Thermophiles

Ann N Y Acad Sci, 2003 Mar, 984, 39 - 52
Closing pulp and paper mill water circuits with membrane filtration; Nuortila-Jokinen J et al.; In this study membrane filtration, ultrafiltration, and nanofiltration alone and as part of hybrid processes are considered as means to purify pulp and paper mill process waters suitable for reuse . Thermophilic aerobic biological treatment, pH adjustment, flocculation, and ozonation were tested as pretreatment methods on pilot or on laboratory scale . The aim was to increase flux and reduce fouling by various pretreatment steps and, thus, increase the competitiveness of the membrane process . The results were also evaluated by comparing the benefits obtained against the costs . It was discovered that benefits could be obtained with all the pretreatments tried . Thermophilic aerobic biology assisted in the removal of organic material and increased flux significantly, but the costs were the highest . The most cost-effective processes, however, seem to be pH-adjusted nanofiltration and flocculation nanofiltration hybrid processes, which is understandable because of their significantly lower investment costs compared to, for example, those of biological process . The pH adjustment increased the electrostatic repulsion between negatively charged solutes and membrane, thereby increasing the flux . Flocculation removed the foulants effectively from the feed and it both increased flux and reduced fouling . Yet, many noteworthy benefits were obtained also with ultrafiltration and ozonation . All of the hybrid processes tested could be applied at various points of the water circuit of an integrated pulp and paper mill for purification purposes . The eventual superiority and cost-effectiveness of the applied process remains to be determined case by case.

Int J Immunopathol Pharmacol, 1999 May, 12(2), 97 - 102
Influence of the oral administration of lactic acid bacteria on iga producing cells associated to bronchus; Perdigon G et al.; Intestinal, respiratory and genitourinary mucosal surfaces are the most important routes of entry for microbial pathogens . The stimulus of the mucosal immunity is not easy because the trigger keys for the activation do not follow the ones of the systemic immune response . In previous works we have demonstrated that some Lactic Acid Bacteria (LAB), when orally administered, can induce an enhance of the gut immune response . Taking into account the concept of a common mucosal response, we studied the effect of orally administered mice with Lactobacillus casei, L . acidophilus, L . rhamnosus, L . delbrueckii subsp . bulgaricus, Streptococcus salivarius subsp . thermophilus and Lactococcus lactis on the IgA secreting cells associated to bronchus . As shown before, oral immunostimulation with LAB induced an increase of the IgA* cells at intestinal level by a dose depending effect . In this study it is also showed that the LAB assayed, with exception of L . acidophilus, were able to enhance IgA+ cells at bronchial level, being also this effect dose dependent . The increment induced by some LAB in the number of IgA+ cells on the mucosa surface of the lower respiratory tract may be very important to prevent bronchus diseases.

Proc Natl Acad Sci U S A, 2003 Jun 10, 100(12), 7003 - 8 Epub 2003 Jun 02.
NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site; Hoffmann B et al.; Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation . We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop . This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop . The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs . From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop . One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis . Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures . In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction . An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme . The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme.

J Theor Biol, 2003 Jun 21, 222(4), 495 - 503
Gene arrangements and branching orders of gram-positive bacteria; Kunisawa T; The availability of complete genomic sequence data allows one to develop new methods of reconstructing phylogenetic trees . A simple method of reconstructing branching orders based on gene transposition (or lateral transfer) is presented . It is argued that specific gene arrangements on four different genomes could determine a branching order . A computer search for such gene arrangements was carried out against gene order data of completely sequenced Gram-positive bacteria . Gene arrangements around ribosomal protein S4 gene, murC (UDP-N-acetylmuramate:alanine ligase) gene and dnaE (DNA polymerase III alpha chain) gene each suggest a branching order in which actinobacteria with a high genomic G+C content first branched off from other Gram-positives with a low G+C content and then a split occurred between Mycoplasma species and a group closely related to Bacillus subtilis . A recently sequenced thermophilic bacterium Thermoanaerobacter tengcongensis is suggested to have branched off from the lineage leading to the low G+C Gram-positives prior to the split between the Mycoplasma and Bacillus groups . By contrast to the indel analysis in which a single evolutionary event of insertion or deletion of a signature sequence is assumed, the present method does not necessarily require such a parsimonious assumption of gene transposition.

J Dairy Sci, 2003 May, 86(5), 1632 - 8
Microstructure and rheology of yogurt made with cultures differing only in their ability to produce exopolysaccharides; Hassan AN et al.; Yogurt was made using an exopolysaccharide-producing strain of Streptococcus thermophilus and its genetic variant that only differed from the mother strain in its inability to produce exopolysaccharides . The microstructure was investigated using confocal scanning laser microscopy, allowing observation of fully hydrated yogurt and the distribution of exopolysaccharide within the protein network . Yogurt made with the exopolysaccharide-producing culture exhibited increased consistency coefficients, but lower flow behavior index, yield stress, viscoelastic moduli and phase angle values than did yogurt made with the culture unable to produce exopolysaccharide . The exopolysaccharides, when present, were found in pores in the gel network separate from the aggregated protein . These effects could be explained by the incompatibility of the exopolysaccharides with the protein aggregates in the milk . Stirring affected the yogurt made with exopolysaccharide differently from yogurt without exopolysaccharide, as it did not exhibit immediate syneresis, although the structural breakdown was increased . The shear-induced microstructure in a yogurt made with exopolysaccharide-producing culture was shown to consist of compartmentalized protein aggregates between channels containing exopolysaccharide, hindering syneresis as well as the buildup of structure after stirring.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 943 - 5 Epub 2003 Apr 25.
Crystallization and preliminary X-ray diffraction analysis of thermophilic imidase from pig liver; Huang CY et al.; Imidase is an enzyme, also known as dihydropyrimidinase (EC 3.5.2.2), hydantoinase, dihydropyrimidine hydrase or dihydropyrimidine amidohydrolase, that catalyzes the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and many other imides . Substrate specificity, metal content and amino-acid sequence all differ significantly between bacterial and mammalian imide-hydrolyzing enzymes . In this study, a thermophilic imidase was isolated from pig liver and crystallized . Two kinds of imidase crystals were grown by the hanging-drop vapour-diffusion method using polyethylene glycol MME 5000 and 2-propanol as precipitants . One belongs to the triclinic P(1) space group, with unit-cell parameters a = 96.35, b = 96.87, c = 154.87 A, alpha = 82.10, beta = 72.54, gamma = 77.19 degrees, and the other belongs to the orthorhombic C222(1) space group, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21 A.

Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 930 - 2 Epub 2003 Apr 25.
Crystallization and preliminary X-ray diffraction analysis of ribosomal protein L11 methyltransferase from Thermus thermophilus HB8; Kaminishi T et al.; Ribosomal proteins are subjected to a variety of post-translational modifications, of which methylation is the most frequently found in all three kingdoms of life . PrmA is the only bacterial enzyme identified to date that catalyzes the methylation of a ribosomal protein . It is responsible for the introduction of nine methyl groups into the N-terminal domain of ribosomal protein L11 . The PrmA protein from Thermus thermophilus HB8 was crystallized and a preliminary X-ray diffraction analysis was performed . A cryocooled crystal diffracted X-rays beyond 1.9 A using synchrotron radiation.

J Biol Chem, 2003 Aug 8, 278(32), 30022 - 7 Epub 2003 May 27.
Crystal structure of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis; Wada T et al.; The crystal structure of the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) kinase from the thermophilic bacterium Thermus thermophilus HB8 has been determined at 1.7-A resolution . This enzyme catalyzes phosphorylation of the 2-hydroxyl group of CDP-ME, the fourth step of the non-mevalonate pathway, which is essential for isoprenoid biosynthesis in several pathogenic microorganisms . Since this pathway is absent in humans, it is an important target for the development of novel antimicrobial compounds . The structure of the enzyme is similar to the structures of mevalonate kinase and homoserine kinase, members of the GHMP superfamily . Lys8 and Asp125 are active site residues in mevalonate kinase that also appear to play a catalytic role in CDP-ME kinase . Both the mevalonate and the non-mevalonate pathways therefore involve closely related kinases with similar mechanisms . Assaying the enzyme showed that CDP-ME kinase will phosphorylate CDP-ME but not 4-(uridine 5'-diphospho)-2-C-methyl-D-erythritol, indicating the substrate pyrimidine moiety is involved in important interactions with the enzyme.

