Appl Microbiol Biotechnol, 1998 Oct, 50(4), 495 - 501 Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population; Gallert C et al.; The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated . For peptone concentrations from 5 g l-1 to 20 g l-1 the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g . 552 mg l-1 day-1 compared to 320 mg l-1 day-1 at 10 g l-1 peptone . The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH3/g peptone in the mesophilic cultures . If 0.5-6.5 g l-1 ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed . The thermophilic biowaste cultures were most active if around 1 g ammonia l-1 was present . Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane . Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a Ki (50%) for NH3 of 92, 95 and 88 mg l-1 at 37 degrees C and 251, 274 and 297 mg l-1 at 55 degrees C respectively . This indicated that the thermophilic flora tolerated significantly more NH3 than the mesophilic flora . In the mesophilic reactor effluent 4.6 x 10(8) peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 x 10(7) cfu/ml was observed. Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1297 - 304 Coprothermobacter platensis sp . nov., a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge; Etchebehere C et al.; A new anaerobic, proteolytic, moderately thermophilic bacterium, strain 3RT, was isolated from a methanogenic mesophilic reactor treating protein-rich wastewater . The cells were Gram-negative, non-spore-forming, non-motile rods . The DNA base composition was 43 mol% G + C . The optimum pH and temperature for growth were 7.0 and 55 degrees C respectively . The bacterium fermented gelatin, casein, bovine albumin, peptone and yeast extract . Glucose, fructose, sucrose, maltose and starch were poorly fermented . The major fermentation products from glucose were acetate, CO2 and H2 and, from gelatin, propionate was also detected . Growth on glucose was stimulated by thiosulfate, which was reduced to sulfide . Sulfate and nitrate were not reduced . 16S rRNA gene analysis revealed that the isolated bacterial strain was phylogenetically related to Coprothermobacter proteolyticus (96.3% sequence similarity), the only known species within the genus . DNA-DNA hybridization analysis demonstrated a very low level of homology, indicating that the isolated strain and C . proteolyticus were not related at species level . Therefore, it is proposed to classify the described strain in the genus Coprothermobacter as a new species, Coprothermobacter platensis . The type strain of C . platensis is strain 3RT (= DSM 11748T). Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1181 - 5 Thermococcus guaymasensis sp . nov . and Thermococcus aggregans sp . nov., two novel thermophilic archaea isolated from the Guaymas Basin hydrothermal vent site; Canganella F et al.; Thermococcus strains TYST and TYT isolated from the Guaymas Basin hydrothermal vent site and previously described were compared by DNA-DNA hybridization analysis with the closest Thermococcus species in terms of physiology and nutritional aspects . On the basis of the new data and taking into consideration the molecular, physiological and morphological traits published previously, it is proposed that strains TYT and TYST should be classified as new species named Thermococcus aggregans sp . nov . and Thermococcus guaymasensis sp . nov., respectively . The type strain of T . aggregans is strain TYT (= DSM 10597T) and the type strain of T . guaymasensis is strain TYST (= DSM 11113T). Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1089 - 93 Streptomyces thermogriseus, a new species of the genus Streptomyces from soil, lake and hot-spring; Xu LH et al.; Many thermophilic actinomycetes were isolated from samples collected from a hot-spring, lake and soil in Yunnan, China . Chemical and molecular classification of four selected strains of thermophilic Streptomyces with an upper limited growth temperature of 65-68 degrees C and autolytic characteristics was carried out . A new species, Streptomyces thermogriseus sp . nov . is described . The type strain is Y-14046T (= CCTCC AA97014T). Protein Sci, 1998 Nov, 7(11), 2431 - 7 Surface salt bridges stabilize the GCN4 leucine zipper; Spek EJ et al.; We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil . Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites . At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges . Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type . An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C . While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles . Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences. FEBS Lett, 1998 Nov 6, 438(3), 195 - 200 Oxidative biodegradation of phosphorothiolates by fungal laccase; Amitai G et al.; Organophosphorus (OP) insecticides and nerve agents that contain P-S bond are relatively more resistant to enzymatic hydrolysis . Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) displayed complete and rapid oxidative degradation of the nerve agents VX and Russian VX (RVX) and the insecticide analog diisopropyl-Amiton with specific activity: k(sp) = 2200, 667 and 1833 nmol min(-1) mg(-1), respectively (pH 7.4, 37 degrees C) . A molar ratio of 1:20 for OP/ABTS and 0.05 M phosphate at pH 7.4 provided the highest degradation rate of VX and RVX . The thermostable laccase purified from the fungus Chaetomium thermophilium (Ct) in the presence of ABTS caused a 52-fold slower degradation of VX with k(sp) = 42 nmol min(-1) mg(-1) . The enzymatic biodegradation products were identified by 31P-NMR and GC/MS analysis. Extremophiles, 1998 Nov, 2(4), 379 - 89 A histidine gene cluster of the hyperthermophile Thermotoga maritima: sequence analysis and evolutionary significance; Thoma R et al.; The sequences of histidine operon genes in hyperthermophiles are informative for understanding high protein thermostability and the evolution of metabolic pathways . Therefore, a cluster of eight his genes from the hyperthermophilic and phylogenetically early bacterium Thermotoga maritima was cloned and sequenced . The cluster has the gene order hisDCBdHAFI-E, lacking only hisG and hisBp, and does not contain intercistronic regions . This compact organization of his genes resembles the his operon of enterobacteria . Sequence analysis downstream of the stop codon of hisI-E identifies a region with a significantly higher cytosine over guanosine content, which is indicative of a rho-dependent termination of transcription of the his operon . Multiple sequence alignments of N1-((5'-phosphoribosyl)-formimino)-5-aminoimidazole-4-carboxyam ide ribonucleotide isomerase (HisA) and of the cycloligase moiety of imidazoleglycerol phosphate synthase (HisF) support the previous assignment of the (beta alpha)8-barrel fold to these proteins . The alignments also reveal a second phosphate-binding motif located in the first halves of both enzymes and thereby support the hypothesis that HisA and HisF have evolved by a sequence of two gene duplication events . Comparison of the amino acid compositions of HisA and HisF from mesophiles and thermophiles shows that the thermostable variants of both enzymes contain a significantly increased number of charged amino acid residues and may therefore be stabilized by additional salt bridges. Res Microbiol, 1998 Oct, 149(9), 631 - 43 Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products; Andrighetto C et al.; Twenty-five strains of thermophilic lactobacilli isolated from yoghurt and from semi-hard and hard cheeses (in parallel with nine type or reference strains) were identified and grouped according to their genetic relatedness . Strains were identified by sugar fermentation patterns using the "API 50 CHL" galleries, by species-specific DNA probes in dot-blot hybridization experiments, by amplification and restriction analysis of the 16S rRNA gene (ARDRA) and by polymerase chain reaction (PCR) using species-specific oligonucleotide primers . Strains were classified as Lactobacillus delbrueckii subsp . lactis and subsp . bulgaricus, L . helveticus, and L . acidophilus . Strains which were atypical by sugar fermentation patterns were also identified . Most of the strains could not be grouped using carbohydrate fermentation profiles . PCR fingerprinting was used to identify DNA profiles for the 25 lactobacilli . Experimentally obtained PCR profiles enabled discrimination of all strains, which were grouped according to the similarities in their combined patterns . In general, the clustering of the strains corresponded well with species delineation obtained by molecular identification . The dendrogram of genetic relatedness enabled the unambiguous identification of most of the strains which were shown to be atypical by the sugar fermentation profile, except for a discrepancy in one L . delbrueckii subsp . lactis strain and one atypical Lactobacillus sp . strain. Eur J Biochem, 1998 Oct 15, 257(2), 372 - 9 Structure-specific binding recognition of a methanogen chromosomal protein; Paradinas C et al.; The archaeon Methanosarcina thermophila expresses large amounts of a small basic protein, called MC1 (methanogen chromosomal protein), which was previously identified as a DNA-binding protein possibly involved in DNA compaction in some methanogenic species . We have investigated the binding of MC1 to various kinds of branched DNA molecules whose double helix axis is severely kinked . We show that MC1 is able to distinguish and to bind preferentially to four-way junctions . This preferential binding is observed in the absence and presence of divalent cations . However, we find that MC1 has a low affinity for bulged DNA structures . These results show how MC1 is able to discriminate between different deformations of the DNA double helix. J Biochem Biophys Methods, 1998 Sep 24, 37(1-2), 69 - 89 Systematic, computer-assisted optimisation of the isolation of Thermus thermophilus 50S ribosomal proteins by reversed-phase high-performance liquid chromatography; Boysen RI et al.; Isolation of the 50S ribosomal proteins from Thermus thermophilus has been achieved for the first time using reversed-phase high-performance liquid chromatography based on the use of the non-end-capped LiChrospher RP-18 sorbent and computer-assisted method development for optimisation of the resolution . The separation approach for these basic ribosomal proteins utilised mobile phases of high ionic strength, to suppress silanophilic interactions with this type of reversed-phase sorbent . These conditions were found to be a key requirement for achieving good resolution with minimal peak-tailing . The retention times of the 50S ribosomal proteins of Thermus thermophilus were observed to be in very close agreement with the values predicted by computer simulation procedures based on linear solvent strength concepts, with an average error of only 0.5%, whilst base-line resolution was achieved for most of the adjacent peak zones . Following N-terminal sequencing, the proteins TthL5, TthL9, TthL18, TthL24, TthL29, TthL32, TthL34, TthL35 and TthL36 of Thermus thermophilus were readily identified . This approach thus provided a readily optimised strategy for the isolation of the 50S ribosomal proteins from Thermus thermophilus and should be generally applicable to the corresponding ribosomal proteins from various other species, as well as other classes of basic proteins present in crude extracts derived from other biological sources. Carbohydr Res, 1998 Aug, 310(4), 269 - 76 The exopolysaccharides produced by Streptococcus thermophilus Rs and Sts have the same repeating unit but differ in viscosity of their milk cultures; Faber EJ et al.; The polysaccharides produced by Streptococcus thermophilus Rs and Sts in skimmed milk consist of D-Gal and L-Rha in a molar ratio of 5:2 . Linkage analysis and 1D/2D NMR (1H and 13C) studies revealed that both polysaccharides have the same branched heptasaccharide repeating unit: {formula: see text} Remarkably, the two strains differ in their effects on the viscosity of stirred milk cultures . The milk culture of S . thermophilus Rs is non-ropy and affords 135 mg/L polysaccharide with an average molecular mass of 2.6 x 10(3) kDa . In contrast, the milk culture of S . thermophilus Sts is ropy and produces 127 mg/L polysaccharide with an average molecular mass of 3.7 x 10(3) kDa . Permeability measurements of non-stirred milk cultures of both strains suggest that both strains have a