Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 467 - 472 Thiobacter subterraneus gen . nov., sp . nov., an obligately chemolithoautotrophic, thermophilic, sulfur-oxidizing bacterium from a subsurface hot aquifer; Hirayama H et al.; A novel, thermophilic, obligately chemolithoautotrophic, sulfur/thiosulfate-oxidizing bacterium was isolated from subsurface geothermal aquifer water (temperature approximately 70 degrees C) in the Hishikari gold mine, Japan . Cells of the isolate, designated strain C55(T), were motile, straight rods with a single polar flagellum . Growth was observed at temperatures between 35 and 62 degrees C (optimum 50-55 degrees C; 60 min doubling time) and pH between 5.2 and 7.7 (optimum pH 6.5-7.0) . High growth rate of strain C55(T) was observed on either thiosulfate or elemental sulfur as a sole energy source, with molecular oxygen as the only electron acceptor . None of the organic compounds tested supported or stimulated growth of strain C55(T) . The G+C content of the genomic DNA was 66.9 mol% . Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C55(T) was affiliated to the beta-Proteobacteria, but was distantly related to recognized genera . On the basis of its physiological and molecular properties, strain C55(T) (=JCM12421(T)=DSM 16629(T)=ATCC BAA-941(T)) is proposed as the type strain of Thiobacter subterraneus gen . nov., sp . nov. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 385 - 389 Silanimonas lenta gen . nov., sp . nov., a slightly thermophilic and alkaliphilic gammaproteobacterium isolated from a hot spring; Lee EM et al.; A moderately thermophilic aerobic bacterium, strain 25-4(T), was isolated from a hot spring at Baekdoo Mountain in Korea . The cells were Gram-negative, motile rods each having a polar flagellum . Analysis of the 16S rRNA gene sequence indicated that the strain represented a new lineage within the family 'Xanthomonadaceae' of the 'Gammaproteobacteria', being most closely related to the genera Thermomonas, Xanthomonas, Luteimonas, Pseudoxanthomonas, Stenotrophomonas and Xylella and having 16S rRNA gene sequence similarities to the most related species of the genera of between 92.9 and 94.4 % . The strain contained Q-8 as the major isoprenoid quinone and had a fatty acid profile with predominant iso-branched fatty acids . Growth occurred at pH 6.0-10, with an optimum at pH 9.0, and at 25-53 degrees C, with an optimum at 47 degrees C . The G+C content of the genomic DNA was 50.7 mol% . On the basis of phylogenetic analyses and its phenotypic characteristics, strain 25-4(T) belongs to a new genus, Silanimonas gen . nov., within the 'Gammaproteobacteria' . The sole species of this genus is Silanimonas lenta sp . nov . (type strain, 25-4(T)=DSM 16282(T)=KCTC 12236(T)). Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 183 - 189 Lebetimonas acidiphila gen . nov., sp . nov., a novel thermophilic, acidophilic, hydrogen-oxidizing chemolithoautotroph within the 'Epsilonproteobacteria', isolated from a deep-sea hydrothermal fumarole in the Mariana Arc; Takai K et al.; A novel thermophilic, acidophilic bacterium, designated strain Pd55(T), was isolated from a self-temperature-recording in situ colonization system deployed in a hydrothermal diffusing flow (maximum temperature of 78 degrees C) at the TOTO caldera in the Mariana Arc . Cells of strain Pd55(T) were motile, short rods with a single polar flagellum . Growth was observed between 30 and 68 degrees C (optimum growth at 50 degrees C; 120 min doubling time) and between (initial) pH 4.2 and 7.0 (optimum at pH 5.2) . The isolate was a strictly anaerobic chemolithoautotroph capable of using molecular hydrogen as sole energy source and carbon dioxide as sole carbon source . Elemental sulfur served as the sole electron acceptor to support growth . The G+C content of the genomic DNA was 34.0 mol% . Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate was related to members of the genera Nautilia and Caminibacter, although it appeared to be a novel lineage prior to the divergence between Nautilia and Caminibacter . Strain Pd55(T) could also be differentiated from Nautilia and Caminibacter species on the basis of its physiological properties . It is, therefore, proposed that strain Pd55(T) (=JCM 12420(T)=DSM 16356(T)) represents the type strain of a novel species in a new genus, Lebetimonas acidiphila gen . nov., sp . nov. Plant Cell Physiol, 2004 Dec, 45(12), 1809 - 1816 PSII-Tc Protein Plays an Important Role in Dimerization of Photosystem II; Iwai M et al.; We cloned and determined the nucleotide sequence of PSII genes, psbB and psbTc, from the thermophilic cyanobacterium, Thermosynechococcus elongatus strain BP-1 . PSII-Tc, encoded by psbTc, is a small membrane-spanning subunit of the PSII core complex of cyanobacteria and plants . However, its role has not been fully elucidated . We generated an insertional disruptant of psbTc and studied the role of the PSII-Tc protein in cyanobacterial PSII . The following observations were made: (i) The psbTc disruptant could grow photoautotrophically at a rate similar to that of wild-type T . elongatus under a wide range of light conditions . (ii) Thylakoids and oxygen-evolving PSII complexes were successfully isolated from the psbTc disruptant as well as the wild type . There was no significant difference in the oxygen evolution activities of cells, thylakoids or PSII complexes between the psbTc disruptant and the wild type . This is in contrast to the lower activities in the other PSII mutants of T . elongatus . (iii) Chromatographic separation of monomeric and dimeric PSII revealed that recovery of dimeric PSII was dramatically reduced in the psbTc disruptant . (iv) SDS-urea-PAGE showed a complete loss of the 4.7-kDa band in the mutant PSII . Since this band in wild-type PSII consists of PSII-M and PSII-Tc, we assume that PSII-Tc is critical for the binding of PSII-M in the PSII complex and is involved directly and indirectly in the dimerization of PSII . These results appear to be in good agreement with the recent structural model of the dimeric PSII complex. FEMS Microbiol Rev, 2005 Jan, 29(1), 3 - 23 Xylanases, xylanase families and extremophilic xylanases; Collins T et al.; Xylanases are hydrolytic enzymes which randomly cleave the beta 1,4 backbone of the complex plant cell wall polysaccharide xylan . Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics . Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent . Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases . Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed . The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented. J Theor Biol, 2005 Apr 7, 233(3), 351 - 362 Epub 2004 Nov 30. Unifying temperature effects on the growth rate of bacteria and the stability of globular proteins; Ratkowsky DA et al.; The specific growth rate constant for bacterial growth does not obey the Arrhenius-type kinetics displayed by simple chemical reactions . Instead, for bacteria, steep convex curves are observed on an Arrhenius plot at the low- and high-temperature ends of the biokinetic range, with a region towards the middle of the growth range loosely approximating linearity . This central region has been considered by microbiologists to be the "normal physiological range" for bacterial growth, a concept whose meaningfulness we now question . We employ a kinetic model incorporating thermodynamic terms for temperature-induced enzyme denaturation, central to which is a term to account for the large positive heat capacity change during unfolding of the proteins within the bacteria . It is now widely believed by biophysicists that denaturation of complex proteins and/or other macromolecules is due to hydrophobic hydration of non-polar compounds . Denaturation is seen as the process by which enthalpic and entropic forces becomes imbalanced both at high and at low temperatures resulting in conformational changes in the enzyme structure that expose hydrophobic amino acid groups to the surrounding water molecules . The "thermodynamic" rate model, incorporating the heat capacity change and its effect on the enthalpy and entropy of the system, fitted 35 sets of data for psychrophilic, psychrotrophic, mesophilic and thermophilic bacteria well, resulting in biologically meaningful estimates for the important thermodynamic parameters . As these results mirror those obtained by biophysicists for globular proteins, it appears that the same or a similar mechanism applies to bacteria as applies to proteins. Biochem J . 2005 Jan 14; {Epub ahead of print} Evidence for cooperativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: fluorescence, kinetic and structural studies of the wild type enzyme and non cooperative N249Y mutant; Giordano A et al.; The interaction of coenzyme with thermostable homotetrameric NAD(H)-dependent alcohol dehydrogenase from the thermoacidophilic sulphur-dependent crenarchaeon Sulfolobus solfataricus (SsADH) and its N249Y (Asn ->249Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophans Trp-95 and Trp-117 to the binding of coenzyme moieties . Fluorescence quenching studies performed at 25 oC show that SsADH exhibits linearity in the NAD(H) binding (the Hill coefficient h close to 1) at pH 9.8 and at moderate ionic strength, in addition to positive cooperativity (h = 2.0-2.4) at pH 7.8 and 6.8, and at pH 9.8 in the presence of salt . Furthermore, NADH binding is positively cooperative below 20 oC (h close to 3) and negatively cooperative at 40-50 oC (h close to 0.7), as determined at moderate ionic strength and pH 9.8 . Steady-state kinetic measurements show that SsADH displays standard Michaelis-Menten kinetics between 35 and 45 oC, but exhibits positive and negative cooperativity for NADH oxidation below (h = 3.3 at 20 oC) and above (h = 0.7 at 70-80 oC) this range of temperatures, respectively . However, N249Y SsADH displays non-cooperative behaviour in coenzyme binding under the same experimental conditions used for the wild type enzyme . In loop 270-275 of the coenzyme domain and segments at the interface of dimer A/B, analyses of the wild type and mutant SsADH structures identified the structural elements involved in the intersubunit communication and suggested a possible structural basis for cooperativity . This is the first report of cooperativity in a tetrameric alcohol dehydrogenase and of temperature-induced cooperativity in a thermophilic enzyme. J Microbiol Methods, 2005 Mar, 60(3), 299 - 313 Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis; Johnson DB et al.; Acidophilic iron-oxidizing microorganisms are important both environmentally and in biotechnological applications . Although, as a group, they are readily detected by their ability to generate ferric iron (resulting in a distinctive color change in liquid media), these microbes highly diverse phylogenetically . Various other characteristics, such as optimum growth temperature, response to organic carbon sources, and cellular morphologies, facilitate, in some cases, identification of isolates to a genus or species level, although this approach has limitations and may give erroneous results . In this study, a combined approach of using physiological traits together with amplified ribosomal DNA restriction enzyme analysis (ARDREA) has been successful in identifying all known acidophilic iron-oxidizing bacteria to the species level . Computer-generated maps were used to identify restriction enzymes that allow the differentiation of the acidophiles, and these were confirmed experimentally using authentic bacterial strains . To test further the validity of this approach, six acidophilic moderately thermophilic iron-oxidizing bacteria isolated from Montserrat (West Indies) were analysed using the ARDREA protocol . Three of the isolates were identified as Sulfobacillus acidophilus-like, and one as Sulfobacillus thermosulfidooxidans-like bacteria . The fifth isolate gave DNA digest patterns that were distinct from all known strains of iron-oxidizing acidophiles . Subsequent sequencing of the 16S rRNA genes of these isolates confirmed the identity of the four Sulfobacillus isolates, and also that the fifth isolate was a novel species . Schematic diagrams showing how ARDREA may be used to rapidly identify all known acidophilic iron-oxidizing bacteria are presented. Br J Biomed Sci, 2004, 61(4), 186 - 9 Phenotypic characterisation of flagellin and flagella of urease-positive thermophilic campylobacters; Sekizuka T et al.; In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C . coli and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) . Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894) . Flagellin components, each consisting of a single peptide, with a heterogeneous molecular mass of approximately 52-63 kDa were demonstrated in the other four UPTC isolates (NCTC12892, NCTC12895, NCTC12896 and NI15F {from Northern Ireland}) and the two Japanese isolates of C . jejuni (JCM2013 and C . coli 27) . The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C . jejuni and C . coli . Flagella were not detected by electron microscopy in the four flagellin-negative UPTC isolates but they were detected in the four flagellin-positive UPTC isolates and the two isolates of C . jejuni and C . coli . Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates. Proteins . 2005 Jan 12; {Epub ahead of print} Crystal structure of TT1662 from Thermus thermophilus HB8: A member of the alpha/beta hydrolase fold enzymes; Murayama K et al.; No abstract. Extremophiles . 2005 Jan 13; {Epub ahead of print} Gene replacement of adenylate kinase in the gram-positive thermophile Geobacillus stearothermophilus disrupts adenine nucleotide homeostasis and reduces cell viability; Counago R et al.; Thermophilic bacteria are of great value for industry and research communities . Unfortunately, the cellular processes and mechanisms of these organisms remain largely understudied . In the present study, we investigate how the inactivation of adenylate kinase (AK) affects the adenine nucleotide homeostasis of a gram-positive moderate thermophile, Geobacillus stearothermophilus strain NUB3621-R . AK plays a major role in the adenine nucleotide homeostasis of living cells and has been shown to be essential for the gram-negative mesophile Escherichia coli . To study the role of AK in the maintenance of adenylate energy charge (EC) and cell viability of G . stearothermophilus, we generated a recombinant strain of this organism in which its endogenous gene coding for the essential protein adenylate kinase (AK) has been replaced with the adk gene from the mesophile Bacillus subtilis . PCR, DNA sequencing and Southern analysis were performed to confirm proper gene replacement and preservation of neighboring genes . The highest growing temperature for recombinant cells was almost 20 degrees C lower than for wild-type cells (56 vs . 75 degrees C) . This temperature-sensitive phenotype was secondary to heat inactivation of B . subtilis AK, as evidenced by enzyme activity assays and EC measurements . At higher temperatures (65 degrees C), recombinant cells also had lower EC values (0.09) compared to wild-type cells (0.45), which reflects a disruption of adenine nucleotide homeostasis following AK inactivation. J Biol Chem . 2005 Jan 12; {Epub ahead of print} Structural and kinetic analyses of arginine residues in the active-site of the acetate kinase from methanosarcina thermophila; Gorrell A et al.; Acetate kinase catalyzes transfer of the gamma phosphate of ATP to acetate . The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K . A., et al . (2001) J Bacteriol 193, 680 - 686) . Here we report two new crystal structure of the M . thermophila enzyme in the presence of substrate and transition state analogs . The enzyme co-crystallized with the ATP analog adenosine 5'-{gamma-thio}triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B . The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B . Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state . Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B suggesting that domain movement contributes to catalysis . The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating this residue is essential for catalysis . Residue Arg(241) was also found adjacent to AlF(3) consistent with a role for stabilization of the transition state . Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis, and also indicated a role in binding acetate. Arch Microbiol . 2005 Jan 12; {Epub ahead of print} Diverse dextranase genes from Paenibacillus species; Finnegan PM et al.; Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2 . Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2 . The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted M(r) of 80.8 kDa . The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted M(r) of 100.1 kDa and 68.3 kDa, respectively . The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides . Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11) . The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins . Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus . The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11) . The reaction catalysed by each enzyme had a distinct temperature and pH dependence. Lett Appl Microbiol, 2005, 40(2), 111 - 6 Production of large multienzyme complex by aerobic thermophilic fungus Chaetomium sp . nov . MS-017 grown on palm oil mill fibre; Ohtsuki T et al.; Abstract t . ohtsuki, suyanto, s . yazaki, s . ui and a . mimura . 2004.Aims: A novel xylanolytic multienzyme complex of the aerobic thermophilic fungus Chaetomium sp . nov . MS-017 was produced on palm oil mill fibre (POMF) and partially characterized . Methods and Results: The assay of the extracellular enzymes of Chaetomium sp . nov . MS-017 on POMF in solid-state fermentation revealed cellulolytic, pectinolytic and extremely high xylanolytic activities . The protein was purified by Sephadex G-200 column chromatography . The SDS-PAGE demonstrated that the purified protein is a complex with at least five xylanolytic, four cellulolytic and eight pectinolytic components . The characterization of the complex at various temperatures showed that the reactivity and stability of the complex are not lost up to 60 degrees C . In addition, the complex was very stable in a wide range of pH (3-9) and at high concentrations (10 mm) of cations and EDTA . The major products of xylan hydrolysis by the purified complex were determined to be xylobiose and xylotriose by thin-layer chromatography . Conclusion: Chaetomium sp . nov . MS-017 preferentially produces a xylanolytic multienzyme complex on POMF in solid-state fermentation . Significance and Impact of the Study: This is the first report on the xylanolytic multienzyme complex produced by an aerobic thermophilic fungus. Environ Microbiol, 2005 Jan, 7(1), 118 - 132 Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments; Nercessian O et al.; Summary To contribute to the identification of methanogens, methanotrophs and sulfate-reducing bacteria (SRB) in microbial communities from the 13 degrees N (East Pacific Rise) and Rainbow (Mid-Atlantic Ridge) hydrothermal vent fields, we investigated the diversity of mcrA, pmoA and dsrAB genes sequences . Clone libraries were obtained using DNA isolated from fragments of diffuse vents, sediment and in situ samplers . The clones were categorized by restriction fragment length polymorphism, and representatives of each group were sequenced . Sequences were related to that of hyperthermophilic (order Methanopyrales and family Methanocaldococcaceae), thermophilic and mesophilic (family Methanococcaceae) methanogens, thermophilic (proposed genus 'Methylothermus') and mesophilic type I methanotrophs, and hyperthermophilic (order Archaeoglobales), thermophilic (order Thermodesulfobacteriales) and mesophilic (family Desulfobulbaceae) SRB . Several of the obtained sequences were distantly related to the genes of cultivated organisms, providing evidence of the existence of novel lineages in the three functional groups . This study provides for the first time an insight into the diversity of several functional genes of deep-sea hydrothermal system microorganisms. Environ Microbiol, 2005 Jan, 7(1), 66 - 77 Lipid biomolecules in silica sinters: indicators of microbial biodiversity; Pancost RD et al.; Summary To explore further the diversity of the microorganisms and their relationship with geothermal sinters, we examined the lipids preserved in six sinters associated with four different hot spring (58-82 degrees C) areas of the Taupo Volcanic Zone (TVZ), New Zealand . These sinters contain microbial remains, but the process of mineralization has rendered them largely unidentifiable . Dominant lipids include free fatty acids, 1,2-diacylglycerophospholipids, 1,2-di-O-alkylglycerols, glycerol dialkylglycerol tetraethers and 1-O-alkylglycerols . These confirm the presence and, in some cases, high abundances of bacteria in all six sinters and archaea in four of the six sinter samples; in addition, the presence of novel macrocyclic diethers and unusual distributions of monoethers and diethers suggest the presence of previously uncharacterized bacteria . The lipid distributions are also markedly dissimilar among the four sites; for example, novel macrocyclic diethers are restricted to the Rotokawa samples while particularly abundant monoethers occur only in the Orakei Korako sample . Thus, biomarkers can provide crucial insight into the complex community structure of thermophilic microorganisms, including both archaea and bacteria, involved with biogenic silica sinter formation. Nucleic Acids Res, 2005 Jan 7, 33(1), 135 - 142 Print 2005. Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties; Blondal T et al.; We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus . One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids . The ORF was cloned, and recombinant protein was expressed, purified and characterized . The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable . The recombinant enzyme exhibits extremely high activity and high ligation efficiency . It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) . The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates . The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis. Appl Environ Microbiol, 2005 Jan, 71(1), 591 - 3 High-Level Overproduction of His-Tagged Tth DNA Polymerase in Thermus thermophilus; Moreno R et al.; A new plasmid for the overexpression of His-tagged thermozymes in Thermus thermophilus was developed . With this plasmid, soluble and active histidine-tagged DNA polymerase from T . thermophilus was overproduced in larger amounts in the thermophile than in Escherichia coli . The protein purified from the thermophile was active in PCR. Appl Environ Microbiol, 2005 Jan, 71(1), 220 - 6 Mercury Adaptation among Bacteria from a Deep-Sea Hydrothermal Vent; Vetriani C et al.; Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater . We collected bacterial biomass at different distances from a diffuse flow vent at 9 degrees N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury {Hg(II)} on vent bacteria . Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 muM Hg(II) and reduced it to elemental mercury {Hg(0)} . However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive . A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus . The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45 degrees C . However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65 degrees C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments . Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance. Appl Environ Microbiol, 2005 Jan, 71(1), 169 - 74 Prevalence and Antimicrobial Resistance of Thermophilic Campylobacter spp . from Cattle Farms in Washington State; Bae W et al.; The prevalence of thermophilic Campylobacter spp . was investigated in cattle on Washington State farms . A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches) . Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR . Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution . Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%) . The most frequently detected resistance was to doxycycline (42.3% of 350 isolates) . Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types . C . jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates) . C . coli isolates were more frequently resistant than C . jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle) . Multiple drug resistance was more frequent in C . coli (51.5%) than in C . jejuni (5.1%) . The results of this study demonstrate that C . jejuni is widely distributed among Washington cattle farms, while C . coli is more narrowly distributed but significantly more resistant. J Biol Chem . 2005 Jan 7; {Epub ahead of print} Engineering a selectable marker for hyperthermophiles: Crystal structure of a thermostable Bleomycin binding protein; Brouns SJ et al.; Limited thermostability of antibiotic resistance markers has restricted genetic research in the field of extremely thermophilic archaea and bacteria . In this study, we have employed directed evolution and selection in the thermophilic bacterium Thermus thermophilus HB27 to find thermostable variants of a bleomycin binding protein from the mesophilic bacterium, Streptoalloteichus hindustanus . In a single selection round, we identified 8 clones bearing 5 types of double mutated genes that provided T . thermophilus transformants with bleomycin resistance at 77 degrees C, while the wild-type gene could only do so up to 65 degrees C . Only 6 different amino acid positions were altered, three of which were glycine residues . All variant proteins were produced in Escherichia coli and analyzed biochemically for thermal stability and functionality at high temperature . A synthetic mutant resistance gene with low GC content was designed which combined 4 substitutions . The encoded protein showed up to 17 degrees C increased thermostability and unfolded at 85 degrees C in the absence of bleomycin, whereas in its presence the protein unfolded at 100 degrees C . Despite these highly thermophilic properties, this mutant was still able to function normally at mesophilic temperatures in vivo . The mutant protein was co-crystallized with bleomycin and the structure of the binary complex was determined to a resolution of 1.5 A . Detailed structural analysis revealed possible molecular mechanisms of thermostabilization and enhanced antibiotic binding, which included the introduction of an intersubunit hydrogen bond network, improved hydrophobic packing of surface indentations, reduction of loop flexibility and a-helix stabilization . The potential applicability of the thermostable selection marker is discussed. Forensic Sci Int, 2004 Dec 2, 146 Suppl, S207 - 9 Forensic entomology and climatic change; Turchetto M et al.; Forensic entomology establishes the postmortem interval (PMI) by studying cadaveric fauna . The PMI today is still largely based on tables of insect succession on human cadavers compiled in the late 19th- or mid-20th centuries . In the last few years, however, the gradual warming of the climate has been changing faunal communities by favouring the presence of thermophilous species . To demonstrate how globalisation and climate change are overcoming geographic barriers, we present some cases of southern and allochthonous species found in north-east Italy during our entomo-forensic investigations. Arch Biochem Biophys, 2005 Feb 15, 434(2), 333 - 43 Distinct metal dependence for catalytic and structural functions in the l-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus; Lee DW et al.; l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose . It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions . The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity . The purified enzymes are homotetramers with a molecular mass of 232kDa and close amino acid sequence identity (67%) . However, they exhibit quite different temperature dependence and metal requirements . B . halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions . Its apparent K(m) values are 36mM for l-arabinose and 167mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4mM(-1)min(-1) (l-arabinose) and 0.4mM(-1)min(-1) (d-galactose) . Unlike B . halodurans AI, G . stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+) . It also has a much higher catalytic efficiency of 4.3mM(-1)min(-1) for d-galactose and 32.5mM(-1)min(-1)for l-arabinose, with apparent K(m) values of 117 and 63mM, respectively . Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B . halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G . stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C) . CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G . stearothermophilus AI, and there is an active tertiary structure for G . stearothermophilus AI at elevated temperatures for its catalytic activity . This is in marked contrast to the mesophilic B . halodurans AI where cofactor coordination is not necessary for proper protein folding . The metal dependence of G . stearothermophilus AI seems to be correlated with their catalytic and structural functions . We therefore propose that the metal ion requirement of the thermophilic G . stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures. J Biotechnol, 2005 Feb 23, 115(4), 355 - 66 Epub 2004 Nov 19. Comparison of mesophilic and thermophilic feruloyl esterases: Characterization of their substrate specificity for methyl phenylalkanoates; Topakas E et al.; The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids . Only 13 out of 26 substrates tested were significant substrates for all the enzymes . Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond . Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates . All four esterases hydrolyzed short chain aliphatic acid (C(2)-C(4)) esters of p-nitrophenol, but not the C(12) ester of laurate . All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid . The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls. J Biotechnol, 2005 Feb 23, 115(4), 345 - 53 Epub 2004 Nov 05. The transformation and toxicity of anthraquinone dyes during thermophilic (55 degrees C) and mesophilic (30 degrees C) anaerobic treatments; Dos Santos AB et al.; We studied in batch assays the transformation and toxicity of anthraquinone dyes during incubations with anaerobic granular sludge under mesophilic (30 degrees C) and thermophilic (55 degrees C) conditions . Additionally, the electron shuttling capacity of the redox mediator anthraquinone-2-sulfonic acid (AQS) and subsequent increase on decolourisation rates was investigated on anthraquinone dyes . Compared with incubations at 30 degrees C, serum bottles at 55 degrees C presented distinctly higher decolourisation rates not only with an industrial wastewater containing anthraquinone dyes, but also with model compounds . Compared with batch assays at 30 degrees C, the first-order rate constant "k" of the Reactive Blue 5 (RB5) was enhanced 11-fold and 6-fold for bottles at 55 degrees C supplemented and free of AQS, respectively . However, the anthraquinone dye Reactive Blue 19 (RB19) demonstrated a very strong toxic effect on volatile fatty acids (VFA) degradation and methanogenesis at both 30 degrees C and 55 degrees C . The apparent inhibitory concentrations of RB19 exerting 50% reduction in methanogenic activity (IC(50)-value) were 55mgl(-1) at 30 degrees C and 45mgl(-1) at 55 degrees C . Further experiments at both temperatures revealed that RB19 was mainly toxic to methanogens, because the glucose oxidizers including acetogens, propionate-forming, butyrate-forming and ethanol-forming microorganisms were not affected by the dye toxicity. Biomacromolecules, 2005 Jan-Feb, 6(1), 262 - 70 Characterization of a New Extracellular Hydrolase from Thermobifida fusca Degrading Aliphatic-Aromatic Copolyesters; Kleeberg I et al.; The paper describes the purification, biochemical characterization, sequence determination, and classification of a novel thermophilic hydrolase from Thermobifida fusca (TfH) which is highly active in hydrolyzing aliphatic-aromatic copolyesters . The secretion of the extracellular enzyme is induced by the presence of aliphatic-aromatic copolyesters but also by adding several other esters to the medium . The hydrophobic enzyme could be purified applying a combination of (NH(4))SO(4)-precipitation, cation-exchange chromatography, and hydrophobic interaction chromatography . The 28 kDa enzyme exhibits a temperature maximum of activity between 65 and 70 degrees C and a pH maximum between pH 6 and 7 depending on the ion strength of the solution . According to the amino sequence determination, the enzyme consists of 261 amino acids and was classified as a serine hydrolase showing high sequence similarity to a triacylglycerol lipase from Streptomyces albus G and triacylglycerol-aclyhydrolase from Streptomyces sp . M11 . The comparison with other lipases and esterases revealed the TfH exhibits a catalytic behavior between a lipase and an esterase . Such enzymes often are named as cutinases . However, the results obtained here show, that classifying enzymes as cutinases seems to be generally questionable. Biomacromolecules, 2005 Jan-Feb, 6(1), 105 - 8 Structural Studies of an Exopolysaccharide Produced by Streptococcus thermophilus THS; Nordmark EL et al.; The structure of an extracellular polysaccharide (EPS) from Streptococcus thermophilus THS has been determined . A combination of component analysis, methylation analysis and NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units . Sequential information was obtained by two-dimensional (1)H,(1)H-NOESY and (1)H,(13)C-HMBC NMR experiments . NMR data indicate different mobility within the EPS with a stiffer backbone and a more flexible side-chain. Res Microbiol, 2005 Jan-Feb, 156(1), 93 - 103 Comparing a ciliate and a fish cell line for their sensitivity to several classes of toxicants by the novel application of multiwell filter plates to Tetrahymena; Dayeh VR et al.; Although ciliated protozoa such as Tetrahymena have many desirable properties as toxicological test organisms, their attributes would be better realized if multiple cultures could be simultaneously exposed to toxicants, quickly washed to terminate toxicant exposure, and conveniently evaluated for changes in cellular functions . Therefore, multiwell filter plates (MWFPs), manufactured primarily for biochemical applications, were used to expose Tetrahymena thermophila to copper, Triton X-100, and gliotoxin and compared to results of exposure in microcentrifuge tubes (MCTs) . For MWFP, removal of toxicant solutions and retention of Tetrahymena in wells was done by placing plates on a manifold and applying pressure with a vacuum pump . Retained cells were resuspended in the same wells and their functions assessed with the fluorescent indicator dyes, Alamar blue to measure energy metabolism, and 5'-carboxyfluorescein diacetate acetoxymethyl ester to evaluate membrane integrity . For MCTs, exposures were terminated by centrifugation, and resuspended Tetrahymena were transferred to conventional multiwell plates for viability assessment with the same fluorescent dyes . Results were measured with a fluorescent multiwell plate reader and dose-response curves were obtained successfully with both procedures . However, MWFPs were much more convenient and rapid, potentially allowing 96 cultures to be processed at a time . Exposing Tetrahymena in MWFPs also allowed the ciliate and a rainbow trout gill cell line, RTgill-W1, to be compared for their sensitivity to toxicants under similar conditions of exposure and by common viability assays . Both cell systems showed toxic responses to Triton X-100 and copper at similar concentrations, but RTgill-W1 was more sensitive to gliotoxin. Appl Microbiol Biotechnol . 2005 Jan 6; {Epub ahead of print} The mechanism of bacterial indigo reduction; Nicholson SK et al.; The reduction of water-insoluble indigo by the recently isolated moderate thermophile, Clostridium isatidis, has been studied with the aim of developing a sustainable technology for industrial indigo reduction . The ability to reduce indigo was not shared with C . aurantibutyricum, C . celatum and C . papyrosolvens, but C . papyrosolvens could reduce indigo carmine (5,5'-indigosulfonic acid), a soluble indigo derivative . The supernatant from cultures of C . isatidis, but not from cultures of the other bacteria tested, decreased indigo particle size to one-tenth diameter . Addition of madder powder, anthraquinone-2,6-disulfonic acid, and humic acid all stimulated indigo reduction by C . isatidis . Redox potentials of cultures of C . isatidis were about 100 mV more negative than those of C . aurantibutyricum, C . celatum and C . papyrosolvens, and reached -600 mV versus the SCE in the presence of indigo, but potentials were not consistently affected by the addition of the quinone compounds, which probably act by modifying the surface of the bacteria or indigo particles . It is concluded that C . isatidis can reduce indigo because (1) it produces an extracellular factor that decreases indigo particle size, and (2) it generates a sufficiently reducing potential. EMBO J . 