J Mol Biol, 2003 Jun 6, 329(3), 403 - 10
Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: mechanistic implications based on the free and cellobiose-bound forms; Hirvonen M et al.; Cellulose, a polysaccharide of beta-1,4-linked D-glucosyl units, is the major component of plant cell walls and one of the most abundant biopolymers in nature . Cellulases (cellobiohydrolases and endoglucanases) are enzymes that catalyse the hydrolysis of cellulose to smaller oligosaccharides, a process of paramount importance in biotechnology . The thermophilic fungus Melanocarpus albomyces produces a 20 kDa endoglucanase known as 20K-cellulase that has been found particularly useful in the textile industry . The crystal structures of free 20K-cellulase and its complex with cellobiose have been determined at 2.0 A resolution . The enzyme, classified into the glycoside hydrolase family 45, exhibits the characteristic six-stranded beta-barrel found before in Humicola insolens endoglucanase V structure . However, the active site in the 20K-cellulase shows a closing of approximately 2.5-3.5A while a mobile loop identified previously in Humicola insolens endoglucanase V and implicated in the catalytic mechanism is well-defined in 20K-cellulase . In addition, the crystal structure of the cellobiose complex shows a shift in the cellobiose position at the substrate-binding cleft . It is therefore proposed that these alterations may reflect differences in the binding mechanism and catalytic action of the enzyme.

Protein Expr Purif, 2003 Jun, 29(2), 223 - 9
High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris; Oledzka G et al.; Aqualysin I is a heat-stable subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile . We report the high-level expression of an aqualysin I protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris . The expression of aqualysin I in P . pastoris was carried out using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter . Pro-aqualysin I (38kDa) as inactive protein was secreted into the medium of shake-flask cultures at a concentration of 1g/L . It was isolated from the culture supernatant by an ammonium sulfate precipitation and one-step anion exchange chromatography in a nearly pure form and was autoproteolytically activiated by heat treatment . A proteolytic activity test indicated that the purified recombinant aqualysin I was properly folded with a specific activity similar to that of the native enzyme . We also explored the possibility of secreting the GAP expressed aqualysin I in P . pastoris by in-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal . However, the levels of secreted pro-aqualysin I particles were approximately 10 times lower, possibly as a consequence of membrane association or to the influence of the alpha-factor secretion signal sequences on the transcription or secretion of aqualysin I . When considering further optimization of the downstream process and culture conditions for high-level production of recombinant aqualysin I by P . pastoris, this expression system is promising for development as an industrial process.

J Biomol NMR, 2003 Jun, 26(2), 131 - 7
NMR structure of the ribosomal protein L23 from Thermus thermophilus; Ohman A et al.; The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel . In this position L23 plays a central role both for protein secretion and folding . We have determined the solution structure of L23 from Thermus thermophilus . Uncomplexed L23 consists of a well-ordered part, with four anti-parallel beta-strands and three alpha-helices connected as beta-alpha-beta-alpha-beta-beta-alpha, and a large and flexible loop inserted between the third and fourth beta-strand . The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry . A comparison with RNA-complexed crystal structures of L23 from T . thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states . However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall . Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.

J Biol Chem, 2003 Aug 8, 278(32), 29515 - 24 Epub 2003 May 24.
MiaB protein from Thermotoga maritima . Characterization of an extremely thermophilic tRNA-methylthiotransferase; Pierrel F et al.; In Escherichia coli, the MiaB protein catalyzes the methylthiolation of N-6-isopentenyl adenosine in tRNAs, the last reaction step during biosynthesis of 2-methylthio-N-6-isopentenyl adenosine (ms2i6A-37) . For the first time the thermophilic bacterium Thermotoga maritima is shown here to contain such a MiaB tRNA-modifying enzyme, named MiaBTm, and to synthesize ms2i6A-37 as demonstrated by an analysis of modified nucleosides from tRNA hydrolysates . The corresponding gene (TM0653) was identified by sequence similarity to the miaB gene cloned and expressed in E . coli . MiaBTm was purified to homogeneity and thoroughly characterized by biochemical and spectroscopic methods . It is a monomer of 443 residues with a molecular mass of 50,710 kilodaltons . Its amino acid sequence shares the CysXXX-CysXXCys sequence with MiaB from E . coli as well as with biotin synthase and lipoate synthase . This sequence was shown to be essential for chelation of an iron-sulfur center and for activity in these enzymes . As isolated, MiaBTm contains both iron and sulfide and an apoprotein form can coordinate up to 4 iron and 4 sulfur atoms per polypeptide chain . UV-visible absorption, resonance Raman, variable temperature magnetic circular dichroism, and EPR spectroscopy of MiaBTm indicate the presence of a {4Fe-4S}+2/+1 cluster under reducing and anaerobic conditions, whereas {3Fe-4S}+1 and {2Fe-2S}+2 forms are generated under aerobic conditions . The redox potential of the {4Fe-4S}+2/+1 transition is -495 +/- 10 mV (versus the normal hydrogen electrode) . Finally, the expression of MiaBTm from T . maritima in an E . coli mutant strain lacking functional miaB gene allowed production of ms2i6A-37 . These results provide further information on the enzymes involved in methylthiolation of tRNAs.

Biochemistry (Mosc), 2003 Apr, 68(4), 429 - 35
Bacillus species LU4 is an effective producer of thermostable site-specific endonuclease BspLU4I, an isoschizomer of AvaI; Svadbina IV et al.; The site-specific endonuclease BspLU4I was discovered in the thermophilic Bacillus species LU4 strain and purified to functionally pure state by chromatography on blue agarose, hydroxyapatite HTP, and heparin-Sepharose columns . Analysis of cleavage patterns of different DNAs with known nucleotide sequences demonstrated that the enzyme recognizes the CPyCGPuG site on the DNA . Cleavage points in the sequence were determined with the elongated primer method . It was shown that the endonuclease is an isoschizomer of AvaI . The final yield of the enzyme is 2.25.10(6) units per g wet biomass.

Bioresour Technol, 2003 Apr, 87(2), 147 - 53
Biomethanation under psychrophilic conditions: a review; Kashyap DR et al.; Anaerobic digestion of animal manure, sewage and other agricultural wastes at psychrophilic temperatures has not been explored as extensively as either mesophilic or thermophilic digestion, probably due to little anticipation of the development of economically attractive systems using this technology . This review article discusses psychrophilic anaerobic digestion studies reported by various researchers using different substrates . The effect of operational parameters such as type of substrate, size of inoculum, concentration of volatile fatty acids, hydraulic retention time and loading rate, on reduction of TS/VS, BOD/COD and biogas yield is discussed in detail.

J Neurosci, 2003 May 15, 23(10), 4369 - 77
Step response analysis of thermotaxis in Caenorhabditis elegans; Zariwala HA et al.; The nematode Caenorhabditis elegans migrates toward a preferred temperature on a thermal gradient . A candidate neural network for thermotaxis in C . elegans has been identified, but the behavioral strategy implemented by this network is poorly understood . In this study, we tested whether thermal migration is achieved by modulating the probability of turning behavior, as in C . elegans chemotaxis . This was done by subjecting unrestrained wild-type, cryophilic, or thermophilic worms to rapid spatially uniform temperature steps (3 degrees C), up or down from the cultivation temperature . Each of the three types of worms we analyzed showed a different pair of responses to the two types of steps . Comparison of wild-type and mutant response patterns suggested a model in which thermal migration involves a unique response to the gradient depending on the orientation of the worm relative to its preferred temperature . Overall, however, turning probability was modulated in a manner consistent with a role for turning behavior in thermal migration . Our results suggest that sensory systems for thermotaxis and chemotaxis may converge on a common behavioral mechanism.

Ann N Y Acad Sci, 2003 Apr, 986, 212 - 8
Heavy metal transport CPx-ATPases from the thermophile Archaeoglobus fulgidus; Arguello JM et al.; PIB-type ATPases transport diverse heavy metals (Cu(+), Ag(+), Cu(2+) . Zn(2+), Cd(2+), Pb(2+), Co(2+)) across membranes . Toward understanding their mechanisms of metal selectivity, we are studying thermophilic archaeal PIB-type ATPases . Like other PIB ATPases, these are characterized by the presence of a cation binding CPX sequence in their 6th transmembrane segment and by cytoplasmic N-terminus metal binding domains (N-MBDs) . CopA and CopB from the thermophile Archaeoglobus fulgidus were cloned and expressed in E . coli . The resulting proteins were purified in a soluble active form . Typical yields were in the order of 3-5 mg of pure protein per liter of bacterial culture . Both enzymes showed maximum activity at 75-85 degrees C . CopA was activated by Ag(+)>Cu(+) while CopB was activated by Cu(2+)>Ag(+)>Cu(+) . The differences in enzyme selectivity can be explained by different consensus sequences in the transmembrane cation binding domain (CopA: CPC, CopB: CPH) . Mutagenesis studies show that the cysteines in the transmembrane CPC site of CopA are necessary for enzyme function, while those in the N-MBD (CXXC), although not essential, are required for maximum enzyme activity . Different from CopA, CopB has a His-rich N-MBD . Removal of this domain reduced enzyme activity without affecting enzyme selectivity . These studies show that these enzymes are an excellent system for structural functional studies directed to explain the mechanisms of metal selectivity by PIB ATPases.

