measuring the changes in far-UV CD, intrinsic fluorescence, anilinonaphthalenesulfonic acid (ANS) binding, and catalytic activity in the presence of nonionic detergent Tween 20 . For both enzymes, the spectral methods revealed a biphasic unfolding transition . The first transition was protein concentration-independent, whereas the second was protein concentration-dependent for both enzymes . The observation suggested a three-state unfolding mechanism with a dimeric intermediate . However, the intermediates of the E . coli and the T . thermophilus IPMDHs seemed to be different from each other . The intermediate of the E . coli IPMDH lost its secondary and tertiary structure more than that of the thermophilic enzyme . E . coli IPMDH lost enzymatic activity through the transition from the native to the intermediate state, though the intermediate of the T . thermophilus enzyme was still active . The unfolding process of E . coli IPMDH can be explained by a sequential unfolding of individual folding domains, while there is only a small structural perturbation in the intermediate of T . thermophilus IPMDH . The higher thermal stability of T . thermophilus IPMDH can be attributed to the increase in the extent of interaction inside the first domain which unfolded prior to the unfolding of the whole molecular structure in E . coli IPMDH. J Mol Evol, 1999 Feb, 48(2), 218 - 35 Phylogeny of organisms investigated by the base-pair changes in the stem regions of small and large ribosomal subunit RNAs; Otsuka J et al.; In order to obtain the evolutionary distance data that are as purely additive as possible, we have developed a novel method for evaluating the evolutionary distances from the base-pair changes in stem regions of ribosomal RNAs (rRNAs) . The application of this method to small-subunit (SSU) and large-subunit (LSU) rRNAs provides the distance data, with which both the unweighted pair group method of analysis and the neighbor-joining method give almost the same tree topology of most organisms except for some Protoctista, thermophilic bacteria, parasitic organisms, and endosymbionts . Although the evolutionary distances calculated with LSU rRNAs are somewhat longer than those with SSU rRNAs, the difference, probably due to a slight difference in functional constraint, is substantially decreased when the distances are converted into the divergence times of organisms by the measure of the time scale estimated in each type of rRNAs . The divergence times of main branches agree fairly well with the geological record of organisms, at least after the appearance of oxygen-releasing photosynthesis, although the divergence times of Eukaryota, Archaebacteria, and Eubacteria are somewhat overestimated in comparison with the geological record of Earth formation . This result is explained by considering that the mutation rate is determined by the accumulation of misrepairs for DNA damage caused by radiation and that the effect of radiation had been stronger before the oxygen molecules became abundant in the atmosphere of the Earth. Int J Food Microbiol, 1998 Dec 22, 45(3), 225 - 8 Lactic acid bacteria growth promoters from Spirulina platensis; Parada JL et al.; Spirulina has been used for many years as human food because of its high protein content and nutritional value . Some strains also produce bioactive substances that may inhibit or promote microbial growth . Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus bulgaricus were grown in rich media, MRS and RM, as well as in minimal saline medium with and without addition of extracellular products obtained from a late log phase culture of Spirulina platensis in Zarrouk medium . In both MRS and RM media, the extracellular products significantly promote the growth of the lactic acid bacteria assayed . This stimulatory effect was observed in media with pH adjusted to 5.3, 6.3 and 7.0 . No effect was observed in minimal saline medium. Appl Environ Microbiol, 1999 Feb, 65(2), 569 - 77 Identification and characterization of a lysis module present in a large proportion of bacteriophages infecting Streptococcus thermophilus; Sheehan MM et al.; A lysis module encoded by the temperate bacteriophage phiO1205 was identified . This lysis module contains a lysin gene, designated lyt51, and two putative holin-encoding genes, designated lyt49 and lyt50 . lyt51 encodes a lytic enzyme specifically directed against streptococcal cell walls . Similar to other phage-encoded lysins, Lyt51 appears to have a modular design in which the N-terminal portion corresponds to its enzymatic activity while the C-terminal region is responsible for its substrate binding specificity . The two putative holin-encoding genes, lyt49 and lyt50, located immediately upstream of lyt51, were identified on the basis of their homology to other identified holin-encoding genes . Expression of lyt49 or lyt50 in Escherichia coli was shown to cause cell death and leakage of the intracellular enzyme isocitrate dehydrogenase into the growth medium without apparent lysis of the cells . Southern blotting experiments demonstrated that at least one of the three components of the identified lysis module is present in all members of a large collection of bacteriophages, indicating that components of this lysis module are widespread among bacteriophages infecting Streptococcus thermophilus. Appl Environ Microbiol, 1999 Feb, 65(2), 367 - 73 Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties; Wyss M et al.; Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed . The biochemical characteristics of an ideal phytase for this application are still largely unknown . To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined . The specific activities of the fungal phytases at 37 degreesC ranged from 23 to 196 U . (mg of protein)-1, and the pH optima ranged from 2.5 to 7.0 . When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product . A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups . When substrate specificity was examined, the A . niger, Aspergillus terreus, and E . coli phytases were rather specific for phytic acid . On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, alpha- and beta-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP . Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A . fumigatus phytase . In contrast, phosphate liberation by A . niger or A . terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is . To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A . fumigatus phytase or A . niger phytase (Natuphos) per kg of feed . Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A . fumigatus phytase than by the A . niger phytase at later stages of incubation. Appl Environ Microbiol, 1999 Feb, 65(2), 359 - 66 Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance; Wyss M et al.; Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi . Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production . The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli . All of the phytases examined are monomeric proteins . While E . coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system . Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts . However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases . When expressed in A . niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant . N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule . Site-directed mutagenesis of A . fumigatus and E . nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack . Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract. Res Microbiol, 1998 Nov-Dec, 149(10), 711 - 22 Genotypic characterization of thermophilic bacilli: a study on new soil isolates and several reference strains; Mora D et al.; A genotypic study using amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA fingerprinting (RAPD) and ribosomal spacer analysis (RSA) in comparison with DNA-DNA reassociation experiments was carried out with 85 thermophilic Bacillus isolates from uncultivated soil of 14 different geographical areas and seventeen reference strains representing defined thermophilic Bacillus species . This approach permitted the attribution of 51% of the new isolates to the Bacillus thermoleovorans group and the identification of 40% of the new isolates as B . "thermodenitrificans" . Moreover, 2 strains were assigned to B . pallidus species and 1 isolate to B . thermosphaericus species . The remaining 6% of our thermophilic isolates from soil, constituting 2 DNA-DNA homology groups, are still unidentified . A detailed genotypic characterization of the heterogeneous species of B . thermoleovorans and B . stearothermophilus was also presented. FEMS Microbiol Lett, 1999 Jan 1, 170(1), 191 - 8 Nisin independent induction of the nisA promoter in Lactococcus lactis during growth in lactose or galactose; Chandrapati S et al.; Nisin biosynthesis is autoregulated extracellularly by the mature and modified peptide . To investigate other regulatory effects on nisin biosynthesis, a transcription fusion of the nisA promoter from Lactococcus lactis ATCC 11454 to the promoterless lacZ gene from Streptococcus thermophilus was constructed . This fusion construct, pDOC99, expressed beta-galactosidase in L . lactis ATCC 11454 growing in M17 medium containing glucose (M17G) . Consistent with the known model for transcription of nisA, pDOC99 did not express beta-galactosidase in the non-nisin producer, L . lactis LM0230 grown in M17G, unless the nisRK genes (cloned in pDOC23) were included in trans and nisin was added to the medium . Growth of this strain in M17 containing lactose or galactose, resulted in nisA transcription, even in the absence of exogenous nisin . This expression was independent of pDOC23 . Furthermore, nisA transcription in L . lactis LM0230(pDOC99) grown in M17G could be induced by the addition of exogenous galactose, with maximum induction occurring at concentrations > 5 mM. Acta Biochim Pol, 1998, 45(3), 661 - 7 Recombinant His-tagged DNA polymerase . II . Cloning and purification of Thermus aquaticus recombinant DNA polymerase (Stoffel fragment); Dabrowski S et al.; The Stoffel DNA fragment, shortened by 12 bp from 5' end, coding for Stoffel DNA polymerase (missing 4 amino acids at N-terminus of Stoffel amino-acids sequence) from the thermophilic Thermus aquaticus (strain YT-1) was amplified, cloned and expressed in Escherichia coli . The recombinant Stoffel fragment contained a polyhistidine tag at the N-terminus (21 additional amino acids) that allowed its single-step isolation by Ni2+ affinity chromatography . The enzyme was characterized and displayed high DNA polymerase activity and thermostability evidently higher than the native Taq DNA polymerase. Acta Biochim Pol, 1998, 45(3), 653 - 60 Recombinant His-tagged DNA polymerase . I . Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase; Dabrowski S et al.; The Tth DNA polymerase gene from the thermophilic Thermus thermophilus (strain HB8) was amplified, cloned and expressed in Escherichia coli . The recombinant DNA polymerase containing a polyhistidine tag at the N-terminus was isolated in a single step by Ni2+ affinity chromatography . The purified recombinant enzyme, showing high polymerase activity contained 43 additional amino-acid residues (including a cluster of six histidine residues inserted for purification of the recombinant protein by metal-affinity chromatography) at N-terminus . The applied overexpression system was very efficient giving 700,000 u of DNA polymerase activity from 1 liter of induced culture . The enzyme was characterized and displayed high DNA polymerase and reverse transcriptase activities and high thermostability as compared to the native Tth DNA polymerase. Biochemistry (Mosc), 1998 Dec, 63(12), 1424 - 9 A site-specific endonuclease from thermophilic strain Bacillus species ZE is a ClaI isoschizomer; Zheleznyakova EN et al.; Strain Bacillus species ZE with a maximal yield of the ClaI isoschizomer was chosen from 12 natural thermophilic strains producing ClaI isomers . The yield is 110 times higher than that of the ClaI prototype endonuclease from Caryophanon latum L . The enzyme is sensitive to DNA dam-methylation and is highly stable under storage. J Nutr, 1999 Jan, 129(1), 77 - 82 Bacteria used for the production of yogurt inactivate carcinogens and prevent DNA damage in the colon of rats; Wollowski I et al.; Lactic acid-producing bacteria prevent carcinogen-induced preneoplastic lesions and tumors in rat colon . Because the mechanisms responsible for these protective effects are unknown, two strains of lactic acid bacteria, Lactobacillus delbrueckii ssp . bulgaricus 191R and Streptococcus salivarius ssp . thermophilus CH3, that are used to produce yogurt, were investigated in vitro and in vivo to elucidate their potential to deactivate carcinogens . Using the "Comet assay" to detect genetic damage, we found that L . bulgaricus 191R applied orally to rats could prevent 1, 2-dimethylhydrazine-induced DNA breaks in the colon in vivo, whereas St . thermophilus CH3 were not effective . However, in vitro, both strains prevented DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in isolated primary rat colon cells . Extracts prepared from milk fermented with St . thermophilus CH3 were as efficient in deactivating MNNG as was L-cysteine . Isolated metabolites arising from bacteria during fermentation in the colon or in milk {L(+) lactate, D(-) lactate, palmitic acid and isopalmitic acid} were not effective . We postulate that thiol-containing breakdown products of proteins, via catalysis by bacterial proteases, could be one mechanism by which MNNG or other carcinogens are deactivated in the gut lumen resulting in reduced damage to colonic mucosal cells. Curr Opin Struct Biol, 1998 Dec, 8(6), 738 - 48 The stability of proteins in extreme environments; Jaenicke R et al.; Three complete genome sequences of thermophilic bacteria provide a wealth of information challenging current ideas concerning phylogeny and evolution, as well as the determinants of protein stability . Considering known protein structures from extremophiles, it becomes clear that no general conclusions can be drawn regarding adaptive mechanisms to extremes of physical conditions . Proteins are individuals that accumulate increments of stabilization; in thermophiles these come from charge clusters, networks of hydrogen bonds, optimization of packing and hydrophobic interactions, each in its own way . Recent examples indicate ways for the rational design of ultrastable proteins. J Exp Biol, 1999 Jan 21, 202(Pt 4), 407 - 416 ATP reception and chemosensory adaptation in Tetrahymena thermophila; Kim MY et al.; Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog &bgr;- &ggr; -methylene ATP are both effective depolarizing chemorepellents in Tetrahymena thermophila . Chemorepellent behavior consists of repeated bouts of backward swimming (avoidance reactions) that can easily be quantified to provide a convenient bioassay for purinergic reception studies . Chemosensory adaptation occurs following prolonged exposure (10 min) to the repellents, and cells regain normal swimming behavior . Adaptation is specific since cells that are behaviorally adapted to either ATP or &bgr;- &ggr; -methylene ATP still retain full responsiveness to the chemorepellents GTP and lysozyme . However, cross adaptation occurs between ATP and &bgr;- &ggr; -methylene ATP, suggesting that they involve the same receptor . Behavioral sensitivity to both ATP and &bgr;- &ggr; -methylene ATP is increased by the addition of Na+, but addition of either Ca2+ or Mg2+ dramatically decreases the response to ATP . These ionic effects are correlated with in vivo ATP hydrolysis, suggesting that divalent ions decrease purinergic sensitivity by activating a Ca2+- or Mg2+-dependent ecto-ATPase to hydrolyze the ATP signal . In vivo {32P}ATP binding studies and Scatchard analysis suggest that the behavioral adaptation is due to a decrease in the number of surface binding sites, as represented by decreased Bmax values . All these changes are reversible (de-adaptation) after 12 min in a repellent-free buffer . Electrophysiological analysis showed that both &bgr;- &ggr; -methylene ATP (10 micromol l-1) and ATP (500 micromol l-1) elicited sustained, reversible depolarizations while GTP (10 micromol l-1) produced a transient depolarization, suggesting that the chemosensory response pathways for ATP and GTP reception may differ . There may be separate ATP and GTP receptors since ATP and GTP responses do not cross-adapt and 'cold' (unlabeled) GTP is not a good inhibitor of {32P}ATP binding . These results suggests that T . thermophila possess high-affinity surface receptors for ATP that are down-regulated during chemosensory adaptation . These ATP receptors may act as chemorepellent receptors to enable T . thermophila to recognize recently lysed cells and avoid a possibly deleterious situation . This is the simplest eukaryotic organism to show an electrophysiological response to external ATP. Biochemistry, 1999 Jan 19, 38(3), 1057 - 65 Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature; Giuffre A et al.; The kinetic properties of the ba3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70 degrees C . Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed . In the O2 reaction, the decay of the F intermediate is significantly slower (k' = 100 s-1 at 5 degrees C) than in the mitochondrial enzyme, with an activation energy E of 10.1 +/- 0.9 kcal mol-1 . The cyanide-inhibited ba3 oxidizes cyt c522 quickly (k approximately 5 x 10(6) M-1 s-1 at 25 degrees C) and selectively, with an activation energy E of 10.9 +/- 0.9 kcal mol-1, but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme . Cyt c552 oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c522-ba3 electrostatic complex . The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days) . Finally, the affinity of reduced ba3 for CO at 20 degrees C (Keq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa3 (Keq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a32+ of ba3 (k' = 0.8 s-1 vs k' = 0.02 s-1 for beef heart aa3 at 20 degrees C) . The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O2 tensions is discussed. Biochimie, 1998 Nov, 80(11), 949 - 57 Structure-function studies on beta-glycosidase from Sulfolobus solfataricus . Molecular bases of thermostability; D'Auria S et al.; beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a thermostable tetrameric protein with a molecular mass of 240 kDa which is stable in the presence of detergents and has a maximal activity above 95 degrees C . An understanding of the structure-function relationship of the enzyme under different chemical-physical conditions is of fundamental importance for both theoretical and application purposes . In this paper we report the effect of basic pH values on the structural stability of this enzyme . The structure of the enzyme was studied at pH 10 and in the temperature range 25-97.5 degrees C using circular dichroism, Fourier-transform infrared and fluorescence spectroscopy . The spectroscopic data indicated that the enzyme stability was strongly affected by pH 10 suggesting that the destabilization of the protein structure is correlated with the perturbation of ionic interactions present in the native protein at neutral pHs . These experiments give support to the observation derived from the 3D-structure, that large ion pair networks on the surface stabilize Sulfolobus solfataricus beta-glycosidase. Biochimie, 1998 Nov, 80(11), 933 - 41 Protein thermostability in extremophiles; Scandurra R et al.; Thermostability of a protein is a property which cannot be attributed to the presence of a particular amino acid or to a post synthetic modification . Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some amino acids and the modulation of the canonical forces found in all proteins such as electrostatic (hydrogen bonds and ion-pairs) and hydrophobic interactions . Proteins produced by thermo and hyperthermophilic microorganisms, growing between 45 and 110 degrees C are in general more resistant to thermal and chemical denaturation than their mesophilic counterparts . The observed structural resistance may reflect a restriction on the flexibility of these proteins, which, while allowing them to be functionally competent at elevated temperatures, renders them unusually rigid at mesophilic temperatures (10-45 degrees C) . The increased rigidity at mesophilic temperatures may find a structural determinant in increased compactness . In thermophilic proteins a number of amino acids are often exchanged . These exchanges with some strategic placement of proline in beta-turns give rise to a stabilization of the protein . Mutagenesis experiments have confirmed this statement . From the comparative analysis of the X-ray structures available for several families of proteins, including at least one thermophilic structure in each case, it appears that thermal stabilization is accompanied by an increase in hydrogen bonds and salt bridges . Thermostability appears also related to a better packing within buried regions . Despite these generalisations, no universal rules can be found in these proteins to achieve thermostability. Nucleic Acids Res, 1999 Feb 1, 27(3), 788 - 94 Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D; Tong J et al.; NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98% . Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity . This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates . The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase . Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+ . While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent . Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp . AK16D ligase remains more specific toward perfectly matched substrate. J Exp Biol, 1999 Feb, 202 (Pt 3), 289 - 300 Pathways of inorganic nitrogen assimilation in chemoautotrophic bacteria-marine invertebrate symbioses: expression of host and symbiont glutamine synthetase Lee RW, Robinson JJ, Cavanaugh CM. Symbioses between chemoautotrophic bacteria and marine invertebrates living at deep-sea hydrothermal vents and other sulfide-rich environments function autotrophically by oxidizing hydrogen sulfide as an energy source and fixing carbon dioxide into organic compounds . For chemoautotrophy to support growth, these symbioses must be capable of inorganic nitrogen assimilation, a process that is not well understood in these or other aquatic symbioses . Pathways of inorganic nitrogen assimilation were investigated in several of these symbioses: the vent tubeworms Riftia pachyptila and Tevnia jerichonana, the vent bivalves Calyptogena magnifica and Bathymodiolus thermophilus, and the coastal bivalve Solemya velum . Nitrate reductase activity was detected in R . pachyptila, T . jerichonana and B . thermophilus, but not in C . magnifica and S . velum . This is evidence for nitrate utilization, either assimilation or respiration, by some vent species and is consistent with the high levels of nitrate availability at vents . The ammonia assimilation enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were detected in all symbioses tested, indicating that ammonia resulting from nitrate reduction or from environmental uptake can be incorporated into amino acids . A complicating factor is that GS and GDH are potentially of both host and symbiont origin, making it unclear which partner is involved in assimilation . GS, which is considered to be the primary ammonia-assimilating enzyme of autotrophs, was investigated further . Using a combination of molecular and biochemical approaches, host and symbiont GS were distinguished in the intact association . On the basis of Southern hybridizations, immunoreactivity, subunit size and thermal stability, symbiont GS was found to be a prokaryote GS . Host GS was distinct from prokaryote GS . The activities of host and symbiont GS were separated by anion-exchange chromatography and quantified . Virtually all activity in symbiont-containing tissue was due to symbiont GS in R . pachyptila, C . magnifica and B . thermophilus . In contrast, no symbiont GS activity was detected in the gill of S . velum, the predominant activity in this species appearing to be host GS . These findings suggest that ammonia is primarily assimilated by the symbionts in vent symbioses, whereas in S . velum ammonia is first assimilated by the host . The relationship between varying patterns of GS expression and host-symbiont nutritional exchange is discussed. J Bacteriol, 1999 Jan, 181(2), 632 - 41 Phosphorylation and functional properties of the IIA domain of the lactose transport protein of Streptococcus thermophilus; Gunnewijk MG et al.; The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIAGlc of Escherichia coli is the best-characterized member . On the basis of these similarities, it was anticipated that IIALacS would be able to perform one or more functions associated with IIAGlc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions . The gene fragment encoding IIALacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography . IIALacS was unable to restore glucose uptake in a IIAGlc-deficient strain, which is consistent with a very low rate of phosphorylation of IIALacS by phosphorylated HPr (HPr approximately P) from E . coli . With HPr approximately P from S . thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIALacS (k1 = 4.3 x 10(2) M-1 s-1) and dephosphorylation of IIALacS approximately P by HPr (k-1 = 1.1 x 10(3) M-1 s-1) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIAGlc and HPr from E . coli . This finding suggests that IIALacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly . On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIAGlc, we constructed a double mutant {IIALacS(I548E/G556D)} that was predicted to have increased phosphoryl transfer activity . Indeed, the phosphorylation rate of IIALacS(I548E/G556D) by HPr approximately P increased (k1 = 4.0 x 10(3) M-1 s-1) and became nearly independent of the source of HPr approximately P (S . thermophilus, Bacillus subtilis, or E . coli) . The increased phosphoryl transfer rate of IIALacS(I548E/G556D) was insufficient to complement IIAGlc in PTS-mediated glucose transport in E . coli . Both IIALacS and IIALacS(I548E/G556D) could replace IIAGlc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form. J Bacteriol, 1999 Jan, 181(2), 434 - 43 Discontinuous occurrence of the hsp70 (dnaK) gene among Archaea and sequence features of HSP70 suggest a novel outlook on phylogenies inferred from this protein; Gribaldo S et al.; Occurrence of the hsp70 (dnaK) gene was investigated in various members of the domain Archaea comprising both euryarchaeotes and crenarchaeotes and in the hyperthermophilic bacteria Aquifex pyrophilus and Thermotoga maritima representing the deepest offshoots in phylogenetic trees of bacterial 16S rRNA sequences . The gene was not detected in 8 of 10 archaea examined but was found in A . pyrophilus and T . maritima, from which it was cloned and sequenced . Comparative analyses of the HSP70 amino acid sequences encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from A . pyrophilus and T . maritima form a thermophilic cluster with that from the green nonsulfur bacterium Thermomicrobium roseum and are unrelated to their counterparts from gram-positive bacteria, proteobacteria/mitochondria, chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts; (ii) the T . maritima HSP70 clusters with the homologues from the archaea Methanobacterium thermoautotrophicum and Thermoplasma acidophilum, in contrast to the postulated unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in HSP70 and gram negativity, or vice versa, absence of insert and gram positivity . Notably, the HSP70 from T . maritima lacks the insert, although T . maritima is phylogenetically unrelated to the gram-positive bacteria . These results, along with the absence of hsp70 (dnaK) in various archaea and its presence in others, suggest that (i) different taxa retained either one or the other of two hsp70 (dnaK) versions (with or without insert), regardless of phylogenetic position; and (ii) archaea are aboriginally devoid of hsp70 (dnaK), and those that have it must have received it from phylogenetically diverse bacteria via lateral gene transfer events that did not involve replacement of an endogenous hsp70 (dnaK) gene. Biosci Biotechnol Biochem, 1998 Oct, 62(10), 2035 - 8 P1 specificity of aqualysin I (a subtilisin-type serine protease) from Thermus