similar effect on the protein-polysaccharide network . Therefore, the only clear difference between both strains, which may cause the difference in ropiness of the milk cultures, is the difference in molecular mass of the polysaccharide. Electrophoresis, 1998 Oct, 19(14), 2528 - 35 Maturation of phagosomes is accompanied by specific patterns of small GTPases; Meyer M et al.; In this study we purified phagosomes of the ciliated protozoan Tetrahymena thermophila to analyze aspects of the maturation pathway of phagocytotic vesicles . Phagosomes were labeled with magnetic microparticles and then purified in high amounts with the help of a permanent magnet . By combining a pulse-chase labeling protocol with the magnetic separation procedure we were able to isolate phagosomes of defined ages, which represent distinct stages of their maturation pathway . GTP-overlay assays showed that a set of small GTPases of the ras superfamily is associated with these phagosomes . Phagosomes isolated at different stages of maturation revealed a change in the pattern of the small GTPases . Some small GTPases identified by the GTPase overlay assays could be aligned to India ink stained protein spots in two-dimensional gels of isolated phagosomes . The results presented here are a first step to identify the members of small GTPases associated with phagosomes during their maturation pathway . Microsequencing of pooled polypeptides by mass-spectrometric techniques will identify the primary structure of these small GTPases. Biochemistry, 1998 Nov 17, 37(46), 16410 - 5 Detergent-mediated reconstitution of membrane proteins; Knol J et al.; The efficiency of reconstitution of the lactose transport protein (LacS) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane insertion . To rationalize these differences, the lipid/detergent structures that are formed in the reconstitution process were studied by cryotransmission electron microscopy . Surprisingly, the two nonionic detergents Triton X-100 and n-dodecyl beta-D-maltoside (DDM) affected the liposome structures in a completely different manner . Preformed liposomes titrated with Triton X-100 maintained their bilayer structure far beyond the onset of solubilization, and transport activity was maximal when LacS was inserted into these structures . With DDM the vesicular structures were already disrupted at the onset of solubilization and these membrane sheets were converted into long threadlike micelles at higher DDM to lipid ratios . Triton X-100 allowed the protein to be reconstituted with the hydrophilic surface exposed to the outside, whereas LacS was incorporated randomly when DDM was used . These differences in orientation are readily explained by the different lipid-detergent structures formed by Triton X-100 and DDM . The orientation of the reconstituted LacS protein is a critical factor for the activity of the protein as the kinetics of translocation is very different for opposite directions of transport. Arch Microbiol, 1998 Oct, 170(5), 389 - 93 The formylmethanofuran dehydrogenase isoenzymes in Methanobacterium wolfei and Methanobacterium thermoautotrophicum: induction of the molybdenum isoenzyme by molybdate and constitutive synthesis of the tungsten isoenzyme; Hochheimer A et al.; Formylmethanofuran dehydrogenase catalyzes the first step in methane formation from CO2 in methanogenic archaea . Methanobacterium wolfei and Methanobacterium thermoautotrophicum have been shown to contain two isoenzymes, a tungsten-containing isoenzyme (Fwd) and a molybdenum-containing isoenzyme (Fmd) . We report here that in both thermophilic organisms the encoding genes are organized in a highly conserved fwdHFGDACB tungsten operon and in an fmdECB molybdenum operon . In both organisms, the tungsten isoenzyme was found to be constitutively transcribed, whereas the transcription of the molybdenum operon was induced by molybdate . Induction by molybdate was not significantly affected by tungstate. Structure, 1998 Nov 15, 6(11), 1433 - 44 High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5; Hilge M et al.; Background: . beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants . These enzymes have potential use in pulp and paper production and are of significant biotechnological interest . Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance . The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC . No three-dimensional structure of a mannan-degrading enzyme has been reported until now . Results: . The crystal structure of the thermostable beta-mannanase from T . fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution . In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively . Conclusions: . Analysis of the -1 subsite of T . fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates . We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite . Compared with the catalytic clefts of family 5 cellulases, the groove of T . fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate . This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan. Gene, 1998 Nov 5, 222(1), 125 - 32 The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus; Tamakoshi M et al.; 3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively . Organization of the leuB, leuC and leuD genes of an extreme thermophile, Thermus thermophilus, was investigated by sequence analysis . Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli . The order was the leuC, leuD, and leuB genes and, in contrast to a previous report, they did not overlap with each other . Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron-sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved. Biochem Biophys Res Commun, 1998 Nov 9, 252(1), 166 - 72 Characterization of a cytochrome P450 from the acidothermophilic archaea Sulfolobus solfataricus; McLean MA et al.; We report the cloning, expression, purification, and molecular characterization of a cytochrome P450 (CYP119) from the thermophilic archaea Sulfolobus solfataricus . This protein displays an absorption spectra in the reduced, oxidized, and carbonyl adduct analogous to those of other P450 enzymes . We demonstrate that P450 (CYP119) exhibits remarkable thermo- and pressure stability, with a melting temperature 40 degrees higher than that of the extensively studied cytochrome P450cam (CYP101) and an optical spectra completely resistant to the formation of the inactive P420 by hydrostatic pressure up to 2 kbar . CO flash photolysis experiments, as well as construction of a CYP119 homology model, suggest an open active site with greater solvent access than P450 (CYP101) and similar to that of P450 (CYP102) . This communication represents the first molecular characterization of an extremophilic cytochrome P450 . FEMS Microbiol Lett, 1998 Nov 1, 168(1), 99 - 104 Application of conductance techniques to the detection of thermophilic Campylobacter spp . in river water; Thomas C et al.; A study of basal medal identified Campylobacter enrichment broth, with (CEB+) and without (CEB) antibiotic supplement, as a suitable medium for the detection and enumeration of Campylobacter jejuni, C . coli and C . lari within aqueous samples via conductance methodology . Despite apparent differences in conductivity profiles between species in the presence of antibiotics, no significant differences (P < 0.05) were detected between detection times for each species tested . CEB+ was successfully employed within a combined enrichment and conductance protocol to the detection of C . jejuni from river water at a concentration of 1 CFU ml-1 from 83% of samples in under 39 h and thus demonstrated an improvement over an applied conventional membrane filtration technique. FEMS Microbiol Lett, 1998 Nov 1, 168(1), 1 - 7 Evidence for substrate binding of a recombinant thermostable xylanase originating from Rhodothermus marinus; Karlsson EN et al.; The xynl encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, beta-glucan and amorphous but not crystalline cellulose . Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only . Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains. Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13561 - 6 A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch; Hang B et al.; Etheno adducts in DNA arise from multiple endogenous and exogenous sources . Of these adducts we have reported that, 1,N6-ethenoadenine (epsilonA) and 3,N4-ethenocytosine (epsilonC) are removed from DNA by two separate DNA glycosylases . We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises epsilonA . The present work is directed toward identifying and purifying the human glycosylase activity releasing epsilonC . HeLa cells were subjected to multiple steps of column chromatography, including two epsilonC-DNA affinity columns, which resulted in >1,000-fold purification . Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the epsilonC activity resides in a 55-kDa polypeptide . This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase . This latter activity copurified to the final column step and was present in the isolated protein band having epsilonC-DNA glycosylase activity . In addition, oligonucleotides containing epsilonC.G or G/T(U), could compete for epsilonC protein binding, further indicating that the epsilonC-DNA glycosylase is specific for both types of substrates in recognition . The same substrate specificity for epsilonC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF. Plasmid, 1998 Nov, 40(3), 190 - 202 Conjugation in archaea: frequent occurrence of conjugative plasmids in Sulfolobus; Prangishvili D et al.; We describe five novel conjugative plasmids (CPs) and two subfamilies, each comprising several closely related variants of CPs isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeon Sulfolobus islandicus, which were obtained by plating of samples from Icelandic solfataras after liquid enrichment . They are related to each other and to the previously described CP pNOB8 from a Japanese Sulfolobus strain in that they share essential functions and limited similarity of genomes as demonstrated by DNA cross-hybridization and sequences . All these plasmids thus form a family of highly efficient self-spreading elements directly transferred from donor into recipient cells . Conjugation is initiated by pair formation, followed by selective transfer of the plasmids into the recipient and expression of transfer functions . Some of these CPs exclude superconjugation of the transcipients with closely related CPs . The novel CPs are stable upon conjugative transfer, but vary upon growth of transcipients . The stability of the CPs is higher in their original hosts or in related S . islandicus strains, than in Sulfolobus solfataricus strain PH1 as recipient . The deletion variant pING3 has lost the ability to transfer itself but is still subject to being transferred by the transfer apparatus of its complete relative, pING6 . The dissection of genes and functions has been initiated by characterizing this incomplete variant . Am J Respir Cell Mol Biol, 1998 Nov, 19(5), 812 - 8 Interleukin-10 modulates the severity of hypersensitivity pneumonitis in mice; Gudmundsson G et al.; Hypersensitivity pneumonitis (HP) is an inflammatory lung disease characterized by granuloma formation . We recently showed that interferon-gamma (IFN-gamma) is essential for inflammation and granuloma formation in HP . Interleukin-10 (IL-10) counteracts many of the biologic effects of IFN-gamma, suggesting that IL-10 modulates inflammation and granuloma formation in HP . We compared the expression of HP in C57BL/6 mice that lack IL-10 (IL-10 knockout {KO}) with that in wild-type (WT) littermates . IL-10 KO and WT mice were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula or to saline alone for 3 wk . The IL-10 KO mice had higher cell counts in their bronchoalveolar lavage fluid (2.85 +/- 0 . 43 x 10(6)) than did WT mice (1.4 +/- 0.3 x 10(6)/ml; P < 0.03), with a more prominent neutrophil response . They also had greater inflammation after antigen exposure than did the WT mice (P < 0 . 