Biochem Biophys Res Commun, 2003 Jun 6, 305(3), 579 - 85
Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii; Reen FJ et al.; The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii . This is the first report of a hemicellulase gene from this novel source . At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids . The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa . The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases . Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside . D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations . The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.

J Basic Microbiol, 2003, 43(3), 202 - 9
Molecular diversity of mesophilic and thermophilic bacteria in a membrane bioreactor determined by fluorescent in situ hybridization with mxaF- and rRNA-targeted probes; Dias JC et al.; An evaluation of the efficiency of treatment of kraft mill foul condensates in a membrane bioreactor was carried out in the laboratory . Efficiency and rate of methanol removal were quantified at operating temperatures of 35, 45 and 55 degrees C . The structure of the bacterial community present in the reactor biomass at the different operating temperatures was evaluated by in situ hybridization of the biomass samples with fluorescently-labelled probes (FISH) targeting the Eubacteria, the alpha, beta and gamma subclasses of the Proteobacteria, the low G + C content Gram-positive bacteria (Bacillus spp.), while community function was evaluated by in situ hybridization with a methanol dehydrogenase gene (mxaF) probe . Methanol removal efficiency decreased from 99.4 to 92%, and removal rate from 2.69 mg MeOH/l x min to 2.49 mg MeOH/l x min when the operating temperature was increased from 35 to 55 degrees C . This decrease in methanol removal was accompanied by a decrease (from 58% to 42%) in the relative proportion of cells that hybridized with the mxaF probe . The relative proportion of Bacillus spp . increased from 5 to 20% while the proportion of members of the alpha subclass of Proteobacteria decreased from 16% to 6% when the bioreactor operating temperature was raised from 35 to 55 degrees C . The relative proportions of bacteria belonging to the beta (22-25%) and gamma (18-20%) subclasses of the Proteobacteria remained relatively constant regardless of operating temperature . Proteobacteria (alpha, beta and gamma subclasses) and Bacillus spp . represented 61, 67 and 71% of the Eubacteria in the biomass sampled at 35, 45 and 55 degrees C, respectively . The FISH technique was shown to be an efficient method for detection of both structural and functional changes in the bacterial communities that could be related to efficiency of methanol removal in a membrane bioreactor operating at different temperatures.

Cell Death Differ, 2003 Jun, 10(6), 634 - 40
Caspase-like activity in programmed nuclear death during conjugation of Tetrahymena thermophila; Kobayashi T et al.; Apoptosis, or programmed cell death, is common in a variety of eucaryotes, from unicellular protozoa to vertebrates . The ciliated protozoan Tetrahymena thermophila has a unique apoptosis-like nuclear death during conjugation, called programmed nuclear death . This death program involves nuclear condensation (pyknosis) and oligonucleosomal DNA fragmentation in the parental macronucleus . Subsequently, the condensed nucleus is entirely resorbed in the autophagosome . Here we demonstrate that caspase-8- and -9-like activity was detected, but no caspase-3-like activity, by in vitro assay during the nuclear resorption process, suggesting that caspase-like activity is associated with both programmed cell death and apoptosis-like nuclear death in Tetrahymena . The use of indicator dye to detect the loss of mitochondrial membrane potential suggested the uptake of mitochondria and the degenerating macronucleus by the autophagosome . An involvement of mitochondria in the programmed nuclear death is discussed.

Protein Sci, 2003 Jun, 12(6), 1195 - 204
Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum; Le Nours J et al.; beta-1,4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin . They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability . Two fungal beta-1,4-galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined . The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing approximately 56% identity . The M . thermophila and H . insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH . The structure of the M . thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 A and 2.14 A resolution, respectively . The structure of the H . insolens galactanase (HIGAL) was determined to 2.55 A resolution . The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the alpha-helices, and a tighter packing . An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum . Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.

J Biochem (Tokyo), 2003 Apr, 133(4), 507 - 13
Compositional changes in RNA, DNA and proteins for bacterial adaptation to higher and lower temperatures; Nakashima H et al.; It is known that in thermophiles the G+C content of ribosomal RNA linearly correlates with growth temperature, while that of genomic DNA does not . Although the G+C contents (singlet) of the genomic DNAs of thermophiles and methophiles do not differ significantly, the dinucleotide (doublet) compositions of the two bacterial groups clearly do . The average amino acid compositions of proteins of the two groups are also distinct . Based on these facts, we here analyzed the DNA and protein compositions of various bacteria in terms of the optimal growth temperature (OGT) . Regression analyses of the sequence data for thermophilic, mesophilic and psychrophilic bacteria revealed good linear relationships between OGT and the dinucleotide compositions of DNA, and between OGT and the amino acid compositions of proteins . Together with the above-mentioned linear relationship between ribosomal RNA and OGT, the DNA and protein compositions can be regarded as thermostability measures for RNA, DNA and proteins, covering a wide range of temperatures . Both the DNA and proteins of psychrophiles apparently exhibit characteristics diametrically opposite to those of thermophiles . The physicochemical parameters of dinucleotides suggested that supercoiling of DNA is relevant to its thermostability . Protein stability in thermophiles is realized primarily through global changes that increase charged residues (i.e., Glu, Arg, and Lys) on the molecular surface of all proteins . This kind of global change is attainable through a change in the amino acid composition coupled with alterations in the DNA base composition . The general strategies of thermophiles and psychrophiles for adaptation to higher and lower temperatures, respectively, that are suggested by the present study are discussed.

J Biochem (Tokyo), 2003 Feb, 133(2), 173 - 80
A novel neutral amino acid transporter from the hyperthermophilic archaeon Thermococcus sp . KS-1; Akahane S et al.; A novel gene encoding a small neutral amino acid transporter was cloned from the genome of the hyperthermophilic archaeon Thermococcus sp . KS-1 by functional cloning using Escherichia coli strain AK430, which is defective in transporting glycine and D-alanine . The cloned gene, snatA, encoded a protein of 216 amino acid residues, SnatA, and was predicted to be a membrane protein with six membrane-spanning segments . E . coli AK430 cells transformed with snatA transported glycine with an apparent K(t) value of 24 micro M, which was one order of magnitude higher than that of other known glycine/alanine transporters, including cycA of E . coli and acp of thermophilic bacterium PS3 . Competition studies revealed that SnatA transported various L-type neutral amino acids, but its substrate specificity was different from that of CycA or ACP . The glycine transport was inhibited by a protonophore, FCCP, or valinomycin plus nigericin, indicating that the process is dependent on an electrochemical potential of H(+) . Homology searches revealed no homology with any transporters known to date . However, several hypothetical genes in prokaryote cells enrolled in the gene bank showed significantly high homology scores, indicating that snatA and its homologues form a family of prokaryotes . To our knowledge, this is the first report on the cloning of a gene of an amino acid transporter from a hyperthermophilic archaeon.

J Mol Biol, 2003 May 30, 329(2), 271 - 82
Thermophilic topoisomerase I on a single DNA molecule; Dekker NH et al.; Control of DNA topology is critical in thermophilic organisms in which heightened ambient temperatures threaten the stability of the double helix . An important role in this control is played by topoisomerase I, a member of the type IA family of topoisomerases . We investigated the binding and activity of this topoisomerase from the hyperthermophilic bacterium Thermotoga maritima on duplex DNA using single molecule techniques, presenting it with various substrates such as (+) plectonemes, (-) plectonemes, and denaturation bubbles . We found the topoisomerase inactive on both types of plectonemes, but active on denaturation bubbles produced at increased stretching forces in underwound DNA . The relaxation rate depended sensitively on the applied force and the protein concentration . These observations could be understood in terms of a preference of the topoisomerase for single-stranded DNA over double-stranded DNA and allowed for a better understanding of activity of the topoisomerase in bulk experiments on circular plasmids . Binding experiments on a single duplex molecule using a mutant unable to perform cleavage confirmed this interpretation and suggested that T.maritima topoisomerase I behaves like an SSB by lowering the denaturation threshold of underwound DNA . Finally, experiments with a unique single-stranded DNA showed that both ends of the cleaved DNA are tightly maintained by the enzyme, supporting an enzyme-bridged mechanism for this topoisomerase.