aquaticus YT-1, using P1-substituted derivatives of Streptomyces subtilisin inhibitor; Tanaka T et al.; Aqualysin I is an alkaline serine protease isolated from Thermus aquaticus YT-1, an extreme thermophile . We have measured the P1-specificity of aqualysin I, using wild-type and five P1-substituted derivatives of Streptomyces subtilisin inhibitor (SSI) . SSIs efficiently inhibited the activity of aqualysin I, with low substrate specificity . Charge and hydrophobicity of side chain of the P1 amino acid residue showed no significant effect to the P1-specificity of this enzyme. J Biochem (Tokyo), 1999 Jan, 125(1), 143 - 50 Cloning of the RNA polymerase alpha subunit gene from Thermus thermophilus HB8 and characterization of the protein; Wada T et al.; The region containing the RNA polymerase alpha subunit (RNAPalpha) gene (rpoA) and the ribosomal protein genes of a thermophilic eubacterial strain, Thermus thermophilus (Tt) HB8, was cloned from a genomic DNA library by Southern hybridization . The gene order in this region is rpl36-rps13-rps11-rps4-rpoA-rpl17, which is identical to that in some other eubacteria . The rpoA gene encodes a 315 amino acid residue protein with a molecular weight of 35,013, the amino acid sequence showing 42% identity to that of Escherichia coli (Ec) . From the results of comparison of the amino acid sequence and the predicted secondary structure of the C-terminal domain of Tt RNAPalpha (Tt alphaCTD) with those of Ec, the overall folding is expected to be similar . However, amino acid residues Asn268 and Cys269 in Ec alphaCTD, which are essential for its interaction with DNA or regulatory proteins, were replaced by His and Ser, respectively, in Tt alphaCTD . By means of a T7-based expression system in Ec cells, Tt RNAPalpha was overexpressed and purified . The high thermostability of Tt RNAPalpha was demonstrated by the CD spectra. J Biochem (Tokyo), 1999 Jan, 125(1), 109 - 14 Effects of C-terminal deletion on the activity and thermostability of orotate phosphoribosyltransferase from Thermus thermophilus; Hamana H et al.; To investigate the role of the C-terminal region on the activity and thermostability of orotate phosphoribosyltransferase (OPRTase, EC 2 . 4.2.10) from Thermus thermophilus, four C-terminal amino acid-deleted OPRTases (1, 2, 3, and 5 residues deleted) were constructed . The activities of all the mutant OPRTases were lower than that of wild-type OPRTase at all temperatures investigated (50-80 degreesC) . V- and EV-OPRTase, mutants with Val and Glu-Val deletions, respectively, showed 63 to 75% of the activity of wild-type OPRTase at the temperatures investigated . EEV- and PLEEV-OPRTase, with Glu-Glu-Val and Pro-Leu-Glu-Glu-Val deletions, respectively, had activities of 22 to 35% of the wild-type . The Km values for orotate of all mutant OPRTases were more than 4-fold higher than that of the wild-type (25 microM) . On the other hand, the Km for PRPP of the wild-type was 34 microM, and there were no significant differences between the wild-type and mutant OPRTases . The kcat values of the V- and EV-OPRTases were similar to that of the wild-type, but those of the EEV- and PLEEV-OPRTases were less than 50% that of the wild-type . The optimum temperature of all mutant OPRTases, 70 degreesC, was 10 degreesC lower than that of the wild-type . The remaining activities of wild-type and V-OPRTase after incubation at 90 degreesC for 20 min were 70 and 60% of the non-treated OPRTase activity, respectively . Although the remaining activity of EV-OPRTase was only 14% of the non-treated OPRTase activity, the addition of 200 mM KCl during heat treatment increased it to 70% . Circular dichroism spectroscopy revealed that V- and EV-OPRTase denature more easily than the wild-type OPRTase . The results suggest that the C-terminal valine and glutamic acid residues are important for the activity and thermostability of T . thermophilus OPRTase. J Biochem (Tokyo), 1999 Jan, 125(1), 48 - 57 Primary structure, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus; Satoh T et al.; The complete primary structure of inorganic pyrophosphatase {EC 3.6 . 1.1} from Bacillus stearothermophilus (ATCC 12016) was determined at the amino acid level by automated Edman degradation . The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18,796 . The amino acid sequence of the enzyme is almost identical to that of thermophilic bacterium PS-3 . Based on the determined primary structure, a PCR-amplified semi-synthetic gene was constructed and expressed in Escherichia coli JM109 . The recombinant Bst . PPase showed the same characteristics and activity as the authentic enzyme, and exhibits higher thermostability than the E . coli enzyme . Furthermore, we prepared tyrosine-substituted variants by site-directed mutagenesis to elucidate the role of two highly conserved tyrosines (Y46 and Y130) . As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected . However, the wild-type and two variants exhibited different thermostability behaviors in the presence or absence of Mg2+ . Therefore, these tyrosines may contribute to the structural integrity of the active site of the enzyme. J Biol Chem, 1999 Jan 15, 274(3), 1581 - 7 Stoichiometrically bound beta-carotene in the cytochrome b6f complex of oxygenic photosynthesis protects against oxygen damage; Zhang H et al.; The cytochrome b6f complex of oxygenic photosynthesis carries out "dark reactions" of electron transfer that link the light-driven reactions of the reaction centers, and coupled proton transfer that generates part of the electrochemical potential utilized for ATP synthesis . In contrast to the bc1 complex of the respiratory chain, with which there are many structural and functional homologies, the b6f complex contains bound pigment molecules . Along with the specifically bound chlorophyll a previously found to be bound stoichiometrically in the dimeric b6f complex, it was found in the present study that beta-carotene is also present in the b6f complex at stoichiometric levels or nearly so . Chlorophyll and carotenoid pigments were quantitatively extracted from b6f complex purified from (i) the thermophilic cyanobacterium, Mastigocladus laminosus, (ii) spinach chloroplasts, and (iii) the green alga, Chlamydomonas reinhardtii . Visible and mass spectra showed the carotenoid to be a beta-carotene of molecular weight = 536, with a stoichiometry of 1 . 0:1 relative to cytochrome f in the highly active M . laminosus complex but somewhat lower stoichiometries, 0.77 and 0.55, in the b6f complex obtained from spinach chloroplasts and C . reinhardtii . A photoprotective function for the beta-carotene was inferred from the findings that the rate of photobleaching of the chlorophyll a bound in the complex was found to vary inversely with beta-carotene content and to decrease markedly in the presence of ambient N2 instead of air . The presence of beta-carotene in the b6f complex, and not in the related bc1 complexes of the mitochondrial respiratory chain and photosynthetic bacteria, suggests that an additional function is to protect the protein complexes in oxygenic photosynthetic membranes against toxic effects of intramembrane singlet O2. J Mol Biol, 1999 Jan 15, 285(2), 689 - 702 Refined crystal structure of a superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius at 2.2 A resolution; Knapp S et al.; The extremely thermostable superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius was crystallized and the three-dimensional structure was determined by X-ray diffraction methods . The enzyme crystallized in the monoclinic spacegroup C2 with the cell dimensions a=168.1 A, b=91.3 A, c=85.7 A, beta=91.4 degrees . The diffraction limit of these crystals was 2.2 A . The crystals were very stable in the X-ray beam and measured diffraction data of a single crystal had a completeness of 99.5 % up to a resolution of 2.2 A.The crystal structure of S . acidocaldarius superoxide dismutase was solved by Patterson search methods using a dimer of Thermus thermophilus superoxide dismutase as a search model . The asymmetric unit accommodates three dimers . Two dimers form a tetramer by using only local symmetries; the third dimer forms a tetramer as well, however, by using the crystallographic 2-fold symmetry.The three-dimensional structure of the S . acidocaldarius dismutase has typical features of tetrameric dismutases . Secondary structure elements as well as residues important for the catalytic activity of the enzyme were found to be highly conserved . The model was refined at a resolution of 2.2 A and yielded a crystallographic R-value of 17.4 % (Rfree=22.3 %) . A structural comparison of the two extremely stable tetrameric dismutases from S . acidocaldarius and Aquifex pyrophilus with the less stable enzyme from T . thermophilus and Mycoplasma tuberculosis revealed the structural determinants which are probably responsible for the high intrinsic stability of S . acidocaldarius dismutase . The most obvious factor which may give rise to the extraordinary thermal stability of S . acidocaldarius dismutase (melting temperature of about 125 degreesC) is the increase in intersubunit ion pairs and hydrogen bonds and, more importantly, the significant reduction of solvent-accessible hydrophobic surfaces, as well as an increase in the percentage of buried hydrophobic residues . J Steroid Biochem Mol Biol, 1998 Oct, 67(2), 163 - 9 Progesterone 6-hydroxylation is catalysed by cytochrome P-450 in the moderate thermophile Bacillus thermoglucosidasius strain 12060; Sideso O et al.; The moderate thermophile, Bacillus thermoglucosidasius, transforms progesterone into four metabolites . These are 6alpha- and 6beta-hydroxyprogesterone, androstenedione and testosterone . This is the first report of bacterial 6alpha-hydroxylation of steroids . The identity of the progesterone metabolites shows that there are three major types of transforming activity in this organism; C-17-C-20 lyase that cleaves the pregnane side chain of the substrate, C-17 oxidoreductase that interconverts the metabolites androstenedione and testosterone, and 6-hydroxylation . 