0001) . There was increased upregulation of IFN-gamma, IL-1, and tumor necrosis factor-alpha (TNF-alpha) mRNAs in the lungs of IL-10 KO mice . Adenovirus-mediated gene transfer of IL-10 to the liver of IL-10 KO mice reduced the inflammation from that seen in WT mice . These studies show that IL-10 has important anti-inflammatory properties in HP, and that lack of this cytokine leads to a more severe granulomatous inflammatory response. Nucleic Acids Res, 1998 Nov 15, 26(22), 5157 - 62 Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli; Buhler C et al.; DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases . DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity . The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively . Here wereport the overexpression in Escherichia coli and the purification of each subunit . The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons . The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation . The B subunit was overexpressed in E.coli and purified in soluble form . When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted . The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA . This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP. J Mol Evol, 1998 Nov, 47(5), 517 - 30 Symbiosis between methanogenic archaea and delta-proteobacteria as the origin of eukaryotes: the syntrophic hypothesis Moreira D, Lopez-Garcia P. We present a novel hypothesis for the origin of the eukaryotic cell, or eukaryogenesis, based on a metabolic symbiosis (syntrophy) between a methanogenic archaeon (methanobacterial-like) and a delta-proteobacterium (an ancestral sulfate-reducing myxobacterium) . This syntrophic symbiosis was originally mediated by interspecies H2 transfer in anaerobic, possibly moderately thermophilic, environments . During eukaryogenesis, progressive cellular and genomic cointegration of both types of prokaryotic partners occurred . Initially, the establishment of permanent consortia, accompanied by extensive membrane development and close cell-cell interactions, led to a highly evolved symbiotic structure already endowed with some primitive eukaryotic features, such as a complex membrane system defining a protonuclear space (corresponding to the archaeal cytoplasm), and a protoplasmic region (derived from fusion of the surrounding bacterial cells) . Simultaneously, bacterial-to-archaeal preferential gene transfer and eventual replacement took place . Bacterial genome extinction was thus accomplished by gradual transfer to the archaeal host, where genes adapted to a new genetic environment . Emerging eukaryotes would have inherited archaeal genome organization and dynamics and, consequently, most DNA-processing information systems . Conversely, primordial genes for social and developmental behavior would have been provided by the ancient myxobacterial symbiont . Metabolism would have been issued mainly from the versatile bacterial organotrophy, and progressively, methanogenesis was lost. Eur J Biochem, 1998 Oct 1, 257(1), 101 - 11 The laminarinase from thermophilic eubacterium Rhodothermus marinus--conformation, stability, and identification of active site carboxylic residues by site-directed mutagenesis; Krah M et al.; A gene (lamR) encoding laminarinase (LamR) was cloned from the marine thermophilic eubacterium Rhodothermus marinus ITI278 . The enzyme purified from recombinant Escherichia coli cells hydrolyses mixed 1,3-1,4-beta-glucans (lichenan, barley and oat beta-glucan) and 1,3-beta-homoglucans (laminarin, curdlan) by an endo type action pattern . The CD spectrum of laminarinase is characteristic for a protein with prevailing beta secondary-structural elements, and the fluorescence spectrum points to a surface localisation of the tryptophan residues . A half-transition concentration of 5.4 M guanidinium chloride was measured for the denaturant-induced unfolding of laminarinase monitoring changes of the ellipticity at 222 nm and the fluorescence . Substitution of acidic residues Glu129, Asp131 and Gln134, which are invariant in family 16 glycosyl hydrolases, caused a severe reduction of beta-glucan-hydrolysing activity suggesting their central role in enzymatic hydrolysis . Deletion of Met133 drastically reduced catalytic activity . Met133 is invariant in family 16 laminarinases but not present in the active-site region of bacterial 1,3-1,4-beta-glucanases which also belong to glycosyl hydrolase family 16 . Replacement of Met133 by Ala, Cys or Trp did not affect activity against 1,3-1,4-beta-polyglucans and 1,3-beta-polyglucans, but in mutant Met133A the rate of hydrolysis of cellobiosyltriose (G1-4G1-3Gr) was increased about 10 times . Hydrolysis of 1,3-beta-oligosaccharides and 1,4-beta-oligosaccharides (DP 2-7) demonstrated the ability of the enzyme to cleave 1,3-beta-linkages and 1,4-beta-linkages in low-molecular-mass carbohydrates independent of the structure of neighbouring linkages . The laminarinase contains five or six subsites for substrate binding according to the action pattern deduced from hydrolysis of labelled and unlabelled curdlan oligosaccharides of different chain length. Appl Environ Microbiol, 1998 Nov, 64(11), 4555 - 65 Use of an intelligent control system To evaluate multiparametric effects on iron oxidation by thermophilic bacteria Stoner DL, Miller KS, Fife DJ, Larsen ED, Tolle CR, Johnson JA. A learning-based intelligent control system, the BioExpert, was developed and applied to the evaluation of multiparametric effects on iron oxidation by enrichment cultures of moderately thermophilic, acidophilic mining bacteria . The control system acquired and analyzed the data and then selected and maintained the sets of conditions that were evaluated . Through multiple iterations, the BioExpert selected sets of conditions that resulted in improved iron oxidation rates . The results obtained with the BioExpert suggested that temperature and pH were coupled, or interactive, parameters . Elevated temperatures (51.5 degreesC) in combination with a moderately high pH (pH 1.84) impaired the growth of and iron oxidation by the enrichment culture . Moderate-to-high oxidation rates were achieved with a relatively high pH in combination with a relatively low temperature or, conversely, with a relatively low pH in combination with a relatively high temperature . The interactive effect of pH and temperature was not apparent from the results obtained in an experiment in which temperature was the only parameter that was varied . When the BioExpert was applied to a mixed culture containing mesophilic and thermophilic bacteria, the computer "learned" that pH 1.8, 45 degreesC, and an inlet iron concentration from 30 to 35 mM were most favorable for iron oxidation . In conclusion, this study demonstrated that the learning-based intelligent control system BioExpert was an effective experimental tool that can be used to examine multiparametric effects on the growth and metabolic activity of mining bacteria. Protein Eng, 1998 Sep, 11(9), 753 - 60 Helix stabilizing factors and stabilization of thermophilic proteins: an X-ray based study; Facchiano AM et al.; We have compared the X-ray structures of 13 thermophilic proteins with their mesophilic homologues, in order to bring out differences in the stability of helices . The energy terms of a helix-coil transition algorithm were used to evaluate helix stability . Helices of thermophilic proteins are more stable than the mesophilic homologues in 69% of cases . This is due mainly to intrinsic helical propensities of amino acids, whereas minor effects are linked to main chain H-bonds, side chain-side chain interactions, capping motifs and charge-dipole effects . Furthermore, the frequency of 10 helix stabilizing factors recognized by appropriate sequence patterns was evaluated . The only factor occurring significantly in the thermostable proteins was the lack of beta branched residues . Other factors do not show a definite trend, although their occurrence in proteins is believed to be important for stability . This is discussed in the light of protein engineering applications. Appl Environ Microbiol, 1998 Nov, 64(11), 4428 - 32 Gene cloning, DNA sequencing, and expression of thermostable beta-mannanase from Bacillus stearothermophilus; Ethier N et al.; A DNA genomic library constructed from Bacillus stearothermophilus, a gram-positive, facultative thermophilic aerobe that secretes a thermostable beta-mannanase, was screened for mannan hydrolytic activity . Recombinant beta-mannanase activity was detected on the basis of the clearing of halos around Escherichia coli colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum . The nucleotide sequence of the mannanase gene, manF, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da . From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases . The manF coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in E . coli as a recombinant fusion protein containing a hexahistidine N-terminal sequence . The fusion protein has thermostability similar to the native enzyme and was purified by Ni2+ affinity chromatography . The values for the kinetic parameters Vmax and Km were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme. Appl Environ Microbiol, 1998 Nov, 64(11), 4423 - 7 Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus; Berka RM et al.; The phyA gene encoding an extracellular phytase from the thermophilic fungus Thermomyces lanuginosus was cloned and heterologously expressed, and the recombinant gene product was biochemically characterized . The phyA gene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa) . The deduced amino acid sequence of PhyA has limited sequence identity (ca . 47%) with Aspergillus niger phytase . The phyA gene was inserted into an expression vector under transcriptional control of the Fusarium oxysporum trypsin gene promoter and used to transform a Fusarium venenatum recipient strain . The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 micromol of inorganic phosphate released per min per mg of protein at pH 6 and 37 degrees C . The Thermomyces phytase retained activity at assay temperatures up to 75 degrees C and demonstrated superior catalytic efficiency to any known fungal phytase at 65 degrees C (the temperature optimum) . Comparison of this new Thermomyces catalyst with the well-known Aspergillus niger phytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range. Appl Environ Microbiol, 1998 Nov, 64(11), 4328 - 32 Construction of a proline-producing mutant of the extremely thermophilic eubacterium Thermus thermophilus HB27; Kosuge T et al.; Growth of Thermus thermophilus HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP) . This result suggested that the gamma-glutamyl kinase (the product of the proB gene) was inhibited by feedback inhibition in T . thermophilus . DHP-resistant mutants were reported previously for Escherichia coli (A . M . Dandekar and S . L . Uratsu, J . Bacteriol . 170:5943-5945, 1988) and Serratia marcescens (K . Omori, S . Suzuki, Y . Imai, and S . Komatsubara, J . Gen . Microbiol . 