2005 Jan 06; {Epub ahead of print} Bacterial chromosome segregation: structure and DNA binding of the Soj dimer - a conserved biological switch; Leonard TA et al.; Soj and Spo0J of the Gram-negative hyperthermophile Thermus thermophilus belong to the conserved ParAB family of bacterial proteins implicated in plasmid and chromosome partitioning . Spo0J binds to DNA near the replication origin and localises at the poles following initiation of replication . Soj oscillates in the nucleoid region in an ATP- and Spo0J-dependent fashion . Here, we show that Soj undergoes ATP-dependent dimerisation in solution and forms nucleoprotein filaments with DNA . Crystal structures of Soj in three nucleotide states demonstrate that the empty and ADP-bound states are monomeric, while a hydrolysis-deficient mutant, D44A, is capable of forming a nucleotide 'sandwich' dimer . Soj ATPase activity is stimulated by Spo0J or the N-terminal 20 amino-acid peptide of Spo0J . Our analysis shows that dimerisation and activation involving a peptide containing a Lys/Arg is conserved for Soj, ParA and MinD and their modulators Spo0J, ParB and MinE, respectively . By homology to the nitrogenase iron protein and the GTPases Ffh/FtsY, we suggest that Soj dimerisation and regulation represent a conserved biological switch. J Biochem (Tokyo), 2004 Nov, 136(5), 629 - 34 A Novel Metal-Activated L-Serine O-Acetyltransferase from Thermus thermophilus HB8; Kobayashi S et al.; l-Cysteine is an important amino acid in terms of its industrial applications . The biosynthesis of l-cysteine in enteric bacteria is regulated through the feedback inhibition by l-cysteine of l-serine O-acetyltransferase (SAT), a key enzyme in l-cysteine biosynthesis . We recently found that l-cysteine is overproduced in Escherichia coli strains expressing a gene encoding feedback inhibition-insensitive SAT . Further improvements in l-cysteine production are expected by the use of SAT with high stability . We report here the sat1 gene encoding SAT of an extreme thermophile, Thermus thermophilus HB8 . The sat1 gene was cloned and overexpressed in E . coli cells based on the genome sequence in T . thermophilus HB8 . The predicted amino acid sequence consists of 295 amino acids and is homologous to other O-acetyltransferase members . In particular, the carboxyl-terminal region shares approximately 30% identities with SATs found in bacteria and plants, despite showing only about 15% identity in the overall sequence . Enzymatic analysis and an atomic absorption study of the purified recombinant proteins revealed that the enzyme is highly activated by Co(2+) or Ni(2+), and contains Zn(2+) and Fe(2+) . These results indicate that the T . thermophilus SAT is a novel type of enzyme different from other members of this protein family. Res Microbiol, 2004 Dec, 155(10), 869 - 83 Physiology of the thermophilic acetogen Moorella thermoacetica; Drake HL et al.; Moorella thermoacetica (originally isolated as Clostridium thermoaceticum) has served as the primary acetogenic bacterium for the resolution of the acetyl coenzyme A (acetyl-CoA) or Wood-Lijungdahl pathway, a metabolic pathway that (i) autotrophically assimilates CO2 and (ii) is centrally important to the turnover of carbon in many habitats . The purpose of this article is to highlight the diverse physiological features of this model acetogen and to examine some of the consequences of its metabolic capabilities. J Struct Biol, 2005 Jan, 149(1), 99 - 110 Crystal structures of the signal transducing protein GlnK from Thermus thermophilus HB8; Sakai H et al.; The Thermus thermophilus HB8 genome encodes a signal transducing PII protein, GlnK . The crystal structures of GlnK have been determined in two different space groups, P2(1)2(1)2(1) and P3(1)21 . The PII protein has the T-loop, which is essential for interactions with receptor proteins . In both crystal forms, three GlnK molecules form a trimer in the asymmetric unit . In one P2(1)2(1)2(1) crystal form, the three T-loops in the trimer are disordered, while in another P2(1)2(1)2(1) crystal form, the T-loop from one molecule in the trimer is ordered . In the P3(1)21 crystal, one T-loop is ordered while the other two T-loops are disordered . The conformations of the ordered T-loops significantly differ between the two crystal forms; one makes the alpha-helix in the middle of the T-loop, while the other has an extension of the beta-hairpin . Two different conformations are captured by the crystal contacts . The observation of multiple T-loop conformations suggests that the T-loop could potentially exhibit "polysterism," which would be important for interactions with receptor proteins . The crystal structures of the nucleotide-bound forms, GlnK.ATP and GlnK.ADP, have also been determined . ATP/ADP binding within a cleft at the interface of two adjacent T . thermophilus GlnK monomers might affect the conformation of the T-loop. Biochem Biophys Res Commun, 2005 Feb 11, 327(2), 382 - 92 Characterization of Aquifex aeolicus RNase E/G; Kaberdin VR et al.; The RNase E/G homologue from the thermophilic eubacterium Aquifex aeolicus has been overexpressed in Escherichia coli, purified, and characterized in vitro . We show that A . aeolicus RNase E/G has a temperature-dependent, endoribonucleolytic activity . The enzyme site-specifically cleaves oligonucleotides and structured RNAs at locations that are partly overlapping or completely different when compared to the positions of E . coli RNase E and RNase G cleavage sites . The efficiency of cleavage by A . aeolicus RNase E/G is dependent on the 5'-phosphorylation status of RNA suggesting differential susceptibility of primary transcripts and their degradative intermediates to the nuclease activity of this enzyme in vivo . Similar to E . coli RNase E, A . aeolicus RNase E/G is able to selectively cleave internucleotide bonds in the 3'-5' direction, and to cut in intercistronic regions of putative tRNA precursors, thus suggesting a common function for RNase E/G homologues in eubacteria. Arch Biochem Biophys, 2005 Feb 1, 434(1), 169 - 77 The Sulfolobus solfataricus electron donor partners of thermophilic CYP119: an unusual non-NAD(P)H-dependent cytochrome P450 system; Puchkaev AV et al.; CYP119 from Sulfolobus solfataricus is the first well-characterized thermophilic cytochrome P450 enzyme . The endogenous substrate for this enzyme is not known but it hydroxylates lauric acid in a reaction supported by surrogate mesophilic electron donors . However, reconstitution of a high-temperature catalytic system requires identification of the normal thermophilic electron donor partners of CYP119 . Here, we describe cloning, expression in Escherichia coli, and characterization of the requisite electron donor partners from S . solfataricus . One is a thermostable ferredoxin and the second a 2-oxoacid-ferredoxin oxidoreductase that utilizes pyruvic acid rather than NAD(P)H as the source of reducing equivalents . CYP119 is the only cytochrome P450 to date known to obtain electrons from a non-NAD(P)H-dependent protein . The two thermophilic partners have been used to reconstitute a catalytic system that hydroxylates lauric acid at 70 degrees C, and the optimal conditions for this system have been defined . This first high-temperature in vitro catalytic system represents an important step in the development of industrially relevant catalysts. Ann Agric Environ Med, 2004, 11(2), 309 - 17 Exposure to airborne microorganisms, dust and endotoxin during flax scutching on farms; Krysinska-Traczyk E et al.; Microbiological air sampling was performed on 5 flax farms located in eastern Poland . Air samples for determination of the concentrations of microorganisms, dust and endotoxin were collected in barns during machine scutching of flax stems by the farmers . The concentrations of mesophilic bacteria ranged from 203.5-698.8 x 10(3) cfu/m3, of Gram-negative bacteria from 27.2-123.4 x 10(3) cfu/m3, of thermophilic actinomycetes from 0.5-2.6 x 10(3) cfu/m3, and of fungi from 23.4-99.8 x 10(3) cfu/m3 . The concentrations of total airborne microorganisms (bacteria + fungi) were within a range of 245.0-741.0 x 10(3) cfu/m3 . The values of the respirable fraction of total airborne microflora on the examined farms were between 45.5-98.3% . Corynebacteria (irregular Gram-positive rods, mostly Corynebacterium spp.) were dominant at all sampling sites, forming 46.8-67.8% of the total airborne microflora . Among Gram-negative bacteria, the most numerous species was Pantoea agglomerans (synonyms: Erwinia herbicola, Enterobacter agglomerans), known to have strong endotoxic and allergenic properties . Among fungi, the allergenic species Alternaria alternata prevailed . Altogether, 25 species or genera of bacteria and 10 species or genera of fungi were identified in the farm air during flax scutching; of these, 11 and 6 species or genera respectively were reported as having allergenic and/or immunotoxic properties . The concentrations of airborne dust ranged within 43.7-648.1 mg/m3 (median 93.6 mg/m3), exceeding on all farms the Polish OEL value of 4 mg/m3 . The concentrations of airborne endotoxin ranged within 16.9-172.1 microg/m3 (median 30.0 microg/m3), exceeding at all sampling sites the suggested OEL value of 0.2 microg/m) . In conclusion, flax farmers performing machine scutching of flax could be exposed to large concentrations of airborne microorganisms, dust and endotoxin, posing a risk of work-related respiratory disease. Ann Agric Environ Med, 2004, 11(2), 233 - 6 Presence of virulent strains of amphizoic amoebae in swimming pools of the city of Szczecin; Gornik K et al.; The studies included 10 public indoor swimming pools and 3 public open-air swimming pools located in the city of Szczecin . In 2003, water samples were collected for detection of virulent amphizoic amoebae strains . In all pools, 16 strains of thermophilic Acanthamoeba spp . were isolated, 5 of which proved virulent for mice . No pathogenic strains were detected in the water sampled in the indoor swimming pools, and the virulent strains, AD 16, AD 148, AD 166, AM 17, and AM 148, were found only in the open-air swimming pools . The post-mortem studies of mice that had been inoculated with these strains revealed the amoebae invasions in brain, lungs, liver, kidneys, and spleen. Acta Biochim Pol, 2004, 51(4), 971 - 81 The effect of mono- and divalent cations on Tetrahymena thermophila telomeric repeat fragment . A photon correlation spectroscopy study; Wlodarczyk A et al.; The structure of the Tetrahymena thermophila telomeric sequence d(TGGGGT)(4) was studied by photon correlation spectroscopy (PCS) in aqueous solution in the presence of NaCl, KCl and SrCl(2) . The sample studied was polydisperse in all conditions studied . Translational diffusion coefficients D(T) describing the diffusion modes observed were determined . On the basis of a comparison between the experimental D(T) values with those calculated assuming the bead model, two forms were identified as telomeric quadruplex structures: monomer and tetramer . In the presence of SrCl(2) formation of aggregates was observed, with a size that reached several micrometres . The relative weighted concentrations of the structures observed for different concentrations of a salt and DNA were determined . The results obtained in the presence of monovalent ions were qualitatively similar and could be presented in a coherent plot in which the concentration of salt was expressed by the number of ions per DNA molecule . A large number of ions per DNA molecule favoured tetramer