6-hydroxylation activity was purified virtually to homogeneity and was shown to be catalysed by a cytochrome P-450 monooxygenase enzyme . This is the first report of a thermostable cytochrome P-450. Biophys J, 1999 Jan, 76(1 Pt 1), 438 - 42 Electron transfer kinetics of caa3 oxidase from Bacillus stearothermophilus: a hypothesis for thermophilicity; Giuffre A et al.; The O2 reaction and the reverse electron transfer of the thermophilic caa3 terminal oxidase of Bacillus stearothermophilus have been studied by laser flash-photolysis . The results show that both reactions, although studied at a temperature of 20 degreesC, far from the optimal temperature of > 60 degreesC for caa3, follow a kinetic behavior essentially identical to that observed with the electrostatic complex between mammalian cyt c and cyt c oxidase . In the O2 reaction cyt a and cyt a3 are very quickly oxidized; cyt a is then re-reduced via CuA, whereas cyt c oxidation is apparently rate-limited by the oxidation of CuA . Upon photodissociation of the mixed valence-CO caa3, reverse electron transfer from the binuclear center to cyt a3+ (tau1 = 3 micros) and CuA2+ (tau2 = 64 micros) is observed, while cyt c is not reduced by any detectable level . These results seem to rule out accounting for enzymatic thermophilicity by altered kinetics of intramolecular electron transfer involving the cyt center in the reduced configuration, which is very fast . On the basis of these results and previous data, we propose that thermophilicity involves an increased activation barrier for the reduction of cyt a3-CuB in the configuration typical of the oxidized site. Appl Environ Microbiol, 1999 Jan, 65(1), 301 - 6 Conditions for vigorous growth on sulfide and reactor-scale cultivation protocols for the thermophilic green sulfur bacterium chlorobium tepidum Mukhopadhyay B, Johnson EF, Ascano M Jr. We describe a reactor-scale cultivation protocol for the fastest-growing and only known thermophilic member of the family Chlorobiaceae, Chlorobium tepidum . We discovered that C . tepidum would grow with sulfide as the sole electron source at rates and with final cell yields comparable to those found with thiosulfate only if the sulfide concentration was maintained below 0.1 mM and the culture redox potential was at -300 +/- 20 mV . Such was also the requirement for growth in a photobioreactor when thiosulfate (optimum level, 12 mM) was used as the preferred electron source . For cultivation of C . tepidum on a 5- to 500-ml scale, we used the system of Balch and Wolfe (Appl . Environ . Microbiol . 32:781-791, 1976) using stopper-sealed serum tubes and bottles as an alternative to the methods commonly used for the cultivation of phototrophic anaerobes and obtained consistent results. Appl Environ Microbiol, 1999 Jan, 65(1), 36 - 40 Potential role of thiobacillus caldus in arsenopyrite bioleaching Dopson M, Lindstrom EB. We investigated the potential role of the three strains of Thiobacillus caldus (KU, BC13, and C-SH12) in arsenopyrite leaching in combination with a moderately thermophilic iron oxidizer, Sulfobacillus thermosulfidooxidans . Pure cultures of T . caldus and S . thermosulfidooxidans were used as well as defined mixed cultures . By measuring released iron, tetrathionate, and sulfur concentrations, we found that the presence of T . caldus KU and BC13 in the defined mixed culture lowered the concentration of sulfur, and levels of tetrathionate were comparable to or lower than those in the presence of S . thermosulfidooxidans . This suggests that T . caldus grows on the sulfur compounds that build up during leaching, increasing the arsenopyrite-leaching efficiency . This result was similar to leaching arsenopyrite with a pure culture of S . thermosulfidooxidans in the presence of yeast extract . Therefore, three possible roles of T . caldus in the leaching environment can be hypothesized: to remove the buildup of solid sulfur that can cause an inhibitory layer on the surface of the mineral, to aid heterotrophic and mixotrophic growth by the release of organic chemicals, and to solubilize solid sulfur by the production of surface-active agents . The results showed that T . caldus KU was the most efficient at leaching arsenopyrite under the conditions tested, followed by BC13, and finally C-SH12. J Food Prot, 1998 Dec, 61(12), 1602 - 8 Persistence of Escherichia coli O157:H7 in dairy fermentation systems; Dineen SS et al.; We examined (i) the persistence of Escherichia coli O157:H7 as a postpasteurization contaminant in fermented dairy products; (ii) the ability of E . coli O157:H7 strains with and without the general stress regulatory protein, RpoS, to compete with commercial starter cultures in fermentation systems; and (iii) the survival of E . coli O157:H7 in the yogurt production process . In commercial products inoculated with 10(3) CFU/ml, E . coli O157:H7 was recovered for up to 12 days in yogurt (pH 4.0), 28 days in sour cream (pH 4.3), and at levels > 10(2) CFU/ml at 35 days in buttermilk (pH 4.1) . For the starter culture competition trials, the relative inhibition of E . coli O157:H7 in the experimental fermentation systems was, in decreasing order, thermophilic culture mixture, Lactobacillus delbrueckii subsp . bulgaricus R110 alone, Lactococcus lactis subsp . lactis D280 alone, Lactococcus lactis subsp . cremoris D62 alone, and Streptococcus thermophilus C90 alone showing the least inhibition . Recovery of the rpoS mutant was lower than recovery of its wild-type parent by 72 h or earlier in the presence of individual starter cultures . No E . coli O157:H7 were recovered after the curd formation step in yogurt manufactured with milk inoculated with 10(5) CFU/ml . Our results show that (i) postprocessing entry of E . coli O157:H7 into fermented dairy products represents a potential health hazard; (ii) commercial starter cultures differ in their ability to reduce E . coli O157:H7 CFU numbers in fermentation systems; and (iii) the RpoS protein appears to most effectively contribute to bacterial survival in the presence of conditions that are moderately lethal to the cell. Eur J Biochem, 1998 Dec 1, 258(2), 837 - 45 Recombinant homo- and hetero-oligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro; Minuth T et al.; The archaeon Pyrodictium occultum is one of the most thermophilic organisms presently known . Previous experiments provided support for the significant contribution of a high-molecular-mass protein complex to the extreme thermotolerance of P . occultum . This protein complex, the 'thermosome', is composed of two subunits, alpha and beta, which form a hexadecameric double ring complex . In order to obtain the thermosome in amounts sufficient for structural and functional investigations, we produced the two subunits jointly and separately in Escherichia coli BL21(DE3) . In all three cases, we isolated soluble, high-molecular-mass double-ring complexes from E . coli BL21(DE3) . On electron micrographs, the recombinant complexes were indistinguishable from each other and from the natural thermosome . To characterize the quaternary structure of the recombinant particles, we used native gel electrophoresis, analytical gel filtration, and analytical ultracentrifugation . Spectral analysis, using absorption, fluorescence emission and far-UV circular dichroism spectroscopy were applied to compare the three recombinant protein complexes with the natural thermosome from P . occultum . All three recombinant complex species exhibit ATPase activity . Furthermore, we could demonstrate that the recombinant complexes slow down the aggregation of citrate synthase, alcohol dehydrogenase, and insulin . Thus, we conclude that the recombinant protein complexes exhibit a chaperone-like activity, interacting with non-native proteins; they do so at temperatures far below the lower physiological limit of growth. FEMS Microbiol Lett, 1998 Dec 15, 169(2), 361 - 7 Lysine is synthesized through the alpha-aminoadipate pathway in Thermus thermophilus; Kosuge T et al.; A 3.8-kb DNA fragment which was able to complement the mutation of a lysine auxotrophic Thermus thermophilus mutant was cloned from T . thermophilus HB27 . Sequence analysis of the 3.8-kb fragment indicated the presence of three open reading frames including a truncated one . The predicted amino acid sequences of two of the three open reading frames showed 55.2% and 45.0% identity with homocitrate synthase and homoaconitate hydratase of Saccharomyces cerevisiae, respectively . These two enzymes act as lysine biosynthetic enzymes through the alpha-aminoadipate pathway which has been reported in S . cerevisiae and fungi . Each of the two open reading frames in T . thermophilus was disrupted by integration of the heat-stable kanamycin nucleotidyltransferase gene . The resulting mutants showed lysine auxotrophy, which could be complemented with alpha-aminoadipate but not with diaminopimelate . These results indicate that