Eur J Biochem, 2003 Jun, 270(11), 2446 - 58
Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms . Universal existence of one chlorophyll a' molecule in Photosystem I; Nakamura A et al.; Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography . To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T . elongatus and Synechocystis sp . PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC . The results were compared with the Chl a/P700 ratio determined spectrophotometrically . The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed . Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio . These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.

Lett Appl Microbiol, 2003, 36(6), 399 - 405
Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase in Streptococcus thermophilus; Monnet C et al.; AIMS: To demonstrate the presence of an active alpha-acetolactate decarboxylase in Streptococcus thermophilus and to investigate its physiological function . METHODS AND RESULTS: Streptococcus thermophilus CNRZ385 contains a gene encoding an alpha-acetolactate decarboxylase . Comparison of the production of alpha-acetolactate and its decarboxylation products, by the parent strain and an alpha-acetolactate decarboxylase-deficient mutant, demonstrated the presence of a control of the pool of alpha-acetolactate by valine, leucine and isoleucine . This control occurs via an allosteric activation of the alpha-acetolactate decarboxylase . Cell-free extracts of S . thermophilus were not able to decarboxylate the isoleucine precursor alpha-acetohydroxybutyrate . CONCLUSIONS: These results strongly suggest that one of the physiological functions of the alpha-acetolactate decarboxylase in S . thermophilus is to regulate leucine and valine biosynthesis by diverting the flux of alpha-acetolactate towards acetoin when the branched-chain amino acids are present at a high concentration . SIGNIFICANCE AND IMPACT OF THE STUDY: Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase may occur in several other micro-organisms and explain some of their growth properties.

Mikrobiologiia, 2003 Mar-Apr, 72(2), 212 - 20
{Microorganisms in heat supply lines and internal corrosion of steel pipes}; Rozanova EP et al.; In laboratory experiments with batch cultures of thermophilic microorganisms isolated from urban heat supply systems, the growth of sulfate-reducing, iron-oxidizing, and iron-reducing bacteria was found to accelerate the corrosion rate of the steel-3 plates used in the pipelines . In the absence of bacteria and dissolved oxygen, minimal, corrosion was determined . The aforementioned microorganisms, as well as sulfur-oxidizing bacteria, were found to be widespread in water and corrosion deposits in low-alloy steel pipelines (both delivery and return) of the Moscow heat networks, as well as in the corrosion deposits on the steel-3 plates in a testing unit supplied with the network water . The microorganisms were found in samples with water pH ranging from 8.1 to 9.6 and a temperature lower than 90 degrees C . Magnetite, lepidocrocite, goethite, X-ray amorphous ferric oxide were the corrosion products identified on the steel-3 plates, as well as siderite, aragonite, and S0 . The effect of microbiological processes on the rate of electrochemical corrosion was evaluated from the accumulation of corrosion deposits and from variation in total and local corrosion of the steel plates in a testing unit.

Mikrobiologiia, 2003 Mar-Apr, 72(2), 161 - 7
{Physiology of organotrophic and lithotrophic growth of the thermophilic iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus}; Gavrilov SN et al.; Growth physiology of the iron-reducing bacteria Thermoterrabacterium ferrireducens and Thermoanaerobacter siderophilus was investigated . The stimulation of the organotrophic growth of T . ferrireducens and T . siderophilus in the presence of Fe(III) was shown to be due to the utilization of ferric iron as an electron acceptor in catabolic processes and not to the effect exerted on the metabolism by Fe(II) or by changes in the redox potential . It was established that Fe(III) reduction in T . ferrireducens is not a detoxication strategy . In T . siderophilus, this process is carried out to relieve the inihibitory effect of hydrogen . T . ferrireducens was shown to be capable of lithoautotrophic growth with molecular hydrogen as electron donor and amorphous ferric oxide as electron acceptor, in the absence of any organic substances . The minimum threshold of H2 consumption was 3 x 10(-5) vol % of H2 . The presence of CO dehydrogenase activity in T . ferrireducens suggests that CO2 fixation in this organism involves the anaerobic acetyl-CoA pathway . T . siderophilus failed to grow under lithoautotrophic conditions . The fact that T . ferrireducens contains c-type cytochromes and T . sidrophilus lacks them confirms the operation of different mechanisms of ferric iron reduction in these species.

Proteomics, 2003 May, 3(5), 647 - 57
Dissecting DNA-protein and protein-protein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection; Snapyan M et al.; The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region . Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions) . Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach . We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E . coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP . We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E . coli RNA polymerase alpha subunit.

Syst Appl Microbiol, 2003 Mar, 26(1), 70 - 5
Thermomonas hydrothermalis sp . nov., a new slightly thermophilic gamma-proteobacterium isolated from a hot spring in central Portugal; Alves MP et al.; Several non-pigmented bacterial isolates, with an optimum growth temperature of about 50 degrees C, were recovered from the hot spring at Sao Gemil in Central Portugal . Phylogenetic analyses of the 16S rRNA gene sequence of strain SGM-6T indicated that this organism represents a new species of the gamma-subclass of the Proteobacteria that is closely related to the newly described slightly thermophilic species Thermomonas haemolytica . The major fatty acids of strains SGM-6T and SGM-7 are C15:0 iso, C16:0 iso, C11:0 iso and C11:0 iso 3OH . Ubiquinone 8 is the major respiratory quinone . The new isolates are strictly organotrophic and aerobic . Strain SGM-6T only assimilated D-glucose, D-maltose, D-cellobiose, D-furanose, L-glutamate, L-glutamine, L-lysine, L-proline, L-ornithine, acetate, L-glutamic acid and pyruvate of sixty-five carbon sources tested . Strain SGM-7 also assimilates L-serine, but does not assimilate L-ornithine . On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strains SGM-6T and SGM-7 represent a new species most closely related to Thermomonas haemolytica for which we propose the name Thermomonas hydrothermalis.

Brain Pathol, 2003 Apr, 13(2), 237 - 9
December 2002: 19-year old male with febrile illness after jet ski accident; Gyori E; The December 2002 COM . A 19-year-old healthy male fell into stagnant water of the intercostal waterway (salt water of South Florida), following a jet ski accident . He sustained minor superficial injuries but engulfed significant quantities of water and sediment . A few days later he developed bifrontal headaches, vomiting, a stiff neck and a temperature of 102 degrees F . A CT scan on admission without contrast was negative . The CSF had markedly elevated white count but bacterial and fungal cultures were negative . He became progressively lethargic . On the fifth day he developed seizure activity . He expired the next day despite antibiotics . Gross examination of the brain at autopsy revealed edema, cerebellar tonsillar herniation and purulent meningitis . Microscopic examination revealed a massive leptomeningeal inflammatory infiltrate composed of neutrophils, lymphocytes, and numerous histiocyte-like cells . The inflammatory infiltrate extended into the cerebral parenchyma in numerous areas also involving the cerebellum, brainstem and ventricular system . Given the exposure to stagnant water (later confirmed to be a man-made fresh water lake), and the numerous histiocytic-like cells, suspicion for an amebic etiology of the disease process was raised and the CDC identified the ameba as Naegleria Fowleri . Infection by Naegleria Fowleri, a free-living ameba, occurs after exposure to polluted water in man-made fresh water lakes, ponds, swimming pools, particularly during the warm weather months when the thermophilic ameba grows well . The pathologic substrate of the infection is an acute hemorrhagic, necrotizing meningo-encephalitis mainly at the base of the brain, brainstem and cerebellum occurring in young, healthy individuals.