formation while a small number favoured the monomer form . A structural phase transition from the monomer to the tetramer induced by a change in the number of ions per DNA molecule was observed . The main difference between the results for Na(+) and K(+) was a greater effectiveness of the K(+) ions in formation of tetramers . The effect of Sr(2+) ions on the structures formed was different than that of the monovalent ions . The results obtained in the presence of Sr(2+) could not be described as a function of the number of ions per DNA molecule. Photochem Photobiol, 2004 Dec, 80(3), 531 - 4 Effects of Low-power Laser Irradiation on Cell Locomotion in Protozoa paragraph sign; Koutna M et al.; Low-power lasers are commonly used in human medicine for treatment of various pathological conditions, but mechanisms of their healing effects are still poorly understood . The results of this study provide information related to these effects at the cellular level . Two different protozoan species, Euglena gracilis and Tetrahymena thermophila, were used to study changes in locomotion behavior in response to low-power lasers . The cells were irradiated at 830 and 650 nm generated by a semiconductor laser (99 J/cm(2), 360 mW) and a laser pointer (0.75 J/cm(2), 5 mW), respectively, and their locomotion was recorded by a TV camera and analyzed using computer software . Exposure to laser light, regardless of the wavelength, resulted in increased cell velocity in both species (P < 0.001) . Exposure to 650 nm produced an equal increase in median cell velocity in both E . gracilis (19.0%) and T . thermophila (18.2%), and some increase persisted in the postirradiation 30 s period . Irradiation by the 830 nm laser resulted in a markedly higher response in Tetrahymena (29.4%) than in Euglena (15.2%), and the two median values remained increased after irradiation was discontinued . Different reactions found in the species studied and some mechanisms underlying the response of cells to radiation are discussed. Chemosphere, 2005 Jan, 58(4), 449 - 58 Fate of polycyclic aromatic hydrocarbons during composting of lagooning sewage sludge; Amir S et al.; The fate of 16 polycyclic aromatic hydrocarbons (PAHs), targeted by the USEPA agency, has been investigated during composting of lagooning sludge . Composting shows efficient decrease of the content and the bioavailability of each PAH . Biodegradation and sorption are suggested as the main mechanisms contributing to this decrease . During the stabilization phase of composting, extensive microbial degradation of PAHs, mainly those with a low number of aromatic rings, was achieved following development of intense thermophilic communities . However, partial sorption of PAH to non-accessible sites temporarily limits the mobility mainly of PAHs with a high number of aromatic rings plus acenaphthene and acenaphthylene, and allows them to escape microbial attack . During the maturation phase, the development of a mesophilic population could play an important role in the degradation of the remaining PAH . During this phase of composting, PAH sequestration and binding of their oxidative metabolites within new-formed humic substances might also explain PAH decrease at the end of composting . The tendency of change of content or bioavailability of various PAH compounds during composting is found to be strongly related to the number of their aromatic rings, their molecular weight and structure. Biochim Biophys Acta, 2005 Jan 7, 1706(1-2), 147 - 57 Purification, characterisation and crystallisation of photosystem II from Thermosynechococcus elongatus cultivated in a new type of photobioreactor; Kern J et al.; The thermophilic cyanobacterium Thermosynechococcus elongatus was cultivated under controlled growth conditions using a new type of photobioreactor, allowing us to optimise growth conditions and the biomass yield . A fast large-scale purification method for monomeric and dimeric photosystem II (PSII) solubilized from thylakoid membranes of this cyanobacterium was developed using fast protein liquid chromatography (FPLC) . The obtained PSII core complexes (PSIIcc) were analysed for their pigment stoichiometry, photochemical and oxygen evolution activities, as well as lipid and detergent composition . Thirty-six chlorophyll a (Chla), 2 pheophytin a (Pheoa), 9+/- 1 beta-carotene (Car), 2.9+/-0.8 plastoquinone 9 (PQ9) and 3.8+/-0.5 Mn were found per active centre . For the monomeric and dimeric PSIIcc, 18 and 20 lipid as well as 145 and 220 detergent molecules were found in the detergent shell, respectively . The monomeric and dimeric complexes showed high oxygen evolution activity with 1/4 O(2) released per 37-38 Chla and flash in the best cases . Crystals were obtained from dimeric PSIIcc by a micro-batch method . They diffract synchrotron X-rays to a maximum resolution of 2.9-A, resulting in complete data sets of 3.2 A resolution. Int J Food Microbiol, 2005 Jan 15, 98(1), 11 - 21 Identification of probiotic microorganisms in South African products using PCR-based DGGE analysis; Theunissen J et al.; Probiotic microorganisms in commercial yoghurts and other food products are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics . In this study, PCR-based DGGE analysis was used for the rapid and accurate identification of probiotic microorganisms from South African yoghurts and lyophilized preparations in capsule and tablet form . To identify the microorganisms present in these products, the DGGE profiles obtained were compared to two reference markers (A and B) composed of five lactobacilli and seven Bifidobacterium species, respectively . The results obtained were confirmed by species-specific PCR, as well as sequence analyses of unknown bands not present in the reference markers . It was found that only 54.5% of the probiotic yoghurts contained the microorganisms stated on the label compared to only a third (33.3%) of the lyophilized probiotic products . Some Bifidobacterium species were incorrectly identified and various microorganisms were detected that were not listed on the label . Sequence analyses confirmed the presence of Streptococcus spp . other than the yoghurt starter, Streptococcus thermophilus, in some of these products and in some instances label information was vague and non-scientific . PCR-based DGGE analyses proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in South African probiotic products. J Fluoresc, 2004 Sep, 14(5), 491 - 8 Protein-based biosensors for diabetic patients; Scognamiglio V et al.; In this article we show the recent progress in the field of glucose sensing based on the utilization of enzymes and proteins as probes for stable and non-consuming fluorescence biosensors . We developed a new methodology for glucose sensing using inactive forms of enzymes such as the glucose oxidase from Aspergillus niger, the glucose dehydrogenase from the thermophilic microorganism Thermoplasma acidophilum, and the glucokinase from the thermophilic eubacterium Bacillus stearothermophilus . Glucose oxidase was rendered inactive by removal of the FAD cofactor . The resulting apo-glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence . 8-Anilino-1-naphthalene sulfonic acid was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence . The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40% . These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the KD of holo-glucose oxidase . The ANS-labeled apo-glucose dehydrogenase from Thermoplasma acidophilum also displayed an approximate 25% decrease in emission intensity upon binding glucose . This decrease can be also used to measure the glucose concentration . The thermophilic apo-glucose dehydrogenase was also stable in the presence of organic solvents, allowing determination of glucose in the presence of acetone . The third enzyme used for glucose sensing was the glucokinase from Bacillus stearothermophilus . A fluorescence competitive assay for the determination of glucose was developed based on the utilization of this thermostable enzyme . Taken together, our results show that enzymes which use glucose as their substrate can be used as reversible and non-consuming glucose biosensors in the absence of required co-factors . Moreover, the possibility of using inactive apo-enzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes. J Ind Microbiol Biotechnol . 2004 Dec 23; {Epub ahead of print} Immobilization of cells with nitrilase activity from a thermophilic bacterial strain; Kabaivanova L et al.; Cells of the moderately thermophilic Bacillus sp . UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques . A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes . Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts . Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles. J Biotechnol, 2005 Jan 26, 115(2), 199 - 210 Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry; Reis A et al.; Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse . Using a mixture of two specific fluorescent stains, DiOC(6)(3) (3,3'-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity . Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC(6)(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane 'ghost cells' (D), stained with both DiOC(6)(3) and PI . Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting 'ghost cells' which were associated with the double stained sub-population . It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions . All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch . A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation . Such information could be used to enhance process efficiency. Water Res, 2005 Jan, 39(1), 171 - 83 Modelling of two-stage anaerobic digestion using the IWA Anaerobic Digestion Model No . 1 (ADM1); Blumensaat F et al.; The aim of the study presented was to implement a process model to simulate the dynamic behaviour of a pilot-scale process for anaerobic two-stage digestion of sewage sludge . The model implemented was initiated to support experimental investigations of the anaerobic two-stage digestion process . The model concept implemented in the simulation software package MATLABtrade mark/Simulink((R)) is a derivative of the IWA Anaerobic Digestion Model No.1 (ADM1) that has been developed by the IWA task group for mathematical modelling of anaerobic processes . In the present study the original model concept has been adapted and applied to replicate a two-stage digestion process . Testing procedures, including balance checks and 'benchmarking' tests were carried out to verify the accuracy of the implementation . These combined measures ensured a faultless model implementation without numerical inconsistencies . Parameters for both, the thermophilic and the mesophilic process stage, have been estimated successfully using data from lab-scale experiments described in literature . Due to the high number of parameters in the structured model, it was necessary to develop a customised procedure that limited the range of parameters to be estimated . The accuracy of the optimised parameter sets has been assessed against experimental data from pilot-scale experiments . Under these conditions, the model predicted reasonably well the dynamic behaviour of a two-stage digestion process in pilot scale. Water Res, 2005 Jan, 39(1), 37 - 46 Bioflocculation of mesophilic and thermophilic activated sludge; Vogelaar JC et al.; Thermophilic activated sludge treatment is often hampered by a turbid effluent . Reasons for this phenomenon are so far unknown . Here, the hypothesis of the temperature dependency of the hydrophobic interaction as a possible cause for diminished thermophilic activated sludge bioflocculation was tested . Adsorption of wastewater colloidal particles was monitored on different flat surfaces as a function of temperature . Adsorption on a hydrophobic surface varied with temperature between 20 and 60 degrees C and no upward or downward trend could be observed . This makes the hydrophobic interaction hypothesis unlikely in explaining the differences in mesophilic and thermophilic activated sludge bioflocculation . Both mesophilic and thermophilic biomass did not flocculate with wastewater colloidal particles under anaerobic conditions . Only in the presence of oxygen, with biologically active bacteria, the differences in bioflocculation behavior became evident . Bioflocculation was shown only to occur with the combination of wastewater and viable mesophilic biomass at 30 degrees C, in the presence of oxygen . Bioflocculation did not occur in case the biomass was inactivated or when oxygen was absent . Thermophilic activated sludge hardly showed any bioflocculation, also under mesophilic conditions . Despite the differences in bioflocculation behavior, sludge hydrophobicity and sludge zetapotentials were almost similar . Theoretical calculations using the DLVO (Derjaguin, Landau, Verweij and Overbeek) theory showed that flocculation is unlikely in all cases due to long-range electrostatic forces . These calculations, combined with the fact that bioflocculation actually did occur at 30 degrees C and the unlikelyness of the hydrophobic interaction, point in the direction of bacterial exo-polymers governing bridging flocculation . Polymer interactions are not included in the DLVO theory and may vary as a function of temperature. Eur J Biochem, 2004 Dec, 271(23-24), 4798 - 803 Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum; Yamashita T et al.; Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan . To help understand d-aspartate biosynthesis in B . bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of d- and l-aspartate, was purified to homogeneity and characterized . The enzyme was a monomer with a molecular mass of 27 kDa . This is the first report showing the presence of a monomeric aspartate racemase . Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus . The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the d-enantiomer . The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells . The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da . The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B . bifidum enzyme. Cell Motil Cytoskeleton, 2005 Feb, 60(2), 96 - 103 High speed sliding of axonemal microtubules produced by outer arm dynein; Seetharam RN et al.; To study dynein arm activity at high temporal resolution, axonemal sliding was measured field by field for wild type and dynein arm mutants of Tetrahymena thermophila . For wt SB255 cells, when the rate of data acquisition was 60 fps, about 5x greater than previously published observations, sliding was observed to be discontinuous with very high velocity sliding (average 196 mum/sec) for a few msec (1 or 2 fields) followed by a pause of several fields . The sliding velocities measured were an order of magnitude greater than rates previously measured by video analysis . However, when the data were analyzed at 12 fps for the same axonemes, consistent with previous observations, sliding was linear as the axonemes extended several times their original length with an average velocity of approximately 10 mum/sec . The pauses or stops occurred at approximately 200 and 300% of the initial length, suggesting that dynein arms on one axonemal doublet were initially active to the limit of extension, and then the arms on the next doublet became activated . In contrast, in a mutant where OADs are missing, sliding observed at 60 fps was continuous and slow (5 mum/sec), as opposed to the discontinuous high-velocity sliding of SB255 and of the mutant at the permissive temperature where OADs are present . High-velocity step-wise sliding was also present in axonemes from an inner arm dynein mutant (KO6) . These results indicate that the high-speed discontinuous pattern of sliding is produced by the mechanochemical activity of outer arm dynein . The rate of sliding is consistent with a low duty ratio of the outer arm dynein and with the operation of each arm along a doublet once per beat . Cell Motil . Cytoskeleton 60:96-103, 2005 . (c) 2004 Wiley-Liss, Inc. Biotechnol Lett, 2004 Oct, 26(20), 1607 - 12 Ethanol production from H2 and CO2 by a newly isolated thermophilic bacterium, Moorella sp . HUC22-1; Sakai S et al.; The thermophilic bacterium, Moorella sp . HUC22-1, newly isolated from a mud sample, produced ethanol from H(2) and CO(2) during growth at 55 degrees C . In batch cultures in serum bottles, 1.5 mM ethanol was produced from 270 mM H(2) and 130 mM CO(2) after 156 h, whereas less than 1 mM ethanol was produced from 23 mM fructose after 33 h . Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were higher in cells grown with H(2) and CO(2) than those grown with fructose . The NADH/NAD(+) and NADPH/NADP(+) ratios in cells grown with H(2) and CO(2) were also higher than those in cells grown with fructose . When the culture pH was controlled at 5 with H(2) and CO(2) in a fermenter, ethanol production was 3.7-fold higher than that in a pH-uncontrolled culture after 220 h. Biotechnol Lett, 2004 Sep, 26(17), 1347 - 51 A new thermostable alpha-L-arabinofuranosidase from a novel thermophilic bacterium; Birgisson H et al.; An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia . Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51 . The gene was cloned into Escherichia coli and its recombinant product expressed and purified . The enzyme appeared to be a hexamer . AraF was optimally active at 70 degrees C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 degrees C . AraF had a Km) value of 0.6 mM and V(max) value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside . AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51. Plant Mol Biol, 2004 May, 55(2), 193 - 207 Gamma carbonic anhydrases in plant mitochondria; Parisi G et al.; Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (gammaCA), a Zn containing enzyme from Methanosarcina thermophila (CAM), were identified and characterized . Evolutionary and structural analyses predict that these genes code for active forms of gammaCA . Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from alpha and gamma proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor . Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome . Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria . Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues . Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models . Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated . Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain gammaCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function. Appl Microbiol Biotechnol, 2005 Jan, 66(4), 393 - 400 Epub 2004 Oct 27. Discovery of a thermostable Baeyer-Villiger monooxygenase by genome mining; Fraaije MW et al.; Baeyer-Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means . However, only a limited number of these atypical monooxygenases are available in recombinant form . Using a recently described protein sequence motif, a putative Baeyer-Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca . Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO . The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted . The best substrate discovered so far is phenylacetone (k(cat) = 1.9 s(-1), K(M) = 59 muM) . The enzyme exhibits moderate enantioselectivity with alpha-methylphenylacetone (enantiomeric ratio of 7) . In addition to Baeyer-Villiger reactions, the enzyme is able to perform sulfur oxidations . Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52 degrees C . This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs. Genes Genet Syst, 2004 Oct, 79(5), 255 - 62 Unusual distribution of mitochondrial large subunit rRNA in the cytosol during conjugation in Tetrahymena thermophila; Kobayashi T et al.; The distribution of mitochondria during conjugation of the ciliated protozoan Tetrahymena thermophila was surveyed using a mitochondrial stain and fluorescence in situ hybridization (FISH) . When the mitochondria-specific stain, Mito-Tracker, was used, the majority of mitochondria were detected in the cortex; their distribution was not changed during conjugation . On the other hand, FISH using mitochondrial large subunit (LSU) rRNA as a probe showed an unusual distribution of signals during conjugation . Unexpectedly, the signals were detected throughout the cytoplasm of conjugating cells . These signals were not observed in pre-mating cells and in exconjugants . The cytosolic localization of mitochondrial rRNA was supported by northern blot analysis using post-mitochondrial RNA fraction at the later stages of conjugation . These observations suggest selec-tive mitochondrial breakdown or transport of LSU rRNA into cytosol . The biological significance of the conjugation-specific appearance of the cytosolic mitochondrial rRNA is discussed. J Biol Chem . 2004 Dec 14; {Epub ahead of print} Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain7; Zhang Z et al.; L-rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms . Glucose-1-phosphate thymidylyltransferase (RmlA; EC2.7.7.24) catalyzes the first reaction of the four-step pathway of L-rhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate . Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain7 . In this study, we report the heterologous expression of the largest homologue (401 residue long ST0452 protein) and characterization of its thermostable activity . RmlA enzymatic activity of this protein was detected from 65 to 100 degrees C, with a half-life of 60 minutes at 95 degrees C and 180 minutes at 80 degrees C . Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein . Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing a-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate, but not a-D-glucosamine-1-phosphate . However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP . Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities . This is the first report of a thermo-stable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities. J Biol Chem . 2004 Dec 14; {Epub ahead of print} Five amino acid residues responsible for the high stability of hydrogenobacter thermophilus cytochrome c552: RECIPROCAL MUTATION ANALYSIS; Oikawa K et al.; Five amino acid residues responsible for extreme stability have been identified in cytochrome c552 (HT c552) from a thermophilic bacterium, Hydrogenobacter thermophilus . The five residues, which are spatially distributed in three regions of HT c552, were replaced with the corresponding residues in the homologous but less stable cytochrome c551 (PA c551) from Pseudomonas aeruginosa . The quintuple HT c552 variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride (GdnHCl) denaturation as that of PA c551, suggesting that the five residues in HT c552 necessarily and sufficiently contribute to the overall stability . In the three HT c552 variants carrying mutation(s) in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol-1, followed by the A7F/M13V ones (-3.3 kJ mol-1), and then the I78V one (-1.5 kJ mol-1) . The order of destabilization in HT c552 was the same as that of stabilization in PA c551 with reverse mutation(s) such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol-1, respectively) . The results of GdnHCl denaturation were consistent with those of thermal denaturation for the same variants . The present study established a method for reciprocal mutation analysis . The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability . A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability. Protein Expr Purif, 2005 Jan, 39(1), 54 - 60 Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration; Turner P et al.; Cyclomaltodextrinases are multidomain and often dimeric proteins from the alpha-amylase family (glycoside hydrolase family 13) which frequently have been very difficult to express in active form in Escherichia coli . To express the soluble form of this type of proteins in larger quantities the expression has to be optimized . We have used and combined two strategies to increase the yield of soluble recombinant cyclomaltodextrinase expressed from a gene originating from the thermophilic Gram-positive bacterium Anoxybacillus flavithermus . One strategy involved tuning of the inducer concentration while the other involved fusion of the gene encoding the target protein to the gene encoding the solubility-enhancing protein NusA . The enzyme activity could be increased 6-7 times solely by