lysine was synthesized through the alpha-aminoadipate pathway and not through the diaminopimelate pathway in T . thermophilus. Biochemistry (Mosc), 1998 Nov, 63(11), 1266 - 70 Thermostable DNA-polymerase from Thermus thermophilus B35: isolation and characterization of some properties; Rechkunova NI et al.; Thermostable Tte DNA-polymerase was isolated from the strain Thermus thermophilus B35 which was found in hot spring water . The enzyme with molecular mass 87 kD was isolated using sequential chromatography on DEAE-Sepharose, hydroxylapatite, hexyl-agarose, and heparin-Sepharose . Biochemical properties of Tte DNA-polymerase are similar to those of Tth DNA-polymerase isolated from Thermus thermophilus HB8; however, practical application of Tte-Pol seems to be more favorable due to higher temperature optimum of this enzyme and lack of restriction endonucleases in the initial strain. Biochemistry (Mosc), 1998 Oct, 63(10), 1216 - 9 Increased functional activity of elongation factor G with G16V mutation in the GTP-binding domain; Martemyanov KA et al.; Oligonucleotide-directed mutagenesis was used to obtain elongation factor G from Thermus thermophilus with the G16V mutation in its GTP-binding domain . Functional studies of the mutated protein and elongation factor G from E . coli were carried out . The data revealed that the G16V mutant retains high thermostability, has an increased ribosome-dependent GTPase activity, and its translation activity in cell-free translation system is equal to that of the factor G from E . coli . The mutated protein with an uncleavable GTP analog also has an increased affinity to the ribosomes. J Bacteriol, 1999 Jan, 181(1), 334 - 7 Peptidoglycan fine structure of the radiotolerant bacterium Deinococcus radiodurans Sark; Quintela JC et al.; Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry . The monomeric subunit was: N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-(gamma)-L-Orn-{(delta)Gly-Gly}-D-Ala-D-Ala . Cross-linkage was mediated by (Gly)2 bridges, and glycan strands were terminated in (1-->6)anhydro-muramic acid residues . Structural relations with the phylogenetically close Thermus thermophilus are discussed. Wei Sheng Wu Xue Bao, 1997 Aug, 37(4), 307 - 11 {Screening of thermophilic superoxide dismatase producing strain and some properties of the enzyme}; Wang Z et al.; A thermophilic SOD producing strain was obtained from the bacteria preserved in our lab . Its content of SOD was 8774u/g fresh cells . The strain can tolerate 0.4% H2O2 and 70 degrees C, and it has outstanding culture characteristics . It was identified as Bacillus stearothermophilus, called B.S 211-15 . Crude SOD was extracted from B.S 211-15, the recovery of total activity was 69.5%, the specific activity was 1793u/mg protein . The enzyme showed fine heat stability, pH stability and good proteinase resistance . The SOD activity didn't decrease after being kept in room temperature for 2 months . The results of inhibition reactions indicated that this enzyme was Fe-SOD. Structure, 1998 Dec 15, 6(12), 1577 - 86 The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance; Unge J et al.; Background: . The ribosomal protein L22 is one of five proteins necessary for the formation of an early folding intermediate of the 23S rRNA . L22 has been found on the cytoplasmic side of the 50S ribosomal subunit . It can also be labeled by an erythromycin derivative bound close to the peptidyl-transfer center at the interface side of the 50S subunit, and the amino acid sequence of an erythromycin-resistant mutant is known . Knowing the structure of the protein may resolve this apparent conflict regarding the location of L22 on the ribosome . Results: . The structure of Thermus thermophilus L22 was solved using X-ray crystallography . L22 consists of a small alpha+beta domain and a protruding beta hairpin that is 30 A long . A large part of the surface area of the protein has the potential to be involved in interactions with rRNA . A structural similarity to other RNA-binding proteins is found, possibly indicating a common evolutionary origin . Conclusions: . The extensive surface area of L22 has the characteristics of an RNA-binding protein, consistent with its role in the folding of the 23S rRNA . The erythromycin-resistance conferring mutation is located in the protruding beta hairpin that is postulated to be important in L22-rRNA interactions . This region of the protein might be at the erythromycin-binding site close to the peptidyl transferase center, whereas the opposite end may be exposed to the cytoplasm. Structure, 1998 Dec 15, 6(12), 1503 - 16 Structures of the psychrophilic Alteromonas haloplanctis alpha-amylase give insights into cold adaptation at a molecular level; Aghajari N et al.; Background: . Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0 degreesC, where the activity of their mesophilic and thermophilic counterparts is drastically reduced . It has generally been assumed that thermophily is associated with rigid proteins, whereas psychrophilic enzymes have a tendency to be more flexible . Results: . Insights into the cold adaptation of proteins are gained on the basis of a psychrophilic protein's molecular structure . To this end, we have determined the structure of the recombinant form of a psychrophilic alpha-amylase from Alteromonas haloplanctis at 2.4 A resolution . We have compared this with the structure of the wild-type enzyme, recently solved at 2.0 A resolution, and with available structures of their mesophilic counterparts . These comparative studies have enabled us to identify possible determinants of cold adaptation . Conclusions: . We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments. Protein Eng, 1998 Oct, 11(10), 925 - 30 Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements; Fiorentino G et al.; The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity . Two putative structural determinants contributing to the higher stability of ADH-hT had been identified by comparison with the less thermostable ADH (ADH-T) from the less thermophilic B . stearothermophilus NCA 1503 . In order to ascertain their role, mutations were designed to eliminate in ADH-hT a salt bridge at the N-terminus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T . Three mutants--Glu11Lys, Pro242Ala, and Glu11Lys/Pro242Ala--were expressed at high level and the proteins purified and characterized . In general, the mutations had little effect on the activity, indicating that they were not disruptive . The thermal resistance was changed displaying quite additive effects. Protein Eng, 1998 Oct, 11(10), 867 - 72 Proteins from thermophilic and mesophilic organisms essentially do not differ in packing; Karshikoff A et al.; The role of the packing density in the elevation of thermal stability of proteins from thermophilic organisms is widely discussed in the literature . In the present study, this issue was reconsidered in the scale of an unbiased set of protein structures . Partial specific volumes, void and cavity volumes were calculated for a set of 80 non-homologous proteins and for 24 proteins from thermophilic organisms and analysed in the context of their possible role in thermal stabilization . The results showed that there is no significant difference between the two sets in respect to the partial specific volume and cavity volume . The proteins from thermophilic organisms showed a slight tendency of increasing void volume, i.e . reducing the packing density . However this observation was not confirmed by the comparison of this parameter for proteins within different structural families . The results suggested that neither the reduction of the packing density nor the reduction of the packing defects can be considered as a common mechanism for increasing the thermal stability of the proteins from thermophilic organisms . Combining the result from this and our previous study we concluded that the electrostatic interactions seem to be a common factor regulating the thermal tolerance of proteins from thermostable organisms. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15218 - 22 Temperature, template topology, and factor requirements of archaeal transcription; Bell SD et al.; Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei . Because some Archaea exist in environments with temperatures of around 100 degreesC, they are likely to have evolved unique strategies for transcriptional control . Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system . Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors . Furthermore, archaeal transcription does not require hydrolysis of the beta-gamma phosphoanhydride bond of ATP . However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled . Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening . These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone . Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea in vivo in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances. Biochemistry, 1998 Dec 15, 37(50), 17402 - 7 Engineering of S2 site of aqualysin I; alteration of P2 specificity by excluding P2 side chain; Tanaka T et al.; Gly101, one of the conserved amino acid residues which was expected to be comprised in half-sphere-shaped S2 site small pocket of aqualysin I, a microbial thermophilic alkaline serine protease, was replaced by alanine, valine, or leucine to alterate the P2 specificity of the enzyme by excluding bulky P2 side chain of the substrate . By the mutation of G101A, the catalytic efficiencies of the enzyme for bulky amino acid residues in P2 site such as valine and leucine drastically decreased by excluding the P2 side chain . By the mutation of G101V, even the side chain of the methyl group of the alanine and the side chain of proline were excluded, while the catalytic efficiency toward glycine residue was retained . The enzyme was altered to be glycine preferable . The mutation of G101L reduced catalytic efficiencies for any substrate including glycine which is corresponding to the main chain of the peptide substrate . The strategies we have adopted in this paper are applicable to all subtilisin-related enzymes. Biochim Biophys Acta, 1998 Nov 10, 1388(2), 437 - 43 High-potential states of blue and purple copper proteins; Wittung-Stafshede P et al.; Electrochemical measurements show that there are high-potential states of two copper proteins, Pseudomonas aeruginosa azurin and Thermus thermophilus CuA domain; these perturbed states are formed in guanidine hydrochloride (GuHCl) solution in which the proteins are still blue (azurin) and purple (CuA) . In each case, the high-potential state forms reversibly . Absorption (azurin, CuA), visible circular dichroism (azurin, CuA), resonance-Raman (CuA), and EPR (CuA) spectra indicate that the structure of the oxidized copper site of each high-potential form is very similar to that of the native protein . It is proposed that GuHCl perturbs one or more H-bonds in the blue or purple copper active site, thereby allowing Cu(I) to adopt a more favorable coordination structure than that in the rigid cavity of the native protein. J Med Microbiol, 1998 Dec, 47(12), 1131 - 5 Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus; Poddar SK et al.; Fifteen randomly selected nasopharyngeal (NP) swab specimens (culture-negative for influenza A virus) were spiked with influenza A virus and the nucleic acids were extracted and subjected to PCR amplification with Thermus aquaticus (Taq) and T . thermophilus (Tth) DNA polymerases . Products of the expected size, and giving equivalent band intensities, were obtained from four specimens with both polymerases . Fox six specimens, less products were obtained with Taq DNA polymerase than with Tth DNA polymerase . Products were detected from five NPs only by PCR with Tth DNA polymerase . The transport medium and the calcium alginate swab fibre of the specimens were shown not to be the source of the inhibitors . The incorporation of 32P-dCTP into cDNA, and the yield of PCR products of cDNA made from control RNA template (purified from H2O spiked virus suspension) were decreased in the presence of inhibitory extracts, showing that both the reverse transcription (RT) and PCR steps in amplification with Taq DNA polymerase were sensitive to the inhibitors . In contrast, Tth DNA polymerase was more resistant to the inhibitors and viral nucleic acid from all the specimens examined could be amplified and detected in a single step by RT-PCR with Tth DNA polymerase. Int J Mol Med, 1998 Sep, 2(3), 283 - 286 Carboxyflavins, novel inhibitors of Taq DNA polymerase; Mizushina Y et al.; Carboxyflavins were found to be potent selective inhibitors of Taq DNA polymerase in a polymerase chain reaction . The inhibitions were dose-dependent, and complete inhibitions were observed at the concentration of 3.0 microM . Carboxyflavins were much less, or not sensitive to the DNA polymerases tested such as calf thymus DNA polymerase a, rat DNA polymerase section sign, human immunodeficiency virus type 1 reverse transcriptase, the Klenow Fragment of E . coli DNA polymerase I and T4 DNA polymerase . To our knowledge, there is no other report of an agent that selectively inhibits only a thermophilic polymerase . Interestingly, the carboxyflavins were able to prevent DNA synthesis in the murine lymphoid leukemia cell line L1210 in vitro; almost complete inhibitory levels were achieved in the range of less than 10 microM. FEMS Microbiol Lett, 1998 Dec 1, 169(1), 185 - 90 Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var . thermoidea to hygromycin B resistance; Dantas-Barbosa C et al.; Germinated conidia of the thermophilic fungus Humicola grisea var . thermoidea were transformed to hygromycin B resistance using the plasmid pAN7.1 . Transformation was achieved using lithium acetate treatment or electroporation . The efficiency of transformation was up to 32 and 25 transformants per microgram of plasmid DNA with the two methods, respectively . Transformants obtained by the lithium acetate method were more stable and showed a high copy number of the hph gene integrated into their genome . The other transformants, from the electroporation procedure, were stable, but unable to grow in the presence of high levels of hygromycin, and detection of the hph gene was only possible by polymerase chain reaction analysis. Mol Pathol, 1998 Jun, 51(3), 160 - 3 A novel, rapid in cell RNA amplification technique for the detection of low copy mRNA transcripts; Uhlmann V et al.; Growing interest now focuses on improvements of in situ polymerase chain reaction (PCR) technology for the detection of DNA and RNA cellular sequences . In this study, reverse transcription PCR in situ hybridisation (RT PCR-ISH) was developed and used to determine gene expression of pyruvate dehydrogenase in a cell model system, using human peripheral blood lymphocytes (PBLs) . The success of in cell RNA amplification depends on the type of cell/tissue fixation, cell permeabilisation, and the efficiency of reverse transcription and cDNA amplification . This paper presents new approaches to overcome the critical aspects of fixation, permeabilisation, and reverse transcription when performing in cell RNA amplification . A novel fixative, "Permeafix", possessing fixative and permeabilisation properties, was used for cell fixation procedures . "Permeafix" obviated the need for pre-amplification proteolysis, facilitating entry of PCR reagents to target sequences within the cell . In addition, a simple on step RNA in cell amplification protocol using recombinant Thermus thermophilus (rTth) DNA polymerase, which reverse transcribes mRNA efficiently to cDNA and then catalyses cDNA amplification, was used . The value of a semi-junctional primer system for in cell gene expression studies, without the need to perform DNase digestion, is demonstrated. Med Lav, 1998 Jul-Aug, 89(4), 301 - 15 {Review of risks of biological agents and preventive measures to safeguard the health of compost production workers}; Giubileo L et al.; A review of studies made in the compost production industry showed the biological agents posing a risk for workers were fungi and thermophile bacteria, gram-negative bacteria and endotoxins, with a prevalent inhalation exposure to airborne contaminated dusts . Medical examinations revealed cases of extrinsic allergic alveolitis due to A . fumigatus, and more frequently irritative and infectious disorders occurring especially in conditions of poor environmental hygiene and macroscopic dust pollution . For the evaluation of the air dispersion of microorganisms, which is high in compost transport and turning operations, at present no exposure limit values are available for biological agents; nevertheless, the concentrations measured were often higher than the limit values proposed for other manufacturing sectors by individual authors and by regulatory agencies in Europe, and were comparable to values observed in other industrial settings for which adverse health effects have been shown . Although the number of studies available are few in number, the results suggest that the hazards posed by microorganisms and the poor environmental hygiene conditions often encountered can undoubtedly be a source of risk for workers, which at present is difficult to establish but significant considering the high airborne concentrations of contaminated dust . Besides technical measures to avoid environmental macroscopic dispersion of dusts, measurement of airborne microbiological contaminants is also recommended . Health surveillance needs to be aimed at identifying subjects with hypersusceptibility to the infectious action of the pathogenetic and/or allergenic agents or with hypersensitivity to the same, and also to periodic control of respiratory organs. Protein Sci, 1998 May, 7(5), 1156 - 63 Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase; Bogin O et al.; A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH . We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline . All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active . The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347 . Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters . Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH . The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain . Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters . The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability. FEBS Lett, 1998 Nov 20, 439(3), 281 - 6 Archaeal cold-adapted proteins: structural and evolutionary analysis of the elongation factor 2 proteins from psychrophilic, mesophilic and thermophilic methanogens; Thomas T et al.; To identify structural features important for low temperature activity in archaeal proteins, elongation factor 2 (EF-2) genes (aef2) were sequenced from psychrophilic, mesophilic and thermophilic methanogens . Scatter plots were used to compare evolutionary distances for EF-2 amino acid sequences vs . 