Extremophiles, 2003 Oct, 7(5), 361 - 70 Epub 2003 May 13.
Desulfurobacterium crinifex sp . nov., a novel thermophilic, pinkish-streamer forming, chemolithoautotrophic bacterium isolated from a Juan de Fuca Ridge hydrothermal vent and amendment of the genus Desulfurobacterium; Alain K et al.; A novel thermophilic, chemolithoautotrophic bacterium, designated as NE1206(T), was isolated from a Juan de Fuca Ridge hydrothermal vent sample (tubes of the annelid polychaete Paralvinella sulfincola attached to small pieces of hydrothermal chimney) . The cells were rod-shaped (1.2-3.5 x 0.4-0.7 microm), occurring as single motile rods or forming macroscopic aggregates visible as pinkish to brownish streamers . The new isolate was anaerobic . It grew between 50 and 70 degrees C (optimum 60-65 degrees C; doubling time approximately 1 h 15 min at 60 degrees C), between pH 5.0 and 7.5 (optimum pH around 6.0-6.5) and at sea salts concentrations between 20 and 40 g l(-1 )(optimum 30 g l(-1)) . Cells grew chemolithoautotrophically in an H(2)/CO(2) atmosphere (80/20, v/v; 200 kPa) . Molecular hydrogen was the sole electron donor used by the strain . Nitrate and elemental sulfur served as electron acceptors, yielding ammonia and hydrogen sulfide, respectively (nitrate reduction supported higher growth rates than sulfur reduction) . The G+C content of the genomic DNA was 36.7+/-0.8 mol% . Phylogenetic analyses of the 16S rRNA gene located the strain within the genus Desulfurobacterium . However, the novel isolate possesses physiological and biochemical characteristics that differ from the previously described species of this genus . We propose that the isolate represents a novel species, Desulfurobacterium crinifex sp . nov . The type strain is NE1206(T) (DSM 15218(T), CIP 107649(T)) . An amendment of the genus Desulfurobacterium description is proposed, based on the phenotypic characteristics of the novel species.

Extremophiles, 2003 Aug, 7(4), 307 - 17 Epub 2003 May 13.
Distribution and phylogenetic diversity of the subsurface microbial community in a Japanese epithermal gold mine; Inagaki F et al.; Distribution and phylogenetic diversity of microbial communities in hot, deep underground environments in the Hishikari epithermal gold mine, southern part of Kyushu, Japan, were evaluated using molecular phylogenetic analyses . Samples included drilled cores such as andesitic volcanic rock (0.95-1.78 Ma) and the oceanic sedimentary basement rock of Shimanto-Supergroup (100 Ma), as well as geothermal hot aquifer waters directly collected from two different sites: AW-site (71.5 degrees C, pH 6.19) and XW-site (85.0 degrees C, pH 6.80) at a depth of 350 mbls (meters below land surface) . Based on PCR-amplified 16S rRNA gene clone analysis, the microbial communities in the drilled cores and the hot aquifer water from the XW-site consisted largely of the 16S rRNA gene sequences, closely related to the sequences often found in marine environments, while the aquifer water from the AW-site contained 16S rRNA gene sequences representing members of Aquificales, thermophilic methanotrophs within the gamma-subdivision of the Proteobacteria and uncultivated strains within the beta-subdivision of Proteobacteria . The cultivable microbial community detected by enrichment cultivation analysis largely matched that detected by the culture-independent molecular analysis.

Appl Microbiol Biotechnol, 2003 May, 61(4), 323 - 8 Epub 2003 Mar 22.
Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum; Aquino AC et al.; An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration . The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE . The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B) . Optima of pH and temperature were 6.0 and 60 degrees C, respectively . In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch . The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity . The alpha-amylase produced by S . thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order . The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses . Maltose and traces of glucose were formed only after 3 h of reaction . These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 356 - 61 Epub 2003 May 13.
2-(2'-Hydroxyphenyl)benzene sulfinate desulfinase from the thermophilic desulfurizing bacterium Paenibacillus sp . strain A11-2: purification and characterization; Konishi J et al.; 2-(2'-Hydroxyphenyl)benzene sulfinate (HPBSi) desulfinase (TdsB), which catalyzes the final step of desulfurization of dibenzothiophene (DBT), was purified from a thermophilic DBT- and benzothiophene (BT)-desulfurizing bacterium: Paenibacillus sp . strain A11-2 . The molecular mass of the purified enzyme was 31 kDa and 39 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting a monomeric structure . The optimal temperature and pH for the reaction involving TdsB was 55 degrees C and the enzyme was more resistant to heat treatment than DszB, a counterpart purified from Rhodococcus erythropolis . The optimum pH for TdsB activity was pH 8 . TdsB converted HPBSi to 2-hydroxybiphenyl (2-HBP) and sulfite stoichiometrically . The Km and kcat values for HPBSi were 0.33 mM and 0.32 s(-1), respectively . TdsB was inactivated by SH reagents such as p-chloromercuribenzoic acid and 5,5'-dithio-bis-2-nitrobenzoic acid, but was not inhibited by chelating reagents such as EDTA and o-phenanthroline . TdsB was also inhibited by o-hydroxystyrene, the final desulfurized product of BT . However, 2-HBP and its derivatives showed only a weak inhibitory effect . TdsB desulfurized 2-(2'-hydroxyphenyl)ethen-1-sulfinate to yield o-hydroxystyrene, but DszB could not . A site-directed mutagenesis study revealed the cysteine residue at position 17 to be essential to the catalytic activity of TdsB.

J Biol Chem, 2003 Jul 25, 278(30), 27525 - 31 Epub 2003 May 12.
Thermotoga maritima 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase: the ancestral eubacterial DAHP synthase?
Wu J, Howe DL, Woodard RW.
The gene encoding the 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase from the thermophilic microorganism Thermotoga maritima was cloned, and the enzyme was overexpressed in Escherichia coli . The purified DAHP synthase displays a homotetrameric structure and exhibits maximal activity at 90 degrees C . The enzyme is extremely thermostable, with 50% of its initial activity retained after incubation for approximately 5 h at 80 degrees C, 21 h at 70 degrees C, and 86 h at 60 degrees C . The enzyme appears to follow Michaelis-Menten kinetics with Km for phosphoenolpyruvate = 9.5-13 microm, Km for d-erythrose 4-phosphate = 57.3-350.1 microm, and kcat = 2.3-7.6 s-1 between 50 degrees C and 70 degrees C . Metal analysis indicates that DAHP synthase as isolated contains Zn2+, and the enzyme is inactivated by treatment with EDTA . The apo-enzyme is partially reactivated by a variety of divalent metals including Zn2+, Cd2+, Mn2+, Cu2+, Co2+, and Ni2+ . These observations suggest that T . maritima DAHP synthase is a metalloenzyme . The activity of T . maritima DAHP synthase is inhibited by two of the three aromatic amino acids (l-Phe and l-Tyr) formed in the Shikimate pathway . This report is the first description of a thermophilic eubacterial DAHP synthase.

Biochemistry (Mosc), 2003 Mar, 68(3), 301 - 8
Thermostable DNA-polymerase from the thermophilic archaeon microorganism Archaeoglobus fulgidus VC16 and its features; Chalov SE et al.; A gene (No . AF0497 GenBank, USA) was cloned from the archaeon Archaeoglobus fulgidus strain found in the water of hot springs . This gene contains an open reading frame of 2346 base pairs which encodes a thermostable DNA-polymerase (762 amino acid residues) . A recombinant protein Afu-pol with molecular weight of 94 kD was isolated in an Escherichia coli strain used as a producer and characterized . By site-directed mutagenesis in the afu-pol gene the amino acid residue Glu170 was replaced with Ala; this resulted in a complete loss of the 3;-5;-exonuclease activity of the enzyme . Thus, the Glu170 residue was suggested to be directly involved in formation of the 3;-5;-exonuclease site . Physicochemical features of the exodeficient enzyme form were studied, and the possible use of Afu(exo(-))-pol in the polymerase chain reaction is shown.

Curr Microbiol, 2003 Apr, 46(4), 265 - 9
Ca2+ -dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris; Bhatnagar K et al.; Ca(2+) enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no . 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no . 1227) . The number of transformants showed a marked increase with increasing concentration of CaCl(2) upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly . Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01-0.04 mM along with optimal CaCl(2) concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca(2+)-stimulated transformation . The results of present investigation suggest the involvement of a Ca(2+)-dependent protein activator in the development of Ca(2+)-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete.