138:693-699, 1992), and their mutated sites in the proB gene were identified . Comparison of the amino acid sequence of T . thermophilus gamma-glutamyl kinase with those of E . coli and S . marcescens mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms . For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the proB gene as reported for the proline-producing mutant of S . marcescens . The mutant, TH401, excreted about 1 mg of L-proline per liter at 70 degreesC after 12 h of incubation . It was also suggested that T . thermophilus had a proline degradation and transport pathway since it was able to grow in minimal medium containing L-proline as sole nitrogen source . In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation . Seven mutants unable to utilize L-proline for their growth were isolated . One of the mutants, TH4017, excreted about 2 mg of L-proline per liter in minimal medium at 70 degreesC after 12 h of incubation. Biochemistry (Mosc), 1998 Sep, 63(9), 1051 - 6 Localization of the binding site for the 3'-terminal sequence of tRNAPhe in subunits of phenylalanyl-tRNA synthetase from Thermus thermophilus; Moor NA et al.; A photoreactive tRNAPhe derivative containing a 4-thiouridine residue at the 3'-end (tRNAPhe-s4U-75) was prepared by tRNA nucleotidyltransferase-mediated incorporation of s4UMP into a tRNAPhe transcript lacking the 3'-terminal dinucleotide . The resulting tRNAPhe-s4U-75 was covalently bound to phenylalanyl-tRNA synthetase from Thermus thermophilus, and all criteria of an affinity modification were met . The main products of modification displaying various electrophoretic mobilities were formed by binding tRNAPhe-s4U-75 to the beta-subunit (major) of the enzyme . These data suggest that the nucleotide found at position 75 of tRNAPhe interacts with the beta-subunit of phenylalanyl-tRNA synthetase. Biochemistry (Mosc), 1998 Sep, 63(9), 1044 - 50 Affinity modification of phenylalanyl-tRNA synthetase from Thermus thermophilus by tRNAPhe transcripts containing 4-thiouridine; Moor NA et al.; Photoreactive derivatives of tRNAPhe containing residues of 4-thiouridine (s4U) were synthesized by the transcription system of T7 RNA polymerase . Complete substitution of s4U for 16 uridine residues ({16s4U}-tRNAPhe) caused a 14-fold decrease in the catalytic efficiency of aminoacylation of the tRNAPhe transcript by phenylalanyl-tRNA synthetase from T . thermophilus . {1s4U}-tRNAPhe obtained by random incorporation of s4U residues with further isolation of s4U-monosubstituted RNA molecules on an affinity gel has the same kinetic parameters in aminoacylation as the tRNAPhe transcript . The s4U-containing tRNAPhe transcripts were shown to bind covalently to phenylalanyl-tRNA synthetase, and the specificity of modification was demonstrated . The modification stoichiometry determined in this work suggests that the enzyme is a functional dimer . The modification labels both alpha- and beta-subunits of the enzyme, which has an oligomeric structure of alpha2beta2, and forms "cross-linking" products of subunits upon modification with {16s4U}-tRNAPhe . The prevalence of modification of the alpha-subunit suggests that tRNA has contacts with the enzyme, which have not been deciphered previously by X-ray analysis. Gene, 1998 Sep 14, 217(1-2), 15 - 23 Sequence analysis and heterologous expression of the groE genes from Thermoanaerobacter sp . Rt8.G4; Truscott KN et al.; The groE homologous genes of the anaerobic thermophile Thermoanaerobacter sp . Rt8.G4 (TRt) have been isolated, sequenced and analysed . The TRt groES and groEL encode subunits of chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) of 94 and 541 amino acids, respectively, and are arranged in that order forming the open reading frames (ORFs) of a bicistronic operon . A controlling inverted repeat of chaperone expression (CIRCE) element lies between the consensus promoter of the operon and TRt groES . At optimum growth temperature (65 degreesC) the chaperonins of TRt are expressed, but production of Cpn60 increases significantly following temperature increases of 3-10 degreesC . Functionally intact recombinant TRt chaperonins were produced in Escherichia coli . However, owing to codon incompatibility, replacement of consecutive AGA codons in the gene encoding TRt Cpn60 was necessary for optimum expression in this heterologous host. Virology, 1998 Oct 25, 250(2), 377 - 87 A short noncoding viral DNA element showing characteristics of a replication origin confers bacteriophage resistance to Streptococcus thermophilus; Foley S et al.; A 302-bp noncoding DNA fragment from the DNA replication module of phage phiSfi21 was shown to protect the Streptococcus thermophilus strain Sfi1 from infection by 17 of 25 phages . The phage-inhibitory DNA possesses two determinants, each of which individually mediated phage resistance . The phage-inhibitory activity was copy number dependent and operates by blocking the accumulation of phage DNA . Furthermore, when cloned on a plasmid, the phiSfi21 DNA acts as an origin of replication driven by phage infection . Protein or proteins in the phiSfi21-infected cells were shown to interact with this phage-inhibitory DNA fragment, forming a retarded protein-DNA complex in gel retardation assays . A model in which phage proteins interact with the inhibitory DNA such that they are no longer available for phage propagation can be used to explain the observed bacteriophage resistance . Genome analysis of phiSfi19, a phage that is insensitive to the inhibitory activity of the phiSfi21-derived DNA, led to the characterisation of a variant putative phage replication origin that differed in 14 of 302 nucleotides from that of phiSfi21 . The variant origin was cloned and exhibited an inhibitory activity toward phages that were insensitive to the phiSfi21-derived DNA . J Biol Chem, 1998 Nov 6, 273(45), 29554 - 64 The novel substrate recognition mechanism utilized by aspartate aminotransferase of the extreme thermophile Thermus thermophilus HB8; Nobe Y et al.; Aspartate aminotransferase (AspAT) is a unique enzyme that can react with two types of substrate with quite different properties, acidic substrates, such as aspartate and glutamate, and neutral substrates, although the catalytic group Lys-258 acts on both types of substrate . The dynamic properties of the substrate-binding site are indispensable to the interaction with hydrophobic substrates (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S . (1997) J . Biochem . (Tokyo) 122, 55-63) . AspATs from various organisms are classified into two subgroups, Ia and Ib . The former includes AspATs from Escherichia coli and higher eukaryotes, whereas the latter includes those from Thermus thermophilus and many prokaryotes . The AspATs belonging to subgroup Ia each have an Arg-292 residue, which interacts with the distal carboxyl groups of dicarboxylic (acidic) substrates, but the functionally similar residue of subgroup Ib AspATs has not been identified . In view of the x-ray crystallographic structure of T . thermophilus AspAT, we expected Lys-109 to be this residue in the subgroup Ib AspATs and constructed K109V and K109S mutants . Replacing Lys-109 with Val or Ser resulted in loss of activity toward acidic substrates but increased that toward the neutral substrate, alanine, considerably . These results indicate that Lys-109 is a major determinant of the acidic substrate specificity of subgroup Ib AspATs . Kinetic analysis of the interactions with neutral substrates indicated that T . thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E . coli AspAT . A flexible active site in the rigid T . thermophilus AspAT molecule may explain its high activity even at room temperature. Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 12832 - 7 Thermus thermophilus: a link in evolution of the tRNA-dependent amino acid amidation pathways; Becker HD et al.; Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNAAsp and tRNAAsn . Aspartate mischarged on tRNAAsn then is converted into asparagine by an omega amidase that differs structurally from all known asparagine synthetases . However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNAAsn to elongation factor Tu is only conferred by conversion of aspartate into asparagine . T . thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNAAsp and an asparaginyl-tRNA synthetase able to charge tRNAAsn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase . In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique . T . thermophilus is able to ensure tRNA aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA . These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the aminoacylation systems. Bioorg Khim, 1998 Aug, 24(8), 593 - 600 {Comparative analysis of interaction sites of Thermus thermophilus and Escherichia coli tRNA(Tyr) with homologous aminoacyl-tRNA synthetases by means of chemical modification and nuclease hydrolysis}; Egorova SP et al.; A nucleotide sequence of tRNA(Tyr) from the extreme thermophile Thermus thermophilus HB-27 living at 75 degrees C was determined . It is 86 nt long and shares a 52% homology with tRNA(Tyr) from Escherichia coli . A comparative analysis of the interaction sites of tRNA(Tyr) from T . thermophilus and E . coli with the cognate aminoacyl-tRNA synthetases was accomplished by the chemical modification and nuclease hydrolysis approaches . The tRNA(Tyr) was shown to interact with the cognate enzyme in the anticodon stem (on the 5'-side), in the anticodon, in the variable stem and loop (on the 5'-side), and in the acceptor stem (on the 3'-side) . These regions are located in the variable stem of the L-form . It was demonstrated that, upon forming the complex E . coli tRNA(Tyr)-cognate synthetase, endonuclease V1 induces additional cleavages of phosphodiester bonds on the 3'-side of the anticodon stem and on the 5'-side of the T-stem . This implies that tRNA may change its conformation when it interacts with the enzyme. Arch Biochem Biophys, 1998 Oct 15, 358(2), 222 - 31 A highly active protein repair enzyme from an extreme thermophile: the L-isoaspartyl methyltransferase from Thermotoga maritima; Ichikawa JK et al.; We show that the open reading frame in the Thermotoga maritima genome tentatively identified as the pcm gene (R . V . Swanson et al., J . Bacteriol . 178, 484-489, 1996) does indeed encode a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) and that this protein repair enzyme displays several novel features . We expressed the 317 amino acid pcm gene product of this thermophilic bacterium in Escherichia coli as a fusion protein with an N-terminal 20 residue hexa-histidine-containing sequence . This protein contains a C-terminal domain of approximately 100 residues not previously seen in this enzyme from various prokaryotic or eukaryotic species and which does not have sequence similarity to any other entry in the GenBank databases . The C-terminal region appears to be required for enzymatic function as no activity is detected in two recombinant constructs lacking this domain . Sedimentation equilibrium analysis indicated that the enzyme is monomeric in solution . The Km values for measured for peptide and protein substrates were found to be intermediate between those of the high-affinity human enzyme and those of the lower-affinity wheat, nematode, and E . coli enzymes . The enzyme was extremely heat stable, with no loss of activity after 60 min at 100 degreesC . Enzyme activity was observed at temperatures as high as 93 degreesC with an optimal activity of 164 nmol/min/mg protein at 85 degreesC . This activity is approximately 18-fold higher than the maximal activities of mesophilic homologs at 37 degreesC . These data suggest that the Thermotoga enzyme has unique features for initiating repair in damaged proteins containing L-isoaspartyl residues at elevated temperatures . Extremophiles, 1998 Aug, 2(3), 339 - 45 Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms; Summit M et al.; DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111 degrees C, 107.5 degrees C, and 100 degrees C (respectively) . Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa . Half-lives of P . strain ES4, P . furiosus, and T . aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa . A pressure of 89 MPa further increased the half-lives of P . strain ES4 and T . aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P . furiosus DNA polymerase did not increase significantly from that at 45 MPa . The decay constant for P . strain ES4 and T . aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm3/mol, respectively . Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state . Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P . strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents. Extremophiles, 1998 Aug, 2(3), 257 - 67 Anaerobic alkalithermophiles, a novel group of extremophiles; Wiegel J; Although some anaerobic and aerobic mesophiles have long been known to grow at alkaline pH (above 9.5), little was known until recently about thermophilic alkaliphiles, termed now alkalithermophiles . This minireview describes presently known and recently validly described anaerobic alkalithermophilic bacteria (pHopt55C > 8.5; Topt > 55 degrees C) and alkalitolerant thermophiles (pHopt55C < 8.5 but pHmax55C above 9.0) . Some of these are widely distributed, but others have been isolated (thus far) only from one specific location . This novel group of anaerobic bacteria is comprised of physiologically different genera and species which, so far, all belong to the Gram-type positive Bacillus-Clostridium phylogenetic subbranch . An interesting feature of these anaerobic alkalithermophiles is that most of the isolates have short doubling times . The fastest growing among them are strains of Thermobrachium celere, with doubling times as short as 10 min while growing above pH 9.0 and above 55 degrees C. Extremophiles, 1998 Aug, 2(3), 207 - 16 Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes; Cowan D et al.; Mesophilic nitrile-degrading enzymes are widely dispersed in the Bacteria and lower orders of the eukaryotic kingdom . Two distinct enzyme systems, a nitrilase catalyzing the direct conversion of nitriles to carboxylic acids and separate but cotranscribed nitrile hydratase and amidase activities, are now well known . Nitrile hydratases are metalloenzymes, incorporating FeIII or CoII ions in thiolate ligand networks where they function as Lewis acids . In comparison, nitrilases are thiol-enzymes and the two enzyme groups have little or no apparent sequence or structural homology . The hydratases typically exist as alpha beta dimers or tetramers in which the alpha- and beta-subunits are similar in size but otherwise unrelated . Nitrilases however, are usually found as homomultimers with as many as 16 subunits . Until recently, the two nitrile-degrading enzyme classes were clearly separated by functional differences, the nitrile hydratases being aliphatic substrate specific and lacking stereoselectivity, whereas the nitrilases are enantioselective and aromatic substrate specific . The recent discovery of novel enzymes in both classes (including thermophilic representatives) has blurred these functional distinctions . Purified mesophilic nitrile-degrading enzymes are typically thermolabile in buffered solution, rarely withstanding exposure to temperatures above 50 degrees C without rapid inactivation . However, operational thermostability is often increased by addition of aliphatic acids or by use of immobilized whole cells . Low molecular stability has frequently been cited as a reason for the limited industrial application of "nitrilases"; such statements notwithstanding, these enzymes have been successfully applied for more than a decade to the kiloton production of acrylamide and more recently to the smaller-scale production of nicotinic acid, R-(-)-mandelic acid and S-(+)-ibuprofen . There is also a rapidly growing catalog of other potentially useful conversions of complex nitriles in which the regioselectivity of the enzyme coupled with the ability to achieve high conversion efficiencies without detriment to other sensitive functionalities is a distinct process advantage. Extremophiles, 1998 Aug, 2(3), 179 - 83 Thermozymes: biotechnology and structure-function relationships; Zeikus JG et al.; Recent findings on the biochemical and molecular features of the following thermozymes are presented, based on their biotechnological use: alpha-amylase and amylopullulanase, used in starch processing; glucose isomerase, used in sweetener production; alcohol dehydrogenase, used in chemical synthesis; and alkaline phosphatase, used in diagnostics . The corresponding genes and recombinant proteins have been characterized in terms of sequence similarities, specific activities, thermophilicity, and unfolding kinetics . Site-directed and nested deletion mutagenesis were used to understand structure-function relationships . All these thermozymes display higher stability and activity than their counterparts currently used in the biotechnology industry. Extremophiles, 1998 Aug, 2(3), 163 - 70 The essence of being extremophilic: the role of the unique archaeal membrane lipids; van de Vossenberg JL et al.; In extreme environments, mainly Archaea are encountered . The archaeal cytoplasmic membrane contains unique ether lipids that cannot easily be degraded, are temperature- and mechanically resistant, and highly salt tolerant . Moreover, thermophilic and extreme acidophilic Archaea possess membrane-spanning tetraether lipids that form a rigid monolayer membrane which is nearly impermeable to ions and protons . These properties make the archaeal lipid membranes more suitable for life and survival in extreme environments than the ester-type bilayer lipids of Bacteria or Eukarya. Extremophiles, 1998 Aug, 2(3), 141 - 8 Histones and nucleosomes in Archaea and Eukarya: a comparative analysis; Pereira SL et al.; Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4 . Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion . Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3 + H4)2 tetramer. Extremophiles, 1998 Aug, 2(3), 131 - 40 Genetic elements in the extremely thermophilic archaeon Sulfolobus; Zillig W et al.; This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory . It does not discuss IS elements and transposons. Microbiology, 1998 Sep, 144 ( Pt 9), 2655 - 65 Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S rRNA gene analysis; Sekiguchi Y et al.; The microbial diversity of two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), which had been treating sucrose/propionate/acetate-based artificial wastewater were compared . 16S rDNA clone libraries were constructed by PCR with a prokaryote-specific primer set, and partial sequencing of the clonal 16S rDNAs was conducted for phylogenetic analysis . Of 115 mesophilic granule and 110 thermophilic granule clones sequenced, 19 and 22%, respectively, were phylogenetically affiliated with the domain Archaea, and the remainder in each case were assigned to the domain Bacteria . Within the domain Archaea, the 16S rDNA clones in both libraries showed relatively close relationships with those of methanogens . Within the Bacteria, a major group represented in the mesophilic clone library was the delta subclass of the Proteobacteria (27%), in which high degrees of relatedness were observed between the clonal 16S rDNA sequences and those of previously identified syntrophic bacteria and sulfate-reducing bacteria . In contrast, in the thermophilic clone library, the Thermodesulfovibrio group (19%), the green non-sulfur bacteria (18%) and the low G + C subclass of the Gram-positive bacteria (18%) were predominant . A significant difference between the two libraries was that no clone affiliated with the Proteobacteria was detected in the thermophilic clone library, whereas the Proteobacteria was detected in the thermophilic clone library, whereas the Proteobacteria was the most predominant group in the mesophilic clones . Thirty-six and 24 different sequences were found in the mesophilic and thermophilic clones, respectively, suggesting that the microbial diversity of the thermophilic granule was lower than that of the mesophilic granule. Klin Padiatr, 1998 Sep-Oct, 210(5), 331 - 9 {Chronic interstitial lung diseases in childhood: bronchopulmonary dysplasia and exogenous allergic alveolitis}; Resch B et al.; Bronchopulmonary dysplasia (BPD) is a chronic lung disease that develops in preterm infants treated with oxygen and positive-pressure ventilation for respiratory distress syndrome . Despite the introduction of new treatment modalities (surfactant therapy, high-frequency oscillation) and improvements in the outcome of critically ill preterm infants, BPD has become an extremely important complication of neonatal intensive care and the most common form of chronic lung disease in infants . Specific pathogenesis, treatment modalities, prognosis, and multidisciplinary approaches to the prevention of BPD are described in detail . Extrinsic allergic alveolitis ("hypersensitivity pneumonitis") is a rare pulmonary disease in childhood due to inhaled organic dust, containing fungal antigens, thermophilic actinomycetes, or avian proteins . Diagnosis is often difficult, but it should be considered in every child with persistent and otherwise unexplained respiratory symptoms. Mol Cell Probes, 1998 Oct, 12(5), 317 - 22 A reverse transcriptase polymerase chain reaction assay for the detection of thermophilic Campylobacter spp; Sails AD et al.; A novel method was developed for the detection of thermophilic enteropathogenic campylobacters based on the detection of mRNA using the reverse transcriptase polymerase chain reaction (RT-PCR) . The RNA extraction method, DNase treatment and RT-PCR assay were shown to be specific for mRNA . The assay is specific for the thermophilic campylobacters Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis and restriction fragment length polymorphism (RFLP) analysis of the 256 bp amplified product with the restriction endonucleases Alu I, Dde I and Dra I revealed distinct species specific patterns . The assay was applied to the detection of C . jejuni cells killed by heating at 72 degreesC for 5 min and mRNA was detected by RT-PCR immediately after heat killing but became undetectable within 4 h when the cells were held at 37 degreesC . The assay therefore can differentiate between viable and dead cells of C . jejuni . Biochemistry, 1998 Oct 20, 37(42), 14788 - 97 Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament; Masui R et al.; Self-assembly of RecA protein in solution and on single-stranded DNA exerts a significant effect on the catalytic activities of this protein . To manipulate the self-association reaction, we examined the effects of various salts on the self-association of RecA from Thermus thermophilus (ttRecA) by circular dichroism spectroscopy and gel-filtration analysis . We showed that the self-association of ttRecA strongly depends on the kind and concentration of the salt, as well as on the protein concentration . Chaotropic ions were especially useful for obtaining RecA in its hexameric and monomeric states . On the basis of these observations, we were able to regulate the oligomeric states of ttRecA and we then examined the activity of RecA in various oligomeric states . Monomeric ttRecA bound to ssDNA and formed a nucleoprotein filament, which showed ssDNA-dependent ATPase activity . These results suggest that the monomeric form of RecA is an intermediate in filament formation on ssDNA. Med Mycol, 1998 Apr, 36(2), 113 - 8 Fatal aortic Myceliophthora thermophila infection in a patient affected by cystic medial necrosis; Farina C et al.; A 22-year-old Italian woman developed fungal aortitis after cardiac surgery for aortic insufficiency . She experienced two episodes of septic embolization and subsequently underwent replacement of the aortic root and initial ascending aorta by a homograft . The lumina of the ascending aorta, aortic arch and the origin of the innominate artery were completely filled with vegetation . From the involved tissue the phaeoid thermophilic hyphomycete Myceliophthora thermophila (Apinis) van Oorschot was isolated in pure culture . This is the second report of isolation of this fungus from humans and the first isolation of a human pathogenic strain of M . thermophila causing fatal vasculitis in a patient affected by cystic medial necrosis . A detailed morphological description of the isolate is also provided. J Biol Chem, 1998 Oct 23, 273(43), 28399 - 407 Group II chaperonin in a thermophilic methanogen, Methanococcus thermolithotrophicus . Chaperone activity and filament-forming ability; Furutani M et al.; A gene encoding 544 amino acids for a subunit of group II chaperonin (thermosome) was cloned from a thermophilic methanogen, Methanococcus thermolithotrophicus . The deduced amino acid sequence showed 66.5, 56.1, and 20.1% similarities to those of Methanopyrus kandleri and Thermoplasma acidophilum and group I chaperonin of Escherichia coli, respectively . We call this chaperonin MTTS (M . thermolithotrophicus thermosome) . The MTTS gene was expressed in E . coli . The purified recombinant MTTS seemed to be monomeric on gel filtration in the absence of Mg2+ and ATP . The monomer assembled to an oligomer (complex) in the presence of 50 mM MgCl2, 0.25 mM ATP, and 0.3 M (NH4)2SO4 . It was eluted immediately before the elution volume of E . coli GroEL tetradecamer on gel filtration with a TSKgel G3000SWXL column . This reconstructed MTTS complex showed the cylindrical structure with two stacked rings in electron microscopy . The MTTS complex formed filamentous structures in the presence of Mg2+ and ATP at the protein concentration above 3.0 mg/ml . This filament formation was reversible . The MTTS filament was dissociated to the complex by dilution to the protein concentration of 0.2 mg/ml, even in the presence of Mg2+ and ATP . The MTTS complex exhibited weak ATPase activity with the hydrolysis rate of 74 mol of ATP hydrolysis/mol of MTTS complex/min at 70 degreesC . The MTTS complex promoted the refolding of chemically denatured thermophilic archaeal citrate synthase and glucose dehydrogenase at 50 degreesC in an ATP-dependent fashion . The analysis of nucleotide specificity of chaperone activity of MTTS suggested that it was coupled with hydrolysis of ATP, CTP, or UTP. J Biol Chem, 1998 Oct 23, 273(43), 28149 - 54 The ferredoxin-dependent conversion of glyceraldehyde-3-phosphate in the hyperthermophilic archaeon Pyrococcus furiosus represents a novel site of glycolytic regulation; van der Oost J et al.; The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya . A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate . In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) . Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene . GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli . Electron paramagnetic resonance analysis of the purified P . furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis . During glycolysis in P . furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed . It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea. DNA Seq, 1998 Mar, 9(1), 31 - 5 The 10Sa RNA gene of Thermus thermophilus; Op De Bekke A et al.; The 10Sa RNA gene of Thermus thermophilus was isolated and sequenced . The tRNA-like structure at the 5' and 3' ends and other secondary structure features of the T . thermophilus 10Sa RNA are similar to E . coli 10Sa RNA . A variant of the sequence motif coding for the tag peptide is located in the centre of T . thermophilus 10Sa RNA. Biochemistry, 1998 Oct 13, 37(41), 14575 - 84 Stabilization of the phospho-aspartyl residue in a two-component signal transduction system in Thermotoga maritima; Goudreau PN et al.; The central signaling pathway in many bacterial regulatory systems involves phosphotransfer between two conserved proteins, a histidine protein kinase, and a response regulator . The occurrence of two-component signaling systems in thermophilic bacteria raises questions of how both the proteins and the labile acyl phosphate of the response regulator are adapted to function at elevated temperatures . Thermotoga maritima HpkA is a transmembrane histidine kinase, and DrrA is its cognate response regulator . Both DrrA and the cytoplasmic region of HpkA (HpkA57) have been expressed in Escherichia coli, purified, and characterized . HpkA57 and DrrA have apparent Tm's of 75 and 90 degreesC, respectively . HpkA57 exhibits ATP-dependent autophosphorylation activity similar to that of histidine kinases from mesophiles, with maximum activity at 70 degreesC . DrrA catalyzes transfer of phosphoryl groups from HpkA57 and exhibits Mg2+-dependent autophosphatase activity, with maximum activity at approximately 80 degreesC . At this temperature, the half-life for phospho-DrrA is approximately 3 min . In the absence of Mg2+, the half-life is 26 min, significantly greater than the half-life of a typical acyl phosphate at 80 degreesC . In the absence of Mg2+, at all temperatures examined, phospho-DrrA exhibits much greater stability than acetyl phosphate . This suggests that the active site of this hyperthermophilic response regulator is designed to protect the phospho-aspartyl residue from hydrolysis. Dev Genet, 1998, 23(2), 151 - 7 Abortive conjugation induced by UV-B irradiation at meiotic prophase in Tetrahymena thermophila; Kobayashi T et al.; Conjugating Tetrahymena were irradiated by ultraviolet-B (UV-B) at various stages of conjugation . When the conjugants were exposed to the UV-B at late meiotic prophase (the stage from pachytene to diplotene), abortive conjugation was induced a high frequencies . After completing meiosis, a significant number of the conjugants showed marked anomalies, i.e., failure of nuclear selection after meiosis, and abortion of the subsequent conjugation process such as a postmeiotic division to form gametic nuclei, nuclear exchange, synkaryon formation, and postzygotic development . The conjugating pairs retained the parental macronucleus and separated earlier as compared with a control . The resultant exconjugants degenerated meiotic products and became amicronucleates . These observations strongly suggest the presence of a UV-sensitive molecule that is expressed specifically at the meiotic prophase and that directs the subsequent development after meiosis. Curr Microbiol, 1998 Nov, 37(5), 295 - 300 Xylose transport by the anaerobic thermophile thermoanaerobacter ethanolicus and the characterization of a D-xylose-binding protein Erbeznik M, Ray M, Dawson KA, Strobel HJ. Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces considerable amounts of ethanol . A protein in xylose-growing cells was solubilized from cell membranes by extraction with octyl-beta-glucoside . Internal peptide sequencing revealed that the protein was the product of a gene, xylF, encoding a putative D-xylose-binding protein . Metabolic labeling with 14C palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine residue . Binding was highly specific for xylose as evident by the lack of competition by sugars with structures similar to xylose . The apparent Kd of the protein for xylose was approximately 1.5 &mgr;M, and this value was very similar to the affinity constant determined for xylose transport by whole cells at low substrate concentrations . Uptake experiments with cells also suggested the presence of a separate low-affinity system . Binding activity varied less than 20% over a pH range of 4-8, and the level of activity was virtually unaffected when temperature was varied between 40 degreesC and 80 degreesC . This is the first biochemical characterization of a D-xylose-binding protein from a thermophilic organism. Biochemistry (Mosc), 1998 Aug, 63(8), 929 - 34 Photoaffinity labeling of DNA polymerase from Thermus thermophilus and DNA template by photoreactive analogs of dCTP; Zakharenko AL et al.; Substrate properties of dCTP analogs N4-{2-(4-azidotetrafluorobenzoylamino)-ethyl}-2;-deoxycytidine-5; -triphosphate (FABdCTP), 5-{N-(4-azidotetrafluorobenzoyl)-3-amino-trans-propen-1-yl} -2;-deoxycytidine-5;-triphosphate (AlFABdCTP), and N4-{2-(2-nitro-5-azidobenzoylamino)-ethyl}-2;-deoxycytidine-5; -triphosphate (NABdCTP) were studied in the reaction of DNA synthesis catalyzed by DNA polymerase from the extremely thermophilic bacterium Thermus thermophilus B 35 (Tte DNA polymerase) . The enzyme was photoaffinity labeled with the mentioned derivatives, NABdCTP being used for the first time . The photoreactive primers containing FABdCTP and AlFABdCTP were synthesized in situ by Tte DNA polymerase and used in the complementary addressed labeling of DNA template . The efficiency of DNA template labeling is shown to be a function of the structure of the photoactive group. Res Microbiol, 1998 Mar, 149(3), 171 - 6 Growth dependence of alpha-glucan phosphorylase activity in Thermus thermophilus; Boeck B et al.; Glycogen from the thermophilic eubacterium Thermus thermophilus has been characterized by enzymatic, chemical and spectroscopic analysis . With an average chain length of seven glucose units, the glycogen from T . thermophilus is one of the most highly branched glycogens known . In contrast to other bacterial species, in T . thermophilus, accumulation of glycogen appears not be affected by low nitrogen concentration . For the first time, alpha-glucan phosphorylase activity and glycogen content were measured throughout the growth cycle of T . thermophilus in order to gain insight into glycogen metabolism . In contrast to the situation that prevails in Escherichia coli, additional carbon sources had no effect on alpha-glucan phosphorylase activity in T . thermophilus . Maximal activity of the thermophilic enzyme was found in the early logarithmic phase of growth, suggesting a function of the alpha-glucan phosphorylase in T . thermophilus as an outgrowth-specific enzyme. Appl Microbiol Biotechnol, 1998 Aug, 50(2), 174 - 80 Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction; Solaiman DK et al.; The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined . A putative open reading frame coding for a replication protein, Rep371, was identified . A characteristic promoter sequence and ribosome-binding site were found upstream of rep371 . Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S . thermophilus cryptic plasmids pST1-No.29 and pST1 respectively . The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89 . A novel 140-nucleotide palindromic minusorigin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371 . A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids . Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria. Acta Crystallogr D Biol Crystallogr, 1998 Jul 1, 54 ( Pt 4), 558 - 69 Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor refined at 2 A resolution; Song S et al.; The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor, South China sea lobster, was determined and refined at 2 A resolution to an R factor of 17.1% and reasonable stereochemistry . The structure refinement has not altered the overall structure of GAPDH from this lobster species . However, some local changes in conformation and the inclusion of ordered solvent model have resulted in a substantial improvement in the accuracy of the structure . Structure analysis reveals that the two subunits including NAD+ in the asymmetric unit are remarkably similar . The thermal differences between the two subunits found in some regions of the NAD+-binding domain may originate from different crystallographic environments rather than from an inherent molecular asymmetry . In this structure, the side chain of Arg194 does not point toward the active site but forms an ion pair with Asp293 from a neighboring subunit . Structural comparisons with other GAPDH's of known structure reveal that obvious contrast exists between mesophilic and thermophilic GAPDH mainly in the catalytic domain with significant conformational differences in the S-loop, beta7-strand and loop 120-125; the P-axis interface is more conserved than the R- and Q-axis interfaces and the catalytic domain is more conserved than the NAD+-binding domain . Some possible factors affecting the thermostability of this enzyme are tentatively analyzed by comparison with the highly refined structures of thermophilic enzymes. Biochem J, 1998 Oct 15, 335 ( Pt 2), 441 - 7 Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus; Faraone-Mennella MR et al.; A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogenate from Sulfolobus solfataricus by means of activity and immunoblot analyses, was purified to electrophoretic homogeneity by a rapid procedure including two sequential affinity chromatographies, on NAD+-agarose and DNA-Sepharose . The latter column selected specifically the poly(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15000-fold . The molecular mass (54-55 kDa) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein . The enzyme was proved to be thermophilic, with a temperature optimum of approx . 80 degreesC, and thermostable, with a half-life of 204 min at 80 degreesC, in good agreement with the requirements of a thermozyme . It displayed a Km towards NAD+ of 154+/-50 microM; in the pH range 6.5-10.0 the activity values were similar, not showing a real optimum pH . The enzyme was able to bind homologous DNA, as evidenced by the ethidium bromide displacement assay . The product of the ADP-ribosylating reaction co-migrated with the short oligomers of ADP-ribose (less than 6 residues) from a eukaryotic source . Reverse-phase HPLC analysis of the products, after digestion with phosphodiesterase I, gave an elution profile reproducing that obtained by the enzymic digestion of the rat testis poly(ADP-ribose) . These results strongly suggest that the activities of the purified enzyme include the elongation step. Appl Environ Microbiol, 1998 Oct, 64(10), 4062 - 4 Use of phospholipid fatty acids and carbon source utilization patterns To track microbial community succession in developing compost; Carpenter-Boggs L et al.; Carbon source utilization and phospholipid fatty acid analyses were used to track the rapidly changing microbial community in composting dairy waste . Microbial abilities to utilize common plant sugars increased during composting . Community phospholipid profiles changed significantly over time . Phospholipids suggested the presence of more thermophiles and fewer bacteria with continued compost development. Appl Environ Microbiol, 1998 Oct, 64(10), 3748 - 53 Capacity of nine thermostable DNA polymerases To mediate DNA amplification in the presence of PCR-inhibiting samples; Abu Al-Soud W et al.; The PCR is an extremely powerful method for detecting microorganisms . However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase from Thermus aquaticus by many components found in complex biological samples . In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases . Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases . The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors . The AmpliTaq Gold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, and Tfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity . The DNA polymerase from Thermotoga maritima (Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples . When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTth from Thermus thermophilus were the most resistant . Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase. Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1032 - 4 Crystallization and preliminary X-ray characterization of aspartate aminotransferase from an extreme thermophile, Thermus thermophilus HB8; Nakai T et al.; Recombinant aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8, has been crystallized in two different crystal forms . The crystals of both forms are orthorhombic and belong to space group P212121 with cell dimensions a = 124.3, b = 113.6 and c = 61.6 A for form I and a = 197.3, b = 109.7 and c = 80.3 A for form II . The crystals of form I and II diffract to 2.1 and 2.5 A resolution, respectively, on a conventional laboratory rotating-anode source . Two heavy-atom derivatives have been identified for form I. Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1002 - 4 Crystallization and preliminary X-ray diffraction studies of DNA polymerase from the thermophilic archaeon Sulfolobus solfataricus; Nastopoulos V et al.; The thermophilic and thermostable family B DNA polymerase from the archaeon Sulfolobus solfataricus (Mr of about 100 kDa) has been crystallized by the hanging-drop vapour-diffusion method at 294 K using ammonium sulfate as precipitant . The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution on a rotating-anode X-ray source . Native data have been collected at 100 K . A heavy-atom derivative search is in progress. Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1539 - 45 Thermostable beta-galactosidase from an extreme thermophile, Thermus sp . A4: enzyme purification and characterization, and gene cloning and sequencing; Ohtsu N et al.; We purified and characterized a thermophilic beta-galactosidase from Thermus sp . A4 isolated from the Atagawa hot spring (Shizuoka, Japan) . The enzyme was monomeric, and its molecular mass was estimated to be 75 kDa by SDS-polyacrylamide gel electrophoresis . The enzyme was extremely thermostable and retained its full activity after incubation at 70 degrees C for 20 h . The Km observed were 5.9 mM for ortho-nitrophenyl beta-D-galactopyranoside and 19 mM for lactose . We cloned and analyzed the complete sequence of the gene encoding this enzyme . It was found to consist of 645 amino acid residues . We propose that this enzyme and seven other unclassified beta-galactosidases are new members of family 42 of the glycosyl hydrolases. J Biol Chem, 1998 Oct 9, 273(41), 26462 - 9 Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila; Becker DF et al.; An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and Mossbauer spectroscopy . The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V . No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M . thermophila . Isf contained a {4Fe-4S}2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82 . The unusual morphology was attributed to microheterogeneity among Isf molecules . The Em value for the 2+/1+ redox couple of the cluster was -0.394 V . Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M . thermophila Isf . In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M . thermoautotrophicum and Methanococcus jannaschii . These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea . It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes. Genetics, 1998 Oct, 150(2), 643 - 50 Tetrahymena mutants with short telomeres; Ahmed S et al.; Telomere length is dynamic in many organisms . Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo . In Tetrahymena thermophila, telomeres become long at 30 degrees, and growth rate slows . A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate . Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation . One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells . Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed . These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation. Nucleic Acids Res, 1998 Oct 15, 26(20), 4657 - 61 Creation of genetic information by DNA polymerase of the thermophilic bacterium Thermus thermophilus; Ogata N et al.; Genetic information encoded in a template of a genome is replicated in a complementary way by DNA polymerase or RNA polymerase with high fidelity; no creation of information occurs in this reaction unless an error occurs . We report here that DNA polymerase of the thermophilic bacterium Thermus thermophilus can synthesize up to 200 kb linear double-stranded DNA in vitro in the complete absence of added primer and template DNAs, indicating that genetic information is actively created by protein . This ab initio DNA synthesis occurs at 74 degrees C and requires magnesium ion . There is a lag time of approximately 1 h and then the reaction proceeds linearly . The synthesized DNAs have a variety of sequences; they are mostly tandem repetitive sequences, e.g . (CATGTATA) n , (TGTATGTATACATACATA) n and (TATACGTA) n . Some degenerate sequences of these basic repeat units are also found . The similar repetitive sequences are found in many natural genes . These results, together with similar results found using DNA polymerase of archaeon Thermococcus litoralis , suggest that creative, non-replicative synthesis of DNA by protein was a driving force for diversification of genetic information at a certain stage of the evolution of life on the early earth. Nucleic Acids Res, 1998 Oct 15, 26(20), 4635 - 44 Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species; Yue M et al.; The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization . The T.thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T.pyriformis , including type I elements, the only known determinant for replication control . To assess the role of conserved sequences in replication control, structural and functional studies were performed on T.pyriformis rDNA . Similar to T.thermophila , replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment . Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T.thermophila rDNA . An in vitro type I element binding activity indistinguishable from the T.thermophila protein, ssA-TIBF, was detected in T.pyriformis extracts . The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries . Despite these similarities, the T.pyriformis 5' NTS failed to support replication in transformed T.thermophila cells, suggesting a more complex genetic organization than previously realized. Nucleic Acids Res, 1998 Oct 15, 26(20), 4566 - 73 Position effect takes precedence over target sequence in determination of adenine methylation patterns in the nuclear genome of a eukaryote, Tetrahymena thermophila; Karrer KM et al.; Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N 6-methyladenine . DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines . In vivo, modification of adenine residues appears to occur exclusively in the sequence 5'-NAT-3', but no consensus sequence for modified sites has been found . In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA . In the novel location on the rDNA minichromosome, the site was unmethylated . The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication . The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation. Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11555 - 60 RNA folding causes secondary structure rearrangement; Wu M et al.; The secondary structure of the P5abc subdomain (a 56-nt RNA) of the Tetrahymena thermophila group I intron ribozyme has been determined by NMR . Its base pairing in aqueous solution in the absence of magnesium ions is significantly different from the RNA in a crystal but is consistent with thermodynamic predictions . On addition of magnesium ions, the RNA folds into a tertiary structure with greatly changed base pairing consistent with the crystal structure: three Watson-Crick base pairs, three G.U base pairs, and an extra-stable tetraloop are lost . The common assumption that RNA folds by first forming secondary structure and then forming tertiary interactions from the unpaired bases is not always correct. Biochemistry, 1998 Sep 29, 37(39), 13475 - 85 Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies; Gruber K et al.; The crystal structure of the thermostable xylanase from Thermomyces lanuginosus was determined by single-crystal X-ray diffraction . The protein crystallizes in space group P21, a = 40.96(4) A, b = 52 . 57(5) A, c = 50.47 (5) A, beta = 100.43(5) degrees, Z = 2 . Diffraction data were collected at room temperature for a resolution range of 25-1.55 A, and the structure was solved by molecular replacement with the coordinates of xylanase II from Trichoderma reesei as a search model and refined to a crystallographic R-factor of 0.155 for all observed reflections . The enzyme belongs to the family 11 of glycosyl hydrolases {Henrissat, B., and Bairoch, A . (1993) Biochem . J . 293, 781-788} . pKa calculations were performed to assess the protonation state of residues relevant for catalysis and enzyme stability, and a heptaxylan was fitted into the active-site groove by homology modeling, using the published crystal structure of a complex between the Bacillus circulans xylanase and a xylotetraose . Molecular dynamics indicated the central three sugar rings to be tightly bound, whereas the peripheral ones can assume different orientations and conformations, suggesting that the enzyme might also accept xylan chains which are branched at these positions . The reasons for the thermostability of the T . lanuginosus xylanase were analyzed by comparing its crystal structure with known structures of mesophilic family 11 xylanases . It appears that the thermostability is due to the presence of an extra disulfide bridge, as well as to an increase in the density of charged residues throughout the protein. J Appl Microbiol, 1998 Aug, 85(2), 224 - 30 The colonization of turkeys by thermophilic campylobacters; Wallace JS et al.; The rate at which five broods of turkey chicks became colonized by thermophilic campylobacters was investigated . Day-old chicks were normally free of campylobacters on arrival on the farm with colonization beginning within 7 d . The carriage rate was 100% by day 14 in three of the broods and by day 21 in the other two . Higher carriage rates were obtained with enrichment procedures than with direct plating . Two broods were investigated over an extended interval for the number of campylobacters shed in their faeces . In brood A, Campylobacters increased from day l to day 14 concomitant with increase in the carriage rate and in the number of chicks with diarrhoea . By day 39, when the birds were sold to other farms, the excretion rate had reached 6 x 10(7) campylobacters g-1 fresh faeces . Brood B . was monitored over 91 d and showed peaks in Campylobacter numbers on days 19 and 75, corresponding to peaks in the number of diarrhoeic samples . The introduction of new birds into the brood resulted in an increase in the Campylobacter population and in the number of birds with diarrhoea . Campylobacter jejuni was the only species isolated but comprised several different biotypes . Analysis of the number of campylobacters at different sites along the gastrointestinal tract of mature turkeys at slaughter showed that numbers increased with distance from the beak and were highest in the caeca. J Appl Microbiol, 1998 Sep, 85(3), 472 - 80 The seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves; Stanley KN et al.; The epidemiology of clinical cases of campylobacter in temperate climates shows a striking seasonality . In the search for a seasonal environmental reservoir changes in the carriage rate and population size of campylobacters in bovine hosts with time have been measured . Most probable number (MPN) methodology was used to enumerate thermophilic campylobacters in samples taken from the small intestines of beef cattle at slaughter and the fresh faeces of four dairy herds and new-born calves . Statistical analyses revealed significant evidence for seasonal periodicity in the data from dairy herds (P = 0.044) . Not only was there a departure from constancy within a 12-month interval but these data revealed a true seasonality, that is, the same periodicity in numbers from one year to the next . Each herd had two peaks per year, in approximately spring and autumn . Peaks coincided in herds on neighbouring farms but those on farms in the north preceded those on farms in the south by 2 and 1 months, respectively (P = 0.0057) . Intestinal carriage by beef cattle at slaughter was 89.4% (n = 360) with an average MPN campylobacters per gram fresh weight (MPN gfw-1) of 6.1 x 10(2) . Average MPN gfw-1 in faeces from the dairy herds and calves were 69.9 (S.D . 