finely tuning the IPTG concentration, but the activity level was very sensitive to the amount of inducer applied . Hence, the IPTG concentration may have to be optimized for every protein under the conditions used . The fusion protein-strategy gave a slightly lower total activity but the level of soluble recombinant protein obtained was in this case significantly less sensitive to the inducer concentration applied . Moreover, the activity could be increased about 2-fold by cleaving off the solubility-tag (NusA) by enterokinase. Mikrobiologiia, 2004 Sep-Oct, 73(5), 716 - 20 {Detection of a cultivated hyperthermophilic archaeon of the genus Sulfophobococcus in a methane tank operated in a thermophilic regime}; Development of a rapid detection and enumeration method for thermophilic bacilli in milk powders; Thermophile Research Unit, Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton 2001, New ZealandThermophilic strains of Geobacillus, Anoxybacillus and Bacillus that are able to grow at 55 degrees C and above are recognized as commonly occurring contaminants during the production of milk powders . In particular, Anoxybacillus flavithermus strain C and Bacillus licheniformis strain F are often the most prevalent . We describe here the development of a TaqMan-based real-time-PCR assay using a small amplicon of the ribosomal 16S rRNA gene for the selective and quantitative detection of thermophilic bacilli in milk powders . We further present an effective, rapid and inexpensive method for the isolation of total bacterial DNA from milk powder for quantitative PCR analysis within 20 min . With this method, the detection of thermophilic bacilli in milk powder can be accomplished within 1 h . The detection limit for reconstituted and inoculated milk was 8 vegetative cfu ml(-1) and 64 spores ml(-1), respectively. J Mol Biol, 2005 Jan 28, 345(4), 681 - 93 A determination of the identity elements in yeast 18 S ribosomal RNA for the recognition of ribosomal protein YS11: the role of the kink-turn motif in helix 11; Dresios J et al.; A description of the site of interaction of YS11, the yeast homolog of eubacterial S17, with 18 S rRNA was obtained by assessing the binding of the ribosomal protein, in a filter retention assay, to oligoribonucleotides that reproduce regions of 18 S rRNA . YS11 binds predominantly to domain I; the Kd value is 113 nM . The dimensions of the YS11 binding site were refined, guided by chemical protection data and by the atomic structure of the Thermus thermophilus 30 S subunit, which has the S17 recognition site in 16 S rRNA . An oligoribonucleotide that mimics helix 11, a phylogenetically conserved region in domain I, binds YS11 with a Kd value of 230 nM; a second oligoribonucleotide that contains only the kink-turn motif in helix 11 binds YS11 with a Kd value of 528 nM . Thus, helix 11 has most of the nucleotides required for the recognition of YS11 . To identify those nucleotides a set of 27 transversion mutations in H11 was constructed and their contribution to the binding of YS11 determined . Mutations of nine nucleotides (U313, C314, A316, G337, C338, G347, U348, U350, and C351) increased the Kd value for YS11 binding by at least eightfold; G325U and U349A mutations increased the Kd value fivefold . Eight of the 11 mutations are in the kink-turn in H11, confirming the critical importance of the motif for YS11 recognition . The other three nucleotides are in the lower stem and the terminal loop of H11, which makes a lesser, but still important, contribution to YS11 binding . The identity elements for YS11 recognition are: A316, G325, G337, G347, U348, U349, U350, and C351 . The effect of the other nucleotides that decrease binding is probably indirect, presumably they affect the conformation of the binding site but do not have contacts to YS11 amino acid residues . The eight identity element nucleotides are in regions of H11 that deviate from A-form geometry and the contacts are predominantly, if not exclusively, to backbone phosphate and sugar oxygen atoms, indicating that YS11 recognizes the shape of the rRNA binding site rather than reading the sequence of nucleotides. Bioresour Technol, 2005 Apr, 96(6), 721 - 30 Effect of aeration rate and waste load on evolution of volatile fatty acids and waste stabilization during thermophilic aerobic digestion of a model high strength agricultural waste; Obeta Ugwuanyi J et al.; Thermophilic aerobic digestion (TAD) is a relatively new, dynamic and versatile low technology for the economic processing of high strength waste slurries . Waste so treated may be safely disposed of or reused . In this work a model high strength agricultural waste, potato peel, was subjected to TAD to study the effects of oxygen supply at 0.1, 0.25, 0.5 and 1.0 vvm (volume air per volume slurry per minute) under batch conditions at 55 degrees C for 156 h on the process . Process pH was controlled at 7.0 or left unregulated . Effects of waste load, as soluble chemical oxygen demand (COD), on TAD were studied at 4.0, 8.0, 12.0 and 16.0 gl(-1) (soluble COD) at pH 7.0, 0.5 vvm and 55 degrees C . Efficiency of treatment, as degradation of total solids, total suspended solids and soluble solid, as well as soluble COD significantly increased with aeration rate, while acetate production increased as the aeration rate decreased or waste load increased, signifying deterioration in treatment . Negligible acetate, and no other acids were produced at 1.0 vvm . Production of propionate and other acids increased after acetate concentration had started to decrease and, during unregulated reactions coincided with the drop in the pH of the slurry . Acetate production was more closely associated with periods of oxygen limitation than were other acids . Reduction in oxygen availability led to deterioration in treatment efficiency as did increase in waste load . These variables may be manipulated to control treated waste quality. Bioresour Technol, 2005 Apr, 96(6), 707 - 19 Effect of digestion temperature and pH on treatment efficiency and evolution of volatile fatty acids during thermophilic aerobic digestion of model high strength agricultural waste; Ugwuanyi JO et al.; Thermophilic aerobic digestion (TAD) of a model agricultural waste, potato peel slurry, at soluble chemical oxygen demand (COD) load equivalent to approximately 8.0 gl(-1), was carried out under batch conditions at 0.5 vvm aeration rate . Digestions were carried out at temperatures of 45, 50, 55, 60 and 65 degrees C (or left unregulated) without pH control to study the effect of digestion temperatures on TAD . The effects of digestion pH on the process were studied at pH 6.0, 7.0, 8.0, 9.0 and 9.5 (and in unregulated control) all at 55 degrees C . Except for digestion at 65 degrees C, which was inoculated extraneously using culture of Bacillus strearothermophilus all reactions were carried out using the populations indigenous to the waste . During digestion at different temperatures, the removal of soluble COD increased with temperature to reach a peak at 60 degrees C before declining slightly, removal of soluble solid (SS) followed similar pattern and reached peak at 65 degrees C being the highest temperature studied, while the degradation of TSS and TS (TSS+TS) decreased with an increase in temperature . Digestion at pH 7.0 was more efficient than at other pH values . Acetate was the predominant volatile fatty acid (VFA) in all the reactions and accounted for up to 90% of the total . Digestion at 60 degrees C led to the greatest accumulation of acetate, and this coincided with the period of highest oxygen uptake, and rapid consumption of soluble carbohydrate . Iso-valerate was also produced at all pH values . Digestion at 55 degrees C and also at pH 7.0 led to rapid and efficient processes with least accumulation of VFA and should be of interest in full-scale processes whenever it is practicable to regulate the digestion pH and temperature . The result of digestion at unregulated pH indicates that gradual adaptation may be used to achieve efficient treatment at elevated pH values . This would be of interest in full-scale processes where it is not practicable to tightly regulate digestion pH, and where the waste is produced at a pH value much higher than neutral. Microb Cell Fact . 2004 Dec 10;3(1):15. The relevance of genetic analysis to dairy bacteria: building upon our heritage; Vadeboncoeur C et al.; Lactic acid bacteria (LAB) are essential for the manufacture of fermented dairy products . Studies on the physiology, biochemistry and genetics of these microorganisms over the last century have contributed considerably to the improvement of fermentation processes and have resulted in better and safer products . Nevertheless, the potential of LAB is far from being maximized . The sophistication of biotechnologies and the availability of complete genome sequences have opened the door to the metabolic engineering of LAB . In this regard, the recent publication of the complete genome sequences of two Streptococcus thermophilus strains will provide a key tool to facilitate the genetic manipulation of this important dairy species. Arch Latinoam Nutr, 2004 Jun, 54(2), 229 - 34 {Use of Phaseolus vulgaris and Vigna sinensis in a fermented dairy drink}; Granito M et al.; The objective of this work was to develop a new kind fermented dairy drink, partially substituted with clear varieties of Phaseolus vulgaris (caraota) and Vigna sinensis (frijol) . The formulation of fermented dairy drinks included sterile extracts of caraota and frijol, as partial substitutes which replaced milk: 10, 20 and 30% . The mixtures were inoculated with 2% of a mixture of Lactobacillus acidophillus, Streptococcus thermophilus and Bifidobacterium sp . and were incubated at 42 degrees C for 7 hours . Mango and guava jams were used as flavorings at 20% . On the basis of the sensorial evaluation the mixtures 10% frijol-mango, 10% frijol-guava, 30% caraota-mango and 20% caraota-guava were selected . In the selected fermented dairy drinks, the levels of protein, soluble and insoluble fiber, available and resistant starches were increased and the protein digestibility was 81% . The technical feasibility of partial substitution of milk with extracts of Phaseolus vulgaris or Vigna sinensis . For the elaboration of a fermented dairy drink similar to the liquid yogurt kind was demonstrated. Extremophiles . 2004 Oct 2; {Epub ahead of print} Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus; Hobel CF et al.; A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli . The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da . The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain . The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C . Due to poor expression of ChiA, a signal peptide-lacking mutant, chiADelta sp, was designed and used subsequently . The optimal temperature and pH for chitinase activity of both ChiA and ChiADeltasp were 70 degrees C and 4.5-5, respectively . The enzyme maintained 100% activity after 16 h incubation at 70 degrees C, with half-lives of 3 h at 90 degrees C and 45 min at 95 degrees C . Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues . The enzyme was fully inhibited by 5 mM HgCl(2), but excess ethylenediamine tetraacetic acid relieved completely the inhibition . The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH . Our results show that the R . marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far. Curr Opin Struct Biol, 2004 Dec, 14(6), 648 - 55 Structural bases of hydrogen tunneling in enzymes: progress and puzzles; Liang ZX et al.; Accumulating experimental evidence suggests that the occurrence of hydrogen tunneling is likely to be widespread in enzyme-catalyzed reactions . The realization that hydrogen can transfer via tunneling mechanisms has far-reaching implications for our understanding of enzyme catalysis involving proton, hydride or hydrogen atom transfer reactions . The current status of the field is highlighted by three enzyme systems that have been under intensive study in recent years, including soybean lipoxygenase-1, thermophilic alcohol dehydrogenase and dihydrofolate reductase . Particular attention has been devoted to the issues of whether protein dynamics modulate hydrogen tunneling probability and whether the tunneling process contributes to the catalytic power of enzymes. J Mol Biol, 2005 Jan 21, 345(3), 501 - 12 Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family; Mandrich L et al.; A superposition between the structures of Alicyclobacillus acidocaldarius esterase 2 (EST2) and Burkholderia cepacia lipase, the latter complexed with a phosphonate inhibitor, allowed us to hypothesize for the EST2 N terminus a role in restricting the access to the active site and therefore in modulating substrate specificity . In order to test this hypothesis we generated by site-directed mutagenesis some truncated versions of EST2 and its double mutant M211S/R215L (S/L) at the N terminus . In parallel, an analysis of the Sulfolobus solfataricus P2 genome allowed us to identify a gene coding for a putative esterase of the HSL family having a natural deletion of the corresponding region . The product of this gene and the above-mentioned EST2 mutants were expressed in Escherichia coli, purified and characterised . These studies support the notion that the N terminus affects substrate specificity other than several other enzyme parameters . Although the deletions afforded a tenfold and 550-fold decrease in catalytic efficiency towards the best substrate pNP-hexanoate at 50 degrees C for EST2 and S/L, respectively, the analysis of the specific activities with different triacylglycerols with respect to pNP-hexanoate showed that their ratios were higher for deleted versus non-deleted enzymes, on all tested substrates . In particular, the above ratios for glyceryl tridecanoate were 30-fold and 14-fold higher in S/L and EST2 deleted forms, respectively, compared with their full-length versions . This behaviour was confirmed by the analysis of the S.solfataricus esterase, which showed similar specific activities on pNP-hexanoate and triacylglycerols; in addition, higher activities on the latter substrates were observed in comparison with EST2, S/L and their deleted forms . Finally, a dramatic effect on thermophilicity and thermostability in the EST2 deleted forms was observed . This is the first report highlighting the importance of the "cap" domain in the HSL family, since the N terminus partly contributes to the building up of this structure. Water Sci Technol, 2004, 50(9), 107 - 14 Anaerobic co-digestion of sewage sludge and food waste using temperature-phased anaerobic digestion process; Kim HW et al.; This study was performed to overcome the low efficiency of anaerobic digestion of sewage sludge and food waste by combining temperature-phased digestion, sequencing batch operation, and co-digestion technology . It was demonstrated that the temperature-phased anaerobic sequencing batch reactor (TPASBR) system for the co-digestion of sewage sludge and food waste resulted in enhanced volatile solids (VS) reduction and methane production rate . At the organic loading rate (OLR) of 2.7 g VS/l/d, the TPASBR system showed the higher VS reduction (61.3%), CH4 yield (0.28 l/g VS(added)) and CH4 production rate (0.41 l CH4/l/d) than those (0.29 l CH4/l/d) of the mesophilic two-stage ASBR (MTSASBR) . In the specific methanogenic activity (SMA) tests on thermophilic biomass of the TPASBR system, the average SMA of acetate (93 ml CH4/gVSS/d) was much higher than those of propionate (46 ml CH4/g VSS/d) and butyrate (76 ml CH4/g VSS/d) . Also, higher specific hydrolytic activity (SHA, 217 mg COD/g VSS/d) of the biomass supported fast hydrolysis under thermophilic conditions . The track study revealed that the most active period of the 24 h cycle was between 6 and 12 h . The enhanced performance of the TPASBR system could be attributed to longer solids retention time, fast hydrolysis, higher CH4 conversion rate, and balanced nutrient condition of co-substrate . It was verified that this combination could be a promising and practical alternative for the simultaneous recycling of two types of organic fraction of municipal solid waste (OFMSW) with high stability. J Biol Chem . 2004 Dec 2; {Epub ahead of print} Characterization of a 5' polynucleotide kinase-3' phosphatase from bacteriophage RM378; Blondal T et al.; A polynucleotide kinase from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus was identified, expressed and purified . This polynucleotide kinase demonstrated to have a 5' kinase domain as well as a 3' phosphohydrolase domain . The RM378 polynucleotide kinase had limited sequence similarity to the 5' kinase domain of the T4 bacteriophage polynucleotide kinase, but apparent homology was not evident within the 3' phosphohydrolase domain . The domain order of RM378 polynucleotide kinase was reversed relative to that of T4 polynucleotide kinase . The RM378 phosphohydrolase domain displayed some sequence similarity with the bacterial poly(A) polymerase family including a HD motif, characteristic of the diverse superfamily of metal dependent HD phosphohydrolases . The RM378 polynucleotide kinase was biochemically characterized and shown to possess 5' kinase activity on RNA and single- and double-stranded DNA at elevated temperatures . It also showed phosphohydrolase activity on 2'-3' cyclic-adenosine monophosphate . This description of the RM378 polynucleotide kinase, along with the recently described RM378 RNA ligase, suggest that the RM378 bacteriophage has to counter similar antiphage mechanism in R . marinus as the T4 phage has to counter in Escherichia coli. Nucleic Acids Res, 2004, 32(21), 6292 - 303 Print 2004. Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus; Takami H et al.; We present herein the first complete genome sequence of a thermophilic Bacillus-related species, Geobacillus kaustophilus HTA426, which is composed of a 3.54 Mb chromosome and a 47.9 kb plasmid, along with a comparative analysis with five other mesophilic bacillar genomes . Upon orthologous grouping of the six bacillar sequenced genomes, it was found that 1257 common orthologous groups composed of 1308 genes (37%) are shared by all the bacilli, whereas 839 genes (24%) in the G.kaustophilus genome were found to be unique to that species . We were able to find the first prokaryotic sperm protamine P1 homolog, polyamine synthase, polyamine ABC transporter and RNA methylase in the 839 unique genes; these may contribute to thermophily by stabilizing the nucleic acids . Contrasting results were obtained from the principal component analysis (PCA) of the amino acid composition and synonymous codon usage for highlighting the thermophilic signature of the G.kaustophilus genome . Only in the PCA of the amino acid composition were the Bacillus-related species located near, but were distinguishable from, the borderline distinguishing thermophiles from mesophiles on the second principal axis . Further analysis revealed some asymmetric amino acid substitutions between the thermophiles and the mesophiles, which are possibly associated with the thermoadaptation of the organism. Biotechnol Prog, 2004 Nov-Dec, 20(6), 1855 - 60 DNA polymerase-catalyzed elongation of repetitive hexanucleotide sequences: application to creation of repetitive DNA libraries; Kurihara H et al.; We demonstrate the elongation of various hexanucleotide sequences with thermophilic DNA polymerase, under isothermal or thermal cyclic reaction conditions . We prepared 10 types of double repeat hexanucleotide duplexes with various GC compositions containing between 0 and 6 GC nucleotides per repeat and incubated these duplexes with thermophilic Taq DNA polymerase and dNTPs at various temperatures . All of the model repetitive short duplexes were elongated under the isothermal incubation conditions, although there were some differences in the elongation efficiencies derived from the GC composition in the repetitive sequences . It was also found that all of the model repetitive duplexes were extended more effectively by a 3-step thermal cyclic reaction involving denaturation, annealing, and extension . On the basis of this technique, we prepared a glutamate-encoding short repetitive duplex and created long repetitive DNAs under isothermal and thermal cyclic reaction conditions . DNA sequencing analysis of the cloned repetitive DNA revealed that well-ordered long repetitive DNAs of various chain lengths were created by this DNA polymerase-catalyzed ligation method, and these were easily cloned into vectors by the TA-cloning method . This method could be useful for obtaining DNAs encoding arbitrary long repetitive amino acid sequences more effectively than the conventional T4 ligase-catalyzed ligation method. Appl Environ Microbiol, 2004 Dec, 70(12), 7236 - 40 Novel physiological features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens; Henstra AM et al.; Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H2 and CO2 . Besides CO, only pyruvate was described as serving as an energy source . Based on 16S rRNA gene sequence similarity, C . hydrogenoformans is closely related to Thermoterrabacterium ferrireducens . T . ferrireducens is like C . hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium . However, it is capable of using various electron donors and acceptors for growth . Growth of C . hydrogenoformans with multiple electron donors and acceptors was tested . C . hydrogenoformans oxidized formate, lactate, glycerol, CO, and H2 with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor . Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor . T . ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H2 from CO . In contrast to what was published before, T . ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors. Appl Environ Microbiol, 2004 Dec, 70(12), 7140 - 7 A group II intron-type open reading frame from the thermophile Bacillus (Geobacillus) stearothermophilus encodes a heat-stable reverse transcriptase; Vellore J et al.; The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR) . However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs . Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements . These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription . Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs . Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10 . The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria . The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified . Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75 degrees C. Nucleic Acids Res, 2004, 32(21), 6176 - 86 Print 2004. A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system; Guy CP et al.; We have identified a novel structure-specific nuclease in highly fractionated extracts of the thermophilic archaeon Methanothermobacter thermautotrophicus (Mth) . The 71 kDa protein product of open reading frame mth1090 is a nuclease with ATPase activity, which we call Nar71 (Nuclease-ATPase in Repair, 71 kDa) . The nar71 gene is located in a gene neighbourhood proposed by genomics to encode a novel DNA repair system conserved in thermophiles . The biochemical characterization of Nar71 presented here is the first analysis from within this neighbourhood, and it supports the insight from genomics . Nuclease activity of Nar71 is specific for 3' flaps and flayed duplexes, targeting single-stranded DNA (ssDNA) regions . This activity requires Mg2+ or Mn2+ and is greatly reduced in ATP . In ATP, Nar71 displaces ssDNA, also with high specificity for 3' flap and flayed duplex DNA . Strand displacement is weak compared with nuclease activity, but in ATPS it is abolished, suggesting that Nar71 couples ATP hydrolysis to DNA strand separation . ATPase assays confirmed that Nar71 is stimulated by ssDNA, though not double-stranded DNA . Mutation of Lys-117 in Nar71 abolished ATPase and nuclease activity, and we describe a separation-of-function mutant (K68A) that has lost ATPase activity but retains nuclease activity . A model of possible Nar71 function in DNA repair is presented. Bioprocess Biosyst Eng . 2004 Nov 24; {Epub ahead of print} High-rate thermophilic methane fermentation on short-chain fatty acids in a down-flow anaerobic packed-bed reactor; Tatara M et al.; In order to maximize the efficiency of methane fermentation on short-chain fatty acids, gro