Appl Environ Microbiol, 2003 May, 69(5), 2985 - 93
Thermophily in the Geobacteraceae: Geothermobacter ehrlichii gen . nov., sp . nov., a novel thermophilic member of the Geobacteraceae from the "Bag City" hydrothermal vent; Kashefi K et al.; Little is known about the microbiology of the "Bag City" hydrothermal vent, which is part of a new eruption site on the Juan de Fuca Ridge and which is notable for its accumulation of polysaccharide on the sediment surface . A pure culture, designated strain SS015, was recovered from a vent fluid sample from the Bag City site through serial dilution in liquid medium with malate as the electron donor and Fe(III) oxide as the electron acceptor and then isolation of single colonies on solid Fe(III) oxide medium . The cells were gram-negative rods, about 0.5 micro m by 1.2 to 1.5 micro m, and motile and contained c-type cytochromes . Analysis of the 16S ribosomal DNA (rDNA) sequence of strain SS015 placed it in the family Geobacteraceae in the delta subclass of the Proteobacteria . Unlike previously described members of the Geobacteraceae, which are mesophiles, strain SS015 was a thermophile and grew at temperatures of between 35 and 65 degrees C, with an optimum temperature of 55 degrees C . Like many previously described members of the Geobacteraceae, strain SS015 grew with organic acids as the electron donors and Fe(III) or nitrate as the electron acceptor, with nitrate being reduced to ammonia . Strain SS015 was unique among the Geobacteraceae in its ability to use sugars, starch, or amino acids as electron donors for Fe(III) reduction . Under stress conditions, strain SS015 produced copious quantities of extracellular polysaccharide, providing a model for the microbial production of the polysaccharide accumulation at the Bag City site . The 16S rDNA sequence of strain SS015 was less than 94% similar to the sequences of previously described members of the Geobacteraceae; this fact, coupled with its unique physiological properties, suggests that strain SS015 represents a new genus in the family Geobacteraceae . The name Geothermobacter ehrlichii gen . nov., sp . nov., is proposed (ATCC BAA-635 and DSM 15274) . Although strains of Geobacteraceae are known to be the predominant Fe(III)-reducing microorganisms in a variety of Fe(III)-reducing environments at moderate temperatures, strain SS015 represents the first described thermophilic member of the Geobacteraceae and thus extends the known environmental range of this family to hydrothermal environments.

Appl Environ Microbiol, 2003 May, 69(5), 2765 - 72
Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin; Dhillon A et al.; The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria . We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment . The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark) . Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica . Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum . Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation . In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments . In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.

J Biol Chem, 2003 Jul 11, 278(28), 25341 - 7 Epub 2003 May 03.
Global conformations, hydrodynamics, and X-ray scattering properties of Taq and Escherichia coli DNA polymerases in solution; Joubert AM et al.; Escherichia coli polymerase 1 (Pol 1) and Thermus aquaticus Taq polymerase are homologous Type I DNA polymerases, each comprised of a polymerase domain, a proofreading domain (inactive in Taq), and a 5' nuclease domain . "Klenow" and "Klentaq" are the large fragments of Pol 1 and Taq and are functional polymerases lacking the 5' nuclease domain . In the available crystal structures of full-length Taq, the 5' nuclease domain is positioned in two different orientations: in one structure, it is extended out into solution, whereas in the other, it is folded up against the polymerase domain in a more compact structure . Analytical ultracentrifugation experiments report s20,w values of 5.05 for Taq, 4.1 for Klentaq, 5.3 for E . coli Pol 1, and 4.6 for Klenow . Measured partial specific volumes are all quite similar, indicating no significant differences in packing density between the mesophilic and thermophilic proteins . Small angle x-ray scattering studies report radii of gyration of 38.3 A for Taq, 30.7 A for Klentaq, and 30.5 A for Klenow . The hydrodynamic and x-ray scattering properties of the polymerases were also calculated directly from the different crystal structures using the programs HYDROPRO (Garcia De La Torre, J., Huertas, M . L., and Carrasco, B . (2000) Biophys J . 78, 719-730) and CRYSOL (Svergun, D . I., Barberato, C., and Koch, M . H . J . (1995) J . Appl . Crystalogr . 28, 768-773), respectively . The combined experimental and computational characterizations indicate that the full-length polymerases in solution are in a conformation where the 5' nuclease domain is extended into solution . Further, the radius of gyration, and hence the global conformation of Taq polymerase, is not altered by the binding of either matched primer template DNA or ddATP.

J Mol Biol, 2003 May 16, 328(5), 1149 - 60
Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities; Suzuki R et al.; Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function . In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity . The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.

Protein Expr Purif, 2003 May, 29(1), 15 - 23
Expression in E . coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3; Wolfrum A et al.; The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3) . The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli . IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR . For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR . Using these procedures we obtained chromatographically pure and biologically active preparations of both T . thermophilus IF1 and IF3.

Water Res, 2003 May, 37(10), 2467 - 77
Selective optimization in thermophilic acidogenesis of cheese-whey wastewater to acetic and butyric acids: partial acidification and methanation; Yang K et al.; For partial acidogenesis of cheese-whey wastewater, a set of experiments were carried out to produce short-chain volatile fatty acids (VFA) in laboratory-scale continuously stirred tank reactors (CSTR) . The maximum rate of acetic and butyric acid production associated with simultaneous changes in hydraulic retention time (HRT), pH, and temperature was investigated, in which the degree of acidification of the whey to the short-chain VFAs was less than 20% of the influent chemical oxygen demand (COD) concentration . Response surface methodology was successfully applied to determine the optimum physiological conditions where the maximum rates of acetic and butyric acid production occurred . These were 0.40-day HRT, pH 6.0 at 54.1 degrees C and 0.22-day HRT, pH 6.5 at 51.9 degrees C, respectively.The optimum conditions for acetic acid production were selected for partial acidification of cheese-whey wastewater because of a higher rate in combined productions of acetic and butyric acids than that at optimum conditions for butyric acid production . A thermophilic two-phase process with the partial acidification followed by a methanation step was operated . Performance of the two-phase process was compared to the single-phase anaerobic system . The two-phase process clearly showed a better performance in management of cheese-whey wastewater over the single-phase system . Maximum rate of COD removal and the rate of methane production in the two-phase process were, respectively, 116% and 43% higher than those of the single-phase system.

Water Res, 2003 May, 37(10), 2269 - 80
Effect of NaCl on thermophilic (55 degrees C) methanol degradation in sulfate reducing granular sludge reactors; Vallero MV et al.; The effect of NaCl on thermophilic (55 degrees C) methanol conversion in the presence of excess of sulfate (COD/SO(4)(2-)=0.5) was investigated in two 6.5L lab-scale upflow anaerobic sludge bed reactors inoculated with granular sludge previously not adapted to NaCl . Methanol was almost completely used for sulfate reduction in the absence of NaCl when operating at an organic loading rate of 5 g CODL(-1)day(-1) and a hydraulic retention time of 10h . The almost fully sulfidogenic sludge consisted of both granules and flocs developed after approximately 100 days in both reactors . Sulfate reducing bacteria (SRB) outcompeted methane producing archaea (MPA) for methanol, but acetate represented a side-product, accounting for maximal 25% of the total COD converted . Either MPA or SRB did not use acetate as substrate in activity tests . High NaCl concentrations (25 gL(-1)) completely inhibited methanol degradation, whereas low salt concentrations (2.5 g NaClL(-1)) provoked considerable changes in the metabolic fate of methanol . The MPA were most sensitive towards the NaCl shock (25 gL(-1)) . In contrast, the addition of 2.5 gL(-1) of NaCl stimulated MPA and homoacetogenic bacteria.

Water Res, 2003 May, 37(10), 2259 - 68
Microbial diversity in a thermophilic aerobic biofilm process: analysis by length heterogeneity PCR (LH-PCR); Tiirola MA et al.; A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site treatment of pulp and paper mill whitewater lining . The microbial diversity in this process was analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA . The primer pair selected for PCR amplification was first evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups . The fragment contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length . The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier elements was found to be diverse in both stages of the SCBP . During normal operating conditions, sequences belonging to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most prominent LH-PCR peak . Samples from the suspended biomass consisted of completely different bacterial populations, which were, however, similar in the serial reactors . The pilot process experienced alkaline shocks, after which Bacillus-like sequences were detected in both the biofilm and suspended biomass . However, when the conditions were reversed, the normal microbial population in the biofilm recovered rapidly without further biomass inoculations . This study shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater processes.

Plasmid, 2003 Mar, 49(2), 188 - 91
Identification and nucleotide sequence analysis of a cryptic plasmid, pRM21, from Rhodothermus marinus; Ernstsson S et al.; Here we report the identification and nucleotide sequence analysis of pRM21, a plasmid isolated from the thermophilic eubacterium Rhodothermus marinus . pRM21 consists of 2935 bp, has a G+C content of 58.2% and one major open reading frame whose deduced product shows significant similarities to RepA proteins from several plasmids, the highest being to the RepA of pSa from Escherichia coli . A region with the characteristics of iteron-containing replicons, three 19 bp repeats, DnaA boxes, an A+T rich region and GATC sequences, was identified . Of 40 additional R . marinus strains screened for plasmids, six (15%) were found to harbour plasmids with the same size and restriction pattern as pRM21.