3) and 3.3 x 10(4) (S.D . 1.7 x 10(2)) . There was no evidence of seasonal periodicity in the size of the campylobacter population in beef cattle at slaughter . Calves were campylobacter free at birth but became colonized with a few days. J Biol Chem, 1998 Oct 2, 273(40), 25721 - 7 Substrate recognition of tRNA (Guanosine-2'-)-methyltransferase from Thermus thermophilus HB27; Hori H et al.; Transfer RNA (guanosine-2'-)-methyltransferase (Gm-methylase, EC 2.1 . 1.32) from Thermus thermophilus HB27 is one of the tRNA ribose modification enzymes . The broad substrate specificity of Gm-methylase has so far been elucidated using various species of tRNAs from native sources, suggesting that the common structures in tRNAs are recognized by the enzyme . In this study, by using 28 yeast tRNAPhe variants obtained by transcription with T7 RNA polymerase, it was revealed that the nucleotide residues G18 and G19 and the D-stem structure are essentially required for Gm-methylase recognition, and that the key sequence for the substrate is pyrimidine (Py)17G18G19 . The other conserved sequences were found not to be essential, but U8, G15, G26, G46, U54, U55, and C56 considerably affected the methylation efficiency . These residues are located within a limited space embedded in the L-shaped three-dimensional structure of tRNA . Therefore, disruption of the three-dimensional structure of the substrate tRNA is necessary for the catalytic center of Gm-methylase to be able to access the target site in the tRNA, suggesting that the interaction of Gm-methylase with tRNA consists of multiple steps . This postulation was confirmed by inhibition experiments using nonsubstrate tRNA variants which functioned as competitive inhibitors against usual substrate tRNAs. Eur J Biochem, 1998 Aug 15, 256(1), 136 - 41 Studies on the Zn-containing S14 ribosomal protein from Thermus thermophilus; Tsiboli P et al.; The S14 ribosomal protein from the thermophilic organism Thermus thermophilus, which contains a zinc-finger-like motif, namely -C-X2-C-X12-C-X2-C- {Tsiboli, P . & Choli, T . (1995) Biol . Chem . Hoppe-Seyler 376, 127-130}, has been overproduced, purified and investigated for its zinc content . According to atomic absorption experiments, the protein contains zinc at a molar ratio of one . Denaturation experiments with simultaneous use of denaturing and chelating agents (guanidine hydrochloride and EDTA), as well as renaturation experiments, have shown both that Zn is strongly bound to the protein and with 1:1 Zn/protein stoicheiometry . These findings provide very strong evidence in support of the participation of the zinc-finger motif and the Zn in the formation of a zinc-finger domain. Eur J Biochem, 1998 Aug 15, 256(1), 97 - 105 Interaction of ribosomal L1 proteins from mesophilic and thermophilic Archaea and Bacteria with specific L1-binding sites on 23S rRNA and mRNA; Kohrer C et al.; In Bacteria and Archaea (formerly Archaebacteria) ribosomal protein L1 has a dual function, as a primary rRNA-binding protein and as a translational repressor which binds to its own mRNA . The L1-binding site on the mRNA exhibits high similarity in both sequence and secondary structure to the binding site for L1 on the 23 S rRNA . A sensitive membrane-filter-binding assay has been used to examine the interactions between ribosomal L1 proteins from different archaeal and bacterial species, and 23S rRNA and mRNA fragments from Methanococcus vannielii containing the MvaL1-binding site . Under standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 500 mM KCl), the apparent dissociation constant Kd of the homologous MvaL1-23S rRNA complex is 5 nM, the apparent dissociation constant Kd of the MvaL1-mRNA complex is 0.15 degrees M . L1 proteins from Escherichia coli (EcoL1) and from the thermophilic Bacterium Thermus thermophilus (TthL1), and from the thermophilic Archaea Methanococcus thermolithotrophicus (MthL1), Methanococcus jannaschii (MjaL1), and Sulfolobus solfataricus (SsoL1) were tested for their affinity to the specific L1-binding sites on the 23 S rRNA and mRNA . In general, the affinity of L1 proteins from thermophilic species to the binding sites on both 23 S rRNA and mRNA is about one order of magnitude higher than that of their mesophilic counterparts . This stronger protein-RNA interaction might make a substantial contribution to the thermal tolerance of ribosomes in thermophilic organisms. Rev Argent Microbiol, 1998 Apr-Jun, 30(2), 73 - 8 {Chitinase production by a strain of Streptomyces griseoruber isolated from the rhizosphere of sugar cane}; Carrillo L et al.; Twenty nine mesophilic and 19 thermophilic actinomycetes from sugar cane phyllosphere, rhizosphere and root-free soil were isolated . Twelve mesophilic and 7 thermophilic strains produced clear zones of hydrolyses on colloidal chitin agar and hydrolysed a chitin colloidal suspension . The rhizosphere strains were significantly more chitinolytic (p < 0.05) than the root-free soil strains . Streptomyces griseoruber 202 produced chitinase in colloidal chitin liquid medium with an activity of 108 micrograms of colloidal chitin hydrolysed/mg protein/hour, at pH 4.6 and 40 degrees C . This strain also produced chitinase in a medium with insect exoskeleton powder. Free Radic Biol Med, 1998 Sep, 25(4-5), 473 - 9 A practical assay of lipoate in biologic fluids and liver in health and disease; Baker H et al.; A procedure for assaying lipoic acid concentration in biologic fluids and tissues was devised using a eukaryotic protozoan Tetrahymena thermophila . T.thermophila has a specific and sensitive (30 pg/ml) requirement for lipoic acid . Unlike humans and other microorganisms, T.thermophila can not synthesize lipoic acid; hence, its requirement for exogenous lipoic acid is specific . The lipoic acid supplied to T . thermophila by the processing of biologic fluids and tissues during the assay procedure, permits the derivation of a practical assay for lipoate concentration as described here . Lipoate concentration in biologic fluids and tissue obtained from healthy humans, compared to those obtained from patients with renal and liver disease, indicate deviations from normal during disease . Absorption chartings of 200 mg of DL-alpha-lipoic acid in humans indicate a peak concentration of lipoate in plasma 2 h after ingestion and then a steady descent of lipoate to a baseline level after 24 h . With this practical assay, it is now possible to chart lipoate's antioxidant activity and therapeutic action during health and disease. Syst Appl Microbiol, 1998 Mar, 21(1), 151 - 61 Characterization of bacteriocins produced by strains from traditional Bulgarian dairy products; Miteva V et al.; A result of extensive screening of over 300 strains from the Collection of ELBY Bulgaricum, PLC, thirty six strains were selected as producers of bacteriocins, active closely related lactic acid bacterial species and some food spoilage bacteria . The selected strains belong to L . helveticus, L . bulgaricus and S . thermophilus, which are rare bacteriocin producers . Nineteen nonidentified producers were characterized by molecular taxonomic approaches--M13 fingerprinting, repetitive PCR, ribotyping and hybridization with species-specific probes, which allowed to affiliate them to the species L . delbrueckii . Several strains were found to harbour plasmids of different size . The estimated activity against food borne pathogens makes the isolated substances perspective as safe food preservatives and the producing strains could be used as components of starters with improved quality. Syst Appl Microbiol, 1998 Mar, 21(1), 12 - 22 F-and V-ATPases in the genus Thermus and related species; Radax C et al.; The discovery of a V-type ATPase in the gram-negative bacterium Thermus thermophilus HB8 (YOKOYAMA et al., J . Biol . Chem . 265, 21946, 1990) was unexpected, since only eukaryotic endomembranes and archaea were thought to contain this enzyme complex, and horizontal gene transfer was suggested to explain the finding . We examined membrane-associated ATPases from representatives of several groups of the genus Thermus . The enzymes were extracted with chloroform and purified by ion exchange chromatography or native gel electrophoresis . One novel Islandic isolate, T . scotoductus SE-1, as well as strain T . filiformis from New Zealand, possessed F-ATPases, as judged by the typical five subunit composition of the F1-moiety, sensitivity to azide, insensitivity to nitrate and a strong crossreaction with antibodies against the F1-ATPase from E . coli . In addition, N-terminal amino acid sequencing of the beta subunit from T . scotoductus SE-1 confirmed its homology with beta subunits from known F-ATPases . In contrast, the same extraction procedure released a V-ATPase from the membranes of T . thermophilus HB27 and T . aquaticus YT-1 . The related species Meiothermus (formerly Thermus) chliarophilus ALT-8 also possessed a V-ATPase . All V-ATPases examined in this study contained larger major subunits than F-ATPases, crossreacted with antiserum against subunit A of the V-ATPase from the archaeon Halobacterium saccharovorum, and the N-terminal sequences of their major subunits were homologous to those of other V-ATPases . Sequences of the 16S rRNA gene clearly placed T . scotoductus SE-1, along with other non-pigmented Thermus strains, as a distinct species close to T . aquaticus . Our results suggested that at least two members of the genus, T . scotoductus SE-1 and T . filiformis, contain an F-ATPase, whereas several others possess a V-ATPase . These data could indicate a greater diversity of the genus Thermus than was previously thought . Alternatively, the genus may consist of species where horizontal gene transfer has occurred and others, where it has not. FEBS Lett, 1998 Aug 28, 434(1-2), 205 - 8 An intact conformation at the tip of elongation factor G domain IV is functionally important; Martemyanov KA et al.; Three variants of Thermus thermophilus EF-G with mutations in the loop at the distal end of its domain IV were obtained . The replacement of His-573 by Ala and double mutation H573A/D576A did not influence the functional activity of EF-G . On the other hand, the insertion of six amino acids into the loop between residues Asp-576 and Ser-577 reduced the translocational activity of EF-G markedly, while its GTPase activity was not affected . It is concluded that the native conformation of the loop is important for the factor-promoted translocation in the ribosome . The functional importance of the entire EF-G domain IV is discussed. FEBS Lett, 1998 Aug 28, 434(1-2), 17 - 22 Electrical current generation and proton pumping catalyzed by the ba3-type cytochrome c oxidase from Thermus thermophilus; Kannt A et al.; Several amino acid residues that have been shown to be essential for proton transfer in most cytochrome c oxidases are not conserved in the ba3-type cytochrome c oxidase from the thermophilic eubacterium Thermus thermophilus . So far, it has been unclear whether the Th . thermophilus ba3-type cytochrome c oxidase can nevertheless function as an electrogenic proton pump . In this study, we have combined charge translocation measurements on a lipid bilayer with two independent methods of proton pumping measurements to show that enzymatic turnover of the Th . thermophilus cytochrome c oxidase is indeed coupled to the generation of an electrocurrent and proton pumping across the membrane . In addition to a 'vectorial' consumption of 1.0 H+/e- for water formation, proton pumping with a stoichiometry of 0.4-0.5 H+/e- was observed . The implications of these findings for the mechanism of redox-coupled proton transfer in this unusual cytochrome c oxidase are discussed. Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 1007 - 13 Petrotoga mobilis sp . nov., from a North Sea oil-production well; Lien T et al.; Rod-shaped, thermophilic bacteria with a sheath-like outer structure (toga) were isolated from hot oilfield water of a North Sea oil reservoir . One of the isolates, designated