16S-rRNA sequences from methanogens growing at diverse temperatures . The absence of a temperature bias for the rate of protein vs . nucleic acid evolution demonstrated the importance of comparing closely related proteins in order to identify changes indicative of thermal adaptation . Three-dimensional modelling of the new EF-2 sequences enabled the identification of amino acid residues that may be important for conferring low temperature activity and included greater structural flexibility produced by fewer salt bridges, less packed hydrophobic cores and the reduction of proline residues in loop structures. Biochemistry, 1998 Nov 24, 37(47), 16757 - 64 The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP; Dou C et al.; The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared . ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO) . Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover . The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present . As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R . S.-F., Grell, E., Senior, A . E . (1993) J . Biol . Chem . 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites . Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min . This was caused by entrapment of MgADP in a single catalytic site . Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM . Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively . During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM . Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO . The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma . This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the gamma subunit within the central cavity of the (alpha beta)3 hexamer. Curr Microbiol, 1999 Jan, 38(1), 43 - 7 Distribution of plasmid-borne stress protein genes in Streptococcus thermophilus and other lactic acid bacteria; Somkuti GA et al.; The presence of heat stress protein genes (hsp) was tested by Southern hybridization analysis in total DNA extracts from species of the genus Streptococcus (47 strains), Lactobacillus (34 strains), Lactococcus (24 strains), and Leuconostoc (5 strains) . The biotinylated hsp16.4 probe prepared from an ORF2 fragment of pER341 (2.8 kb) tested positively with restricted DNA extracts of seven Streptococcus thermophilus strains and a single strain of Lactococcus lactis subsp . cremoris . In all positive S . thermophilus strains, the hsp was located on plasmids ranging from ca . 2.8 kb to 11 kb in size, while hsp was present in a 7.5-kb plasmid in Lactococcus lactis subsp . cremoris . Southern blots with a rep probe showed that all hsp16.4+ plasmids in S . thermophilus strains also shared homology with the replication function (rep) of pER341, suggesting the common origin of these plasmids. Biotechnol Prog, 1998 Nov-Dec, 14(6), 963 - 5 Production of amino acids by yogurt bacteria; Beshkova DM et al.; The dynamics of free amino acid production by the selected strains Streptococcus thermophilus 13a and Lactobacillus bulgaricus 2-11 were studied in pure and mixed cultivations during yogurt starter culture manufacture . L . bulgaricus 2-11 showed the highest activity for producing free amino acids with high individual concentrations over the first hour of growth (50% of the total amount) . By the end of milk's full coagulation (4.5 h), 70% of the total amount of amino acids was released . S . thermophilus 13a showed poor proteolytic properties and consumed up to 70% of the free amino acids produced by L . bulgaricus 2-11 in the process of coagulation of milk with the mixed culture. Science, 1998 Oct 9, 282(5387), 259 - 64 A preorganized active site in the crystal structure of the Tetrahymena ribozyme; Golden BL et al.; Group I introns possess a single active site that catalyzes the two sequential reactions of self-splicing . An RNA comprising the two domains of the Tetrahymena thermophila group I intron catalytic core retains activity, and the 5.0 angstrom crystal structure of this 247-nucleotide ribozyme is now described . Close packing of the two domains forms a shallow cleft capable of binding the short helix that contains the 5' splice site . The helix that provides the binding site for the guanosine substrate deviates significantly from A-form geometry, providing a tight binding pocket . The binding pockets for both the 5' splice site helix and guanosine are formed and oriented in the absence of these substrates . Thus, this large ribozyme is largely preorganized for catalysis, much like a globular protein enzyme. J Dairy Sci, 1998 Nov, 81(11), 2804 - 16 Ingredient supplementation effects on viability of probiotic bacteria in yogurt; Dave RI et al.; The present investigation studied the effects of cysteine, whey powder, whey protein concentrate, acid casein hydrolysates, or tryptone on the viability of Streptococcus thermophilus, Lactobacillus acidophilus, and bifidobacteria . Changes in pH, titratable acidity, redox potential, and viability of bacteria were monitored during 24 h of fermentation and refrigerated storage (4 degrees C) of yogurt for 35 d . The incubation time that was needed to reach pH 4.5 was considerably affected by the added ingredients . Also, the drop in pH or the increase in acidity and redox potential was dependent on the added ingredients . The addition of cysteine, whey protein concentrate, acid casein hydrolysates, or tryptone improved the viability of bifidobacteria to a variable extent, but whey powder failed to improve their viability . The morphology of S . thermophilus, as shown by electron microscopy, was affected by cysteine at 500 mg/L, possibly as a result of reduced redox potential . Sodium dodecyl sulfate-PAGE and amino acid analyses suggested that the nitrogen source in the form of peptides and amino acids improved the viability of bifidobacteria in yogurt made with a commercial ABT (Lactobacillus acidophilus, bifidobacteria, and Streptococcus thermophilus) starter culture, which showed a dramatic decline in the counts of this organism in previous studies. FEMS Microbiol Lett, 1998 Nov 15, 168(2), 213 - 9 Comparison of Streptococcus thermophilus strains by pulse field gel electrophoresis of genomic DNA; O'Sullivan TF et al.; Pulse field gel electrophoresis (PFGE) was utilised to compare the genomes of 16 Streptococcus thermophilus cultures from yoghurt, cheese, laban and dahi after digestion with the restriction endonucleases, SfiI, SmaI and BssHII . PFGE profiles could be used for strain identification and were also useful in predicting relatedness of certain strains . Genetic variations between specific morphotypes of a highly proteolytic culture were not detectable by PFGE in this study . Statistical analysis of SmaI restriction patterns enabled the clustering of strains into two groups which corresponded with biochemical properties of the strains examined and suggested that PFGE profiles could be useful in predicting biochemical characteristics. J Biomol Struct Dyn, 1998 Oct, 16(2), 397 - 411 Native protein fluctuations: the conformational-motion temperature and the inverse correlation of protein flexibility with protein stability; Tang KE et al.; We study the fluctuations of native proteins by exact enumeration using the HP lattice model . The model fluctuations increase with temperature . We observe a low-temperature point, below which large fluctuations are frozen out . This prediction is consistent with the observation by Tilton et al . {R . F . Tilton, Jr., J . C . Dewan, and G . A . Petsko, Biochemistry 31, 2469 (1992)}, that the thermal motions of ribonuclease A increase sharply above about 200 K . We also explore protein "flexibility" as defined by Debye-Waller-like factors and solvent accessibilities of core residues to hydrogen exchange . We find that proteins having greater stability tend to have fewer large fluctuations, and hence lower flexibilities . If flexibility is necessary for enzyme catalysis, this could explain why proteins from thermophilic organisms, which are exceptionally stable, may be catalytically inactive at normal temperatures. J Biomol Struct Dyn, 1998 Oct, 16(2), 341 - 5 Sequence periodicity in complete genomes of archaea suggests positive supercoiling; Herzel H et al.; The topological state of genomic DNA is of importance for its replication, recombination and transcription . The wrapping of the DNA around nucleosomes is associated with sequence periodicities (Trifonov and Sussman, Proc . Natl . Acad . Sci . USA, 77, pp . 3816-20) . Recently, also the negative supercoiling of eubacterial DNA was related to 11 base pair (bp) periodicity (Herzel et al . Physica A, 249, pp . 449-59) . Archaeal plasmids and a virus-like particle from Sulfolobus are positively supercoiled, but the superhelical conformation of archaeal genomic DNA is still uncertain . The problem of superhelicity can now be addressed via a comparative statistical analysis of the available complete genomes . For this purpose one has to look for periodicities which are in phase with the helical repeat of 10-11 bp . Similar periodicities are induced, however, by the amphipatic character of alpha-helices of encoded proteins (Zhurkin, Nucl . Acids Res., 9, pp . 1963-71) . We show that these protein-induced periodicities are extended over a few periods only . The periods of additional long-ranging oscillations deviate significantly from the value for free DNA . A period of 11 bp in Eubacteria reflects negative supercoiling, whereas the significantly different period of thermophilic Archaea close to 10 bp suggests positive supercoiling of archaeal genomes.
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