Biosci Biotechnol Biochem, 2003 Mar, 67(3), 639 - 42
Continuous cell-free protein synthesis directed by messenger DNA and catalyzed by extract of Thermus thermophilus HB27; Uzawa T et al.; Polypeptide synthesis directed by DNA as the messenger in a cell-free system of Thermus thermophilus was investigated . Polypeptides were synthesized with the addition of neomycin in the presence of DNA catalyzed by the cell extract . The stability of messenger DNA was greater than that of messenger RNA . Continuous cell-free translation with messenger DNA produced polypeptides at the rate of more than 8 microg/h in the presence of spermine.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 557 - 66
Hydrogen production from paper sludge hydrolysate; Kadar Z et al.; The main objective of this study was to develop a system for the production of "renewable" hydrogen . Paper sludge is a solid industrial waste yielding mainly cellulose, which can be used, after hydrolysis, as a feedstock in anaerobic fermentation by (hyper)thermophilic organisms, such as Thermotoga elfii and Caldicellulosiruptor saccharolyticus . Tests on different medium compositions showed that both bacteria were able to produce hydrogen from paper sludge hydrolysate, but the amount of produced hydrogen and the requirement for other components differed . Hydrogen production by T . elfii strongly depended on the presence of yeast extract and salts . By contrast, C . saccharolyticus was less dependent on medium components but seemed to be inhibited by a component present in the sludge hydrolysate . Utilization of xylose was preferred over glucose by C . saccharolyticus.

Plant Cell Physiol, 2003 Apr, 44(4), 447 - 50
Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants; Orlova IV et al.; The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter . Expression of the desaturase was confirmed by Western blotting . Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants . Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments . Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants . The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5724 - 9 Epub 2003 Apr 28.
An unusual mechanism of bacterial gene expression revealed for the RNase P protein of Thermus strains; Feltens R et al.; The RNase P protein gene (rnpA) completely overlaps the rpmH gene (encoding ribosomal protein L34) out of frame in the thermophilic bacterium Thermus thermophilus . This results in the synthesis of an extended RNase P protein (C5) of 163 aa and, by inference, of 240 aa in the related strain Thermus filiformis . Start codons of rnpA and rpmH, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between L34 and C5 translation and, accordingly, between ribosome and RNase P biosynthesis . Within the sequence encoding the N-terminal extensions and downstream of rpmH, several Thermus species exhibit in-frame deletionsinsertions, suggesting relaxed constraints for sequence conservation in this region . Roughly the N-terminal third of T . thermophilus C5 was further shown to be dispensable for RNase P function in vitro by using a precursor tRNA(Gly) substrate from the same organism . Taken together, these data reveal a mode of gene expression that is to our knowledge unprecedented in bacteria.

Biophys J, 2003 May, 84(5), 2831 - 40
Mechanically probing the folding pathway of single RNA molecules; Gerland U et al.; We study theoretically the denaturation of single RNA molecules by mechanical stretching, focusing on signatures of the (un)folding pathway in molecular fluctuations . Our model describes the interactions between nucleotides by incorporating the experimentally determined free energy rules for RNA secondary structure, whereas exterior single-stranded regions are modeled as freely jointed chains . For exemplary RNA sequences (hairpins and the Tetrahymena thermophila group I intron), we compute the quasiequilibrium fluctuations in the end-to-end distance as the molecule is unfolded by pulling on opposite ends . Unlike the average quasiequilibrium force-extension curves, these fluctuations reveal clear signatures from the unfolding of individual structural elements . We find that the resolution of these signatures depends on the spring constant of the force-measuring device, with an optimal value intermediate between very rigid and very soft . We compare and relate our results to recent experiments by Liphardt et al . (2001).

Mol Genet Genomics, 2003 Apr, 269(1), 109 - 15 Epub 2003 Feb 22.
Identification and characterization of a Hsp70 (DnaK) chaperone system from Meiothermus ruber; Pleckaityte M et al.; We have cloned the genes encoding the chaperones of Meiothermus ruber, Hsp70 (Mru.Hsp70), Hsp40 (Mru.Hsp40) and Hsp22 (Mru.Hsp22) . The genes hsp70, hsp22 and hsp40 of M . ruber are organized into an operon . The amino acid sequences of the three M . ruber chaperones show strong similarity with the heat shock proteins of Thermus thermophilus . Both Mru.Hsp40 and its homolog from T . thermophilus lack a cysteine-rich region . However, recombinant Mru.Hsp70 and Mru.Hsp40 associate in an ATP-dependent manner, and assemble into a complex in the absence of other proteins, unlike their counterparts from T . thermophilus, which require DafA for assembly . The analysis revealed that Mru.Hsp70 and Mru.Hsp40 assemble as monomers into the complex, although their homologs from T . thermophilus enter the complex as trimers . The Mru.Hsp70 and Mru.Hsp40 complex increases the spontaneous rate of refolding of denatured mitochondrial malate dehydrogenase by tenfold.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 401 - 6
Microvirga subterranea gen . nov., sp . nov., a moderate thermophile from a deep subsurface Australian thermal aquifer; Kanso S et al.; A strictly aerobic bacterium, strain Fail4T, was isolated from free-flowing geothermal waters of a bore (bore register no . 3768) tapping the Great Artesian Basin of Australia . The non-sporulating, Gram-negative cells of strain Fail4T produced light-pink colonies, were rod-shaped (1 x 1.5-4 microm) and were motile by a single polar flagellum . Strain Fail4T grew optimally at 41 degrees C at a pH of 7.0 and had an absolute requirement for yeast extract . The strain grew on casein hydrolysate, tryptone, gelatin, xylose and acetate in a medium supplemented with 0.06 or 0.006% yeast extract . Weak acid production was detected from glucose and arabinose . Catalase was produced . Nitrite was produced from nitrate . Strain Fail4T was sensitive to antibiotics that inhibit growth of bacteria . The G + C content was 63.5 +/- 0.5 mol% . Strain Fail4T was a member of the class 'Alphaproteobacteria', phylum Proteobacteria, placed almost equidistantly between Methylobacterium species, Chelatococcus asaccharovorans and Bosea thiooxidans (similarity value of 93%) as its nearest phylogenetic relatives . Phylogenetic and phenotypic evidence suggest that strain Fail4T (=ATCC BAA-295T = DSM 14364T) should be placed as the type strain of a species in a newly created genus, for which the name Microvirga subterranea gen . nov., sp . nov . is proposed.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 377 - 80
Thermoleophilum album and Thermoleophilum minutum are culturable representatives of group 2 of the Rubrobacteridae (Actinobacteria); Yakimov MM et al.; Analysis of the morphological and genotypic properties of three obligately thermophilic strains of Thermoleophilum album and Thermoleophilum minutum, originally described as green non-sulfur bacteria, indicates that these taxa belong to the Rubrobacter subdivision of the Actinobacteria . EM of the cell wall clearly showed morphology typical of Gram-positive bacteria . A comparison of 16S rRNA gene sequences, including signature nucleotide pairs and secondary structural features considered diagnostic for the subclass Rubrobacteridae, revealed that the three strains of Thermoleophilum were highly similar and should be considered as members of group 2 of this subclass, represented up to now only by uncultured organisms.

Occup Environ Med, 2003 May, 60(5), 336 - 42
Effects of bioaerosol polluted outdoor air on airways of residents: a cross sectional study; Herr CE et al.; BACKGROUND: Bioaerosol pollution of workplace and home environments mainly affects airways and mucous membranes . The effect of environmental outdoor residential bioaerosol pollution, for example, livestock holdings, farming, and waste disposal plants, is unclear . AIMS: To investigate the perceived health of residents living in areas with measurable outdoor bioaerosol pollution (for example, spores of Aspergillus fumigatus and actinomycetes), and effects of accompanying odours . METHODS: In a cross sectional study, double blinded to ongoing microbial measurements, doctors collected 356 questionnaires from residents near a large scale composting site, and from unexposed controls in 1997 . Self reported prevalence of health complaints during the past year, doctors' diagnoses, as well as residential odour annoyance were assessed . Microbiological pollution was measured simultaneously in residential outdoor air . RESULTS: Concentrations of >10(5) colony forming units of thermophilic actinomycetes, moulds, and total bacteria/m(3) air were measured 200 m from the site, dropping to near background concentrations within 300 m . Positive adjusted associations were observed for residency within 150-200 m from the site versus unexposed controls for self reported health complaints: "waking up due to coughing", odds ratio (OR) 6.59 (95% confidence interval (CI) 2.57 to 17.73); "coughing on rising or during the day", OR 3.18 (95% CI 1.24 to 8.36); "bronchitis", OR 3.59 (95% CI 1.40 to 9.4); and "excessive tiredness", OR 4.27 (95% CI 1.56 to 12.15) . Reports of irritative airway complaints were associated with residency in the highest bioaerosol exposure, 150-200 m (versus residency >400-500 m) from the site, and period of residency more than five years, but not residential odour annoyance . Lifetime prevalence of self reported diseases did not differ with exposure . CONCLUSIONS: Bioaerosol pollution of residential outdoor air can occur in concentrations found in occupational environments . For the first time residents exposed to bioaerosol pollution were shown to report irritative respiratory complaints similar to mucous membrane irritation independently of perceived odours.

J Biol Chem, 2003 Jul 11, 278(28), 25926 - 32 Epub 2003 Apr 22.
The crystal structure of a novel unmethylated form of C-phycocyanin, a possible connector between cores and rods in pycobilisomes; Adir N et al.; A novel fraction of c-phycocyanin from the thermophilic cyanobacterium Thermosynechcoccus vulcanus, with an absorption maxima blue-shifted to 612 nm (PC612), has been purified from allophycocyanin and crystallized . The crystals belong to the P63 space group with cell dimensions of 153 A x 153 A x 59 A with a single (alphabeta) monomer in the asymmetric unit, resulting in a solvent content of 65%, and diffract to 2.7 A . The PC612 crystal structure has been determined by molecular replacement and refined to a crystallographic R-factor of 20.9% (Rfree = 27.8%) . The crystal packing in this form shows that the PC612 form of phycocyanin does not associate into hexamers and that its association with adjacent trimers in the unit cell is very different from that found in a previously determined structure of the normal form of T . vulcanus phycocyanin, which absorbs at 620 nm . Analysis of the PC612 structure shows that the alpha subunits, which typically form the interface between two trimers within a hexamer, have a high degree of flexibility, as indicated by elevated B-factors in portions of helices B, E, and G . Examination of calculated electron density omit maps shows that unlike all other structures of phycobiliproteins determined so far, the Asnbeta72 residue is not methylated, explaining the blue-shift in its absorption spectra . On the basis of the results presented here, we suggest that this new form of trimeric phycocyanin may constitute a special minor component of the phycobilisome and may form the contact between the phycocyanin rods and the allophycocyanin core.

Acta Microbiol Pol, 2002, 51(4), 353 - 66
Lipase production from a novel thermophilic Bacillus sp.: application of Plackett-Burman design for evaluating culture conditions affecting enzyme formation; Abdel-Fattah YR et al.; A novel thermophilic Bacillus sp . capable of producing lipase was locally isolated . Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Bacillus thermoleovorans . Plackett-Burman experimental design was used to evaluate cultural conditions affecting lipase production process . Fifteen variables and four dummy variables were examined in the experimental design . Tween 80, temperature, olive oil, aeration, beef extract and inoculum age were found to be the highest positive significant variables affecting lipase activity, whereas pH and calcium chloride were the highest negative significant variables . Moreover, Tween 80, temperature and olive oil positively affected lipase specific activity . On the other hand, gum arabic, inoculum size and calcium chloride had the highest negative effect on lipase specific activity . This study would improve the further optimization steps on the bioprocess development track.

Chromosoma, 2003 Apr, 111(7), 429 - 37 Epub 2003 Mar 11.
Loss of cap structure causes mitotic defect in Tetrahymena thermophila telomerase mutants; Petcherskaia M et al.; Mutation of the telomeric repeat sequence has severe cellular consequences in a variety of systems . A Tetrahymena thermophila telomerase template mutant, ter1-43AA, displays an acute mitotic chromosome segregation defect . In the study described here we investigated the molecular basis for this lethality . Although cloned ter1-43AA macronuclear telomeres had long tracts of wild-type G4T2 repeats, they were capped by a mixture of G4T3 repeats, shown previously to be non-lethal, and G4T4 repeats, the telomeric sequence normally found in hypotrichous ciliates such as Oxytricha . To test further the functionality of the G4T4 repeat sequence in T . thermophila, we devised a new template mutation, ter1-44+AA, that resulted in more uniform synthesis of this sequence at telomere caps in vivo . The ter1-44+AA mutant displayed the most severe mitotic defect reported to date, with up to 85% of the population having micronuclei in anaphase, providing firm evidence that the hypotrich repeat sequence is not functional in Tetrahymena . Surprisingly, in spite of the telomeric sequence mutation, neither the ter1-43AA nor ter1-44+AA mutant displayed any significant loss of telomere length regulation . These results demonstrate that loss of telomere cap integrity, rather than length regulation, leads to the anaphase defect.

J Biol Chem, 2003 Jul 4, 278(27), 24255 - 8 Epub 2003 Apr 21.
Rotation of the proteolipid ring in the V-ATPase; Yokoyama K et al.; V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria . We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K . (2003) Proc . Natl . Acad . Sci . U . S . A . 100, 2312-2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V0 sector, has remained to be proven . V0V1-ATPase that retained sensitivity to N',N'-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V1, and decorated with a bead attached to a proteolipid subunit of V0 . Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclockwise viewed from the membrane side . The rotation proceeded at approximately 3.0 rev/s in average at 4 mm ATP and was abolished by N',N'-dicyclohexylcarbodiimide treatment . Thus, the rotation of the central stalk in V1 accompanies rotation of a proteolipid ring of V0 in the functioning V0V1-ATPase.

J Biol Chem, 2003 Jul 4, 278(27), 24308 - 13 Epub 2003 Apr 21.
Stereoselective in vitro formation of c-type cytochrome variants from Hydrogenobacter thermophilus containing only a single thioether bond; Daltrop O et al.; In vitro formation of Hydrogenobacter thermophilus cytochrome c552 has previously been demonstrated (Daltrop, O., Allen, J . W . A., Willis, A . C., and Ferguson, S . J . (2002) Proc . Natl . Acad . Sci . U . S . A . 99, 7872-7876) . Now we report that the single cysteine variants of H . thermophilus c552, which bind heme via a single thioether bond, also form in vitro . Furthermore, reaction of the apocytochromes containing either AXXCH or CXXAH in the binding motif with 2-vinyldeuteroheme and 4-vinyldeuteroheme resulted predominantly in covalent attachment between Cys-11 and the 2-vinyl moiety and Cys-14 and the 4-vinyl functionality . This observation shows that the covalent attachment of heme to apocytochrome is stereoselective, indicating that the initial non-covalent complexes between apoprotein and heme have to be in the correct stereochemical orientation for preferential promotion of thioether bond formation . Additionally, the heme derivatives 2-vinyldeuteroheme and 4-vinyldeuteroheme were reacted with wild-type H . thermophilus c552 to yield another modification of cytochromes containing only one thioether bond . These results show that the formation of the two thioether bonds in typical c-type cytochromes can occur independently from one another . Aspects of rotational isomerism of heme in heme-proteins are discussed.

J Mol Biol, 2003 May 2, 328(3), 737 - 47
Functional role of C(alpha)-H...O hydrogen bonds between transmembrane alpha-helices in photosystem I; Loll B et al.; The crystal structure at 2.5A resolution of the membrane-intrinsic, homotrimeric photosystem I (PSI) isolated from the thermophilic cyanobacterium Synechococcus elongatus shows that each monomer is composed of 12 protein subunits of which nine are embedded in the membrane and feature a total of 34 transmembrane alpha-helices (TMH) . Hence, PSI provides an ideal case to study "conventional" and C(alpha)-H...O hydrogen bonds between TMH engaged in intra- and intersubunit interactions . Of the total of 75 C(alpha)-H...O hydrogen bonds between TMHs, 72 are intrasubunit and only three are intersubunit . The two largest subunits PsaA and PsaB are each folded into 11 TMHs showing 29 and 24 intrasubunit C(alpha)-H...O hydrogen bonds, respectively, that are not distributed randomly but many of them flank chlorophyll a (Chl a) co-ordinating amino acids, suggesting stabilisation of these structural segments . As major constituent of the trimerisation domain, subunit PsaL is located next to the 3-fold axis relating the three monomers of PSI . PsaL features a unique number of 19 intrasubunit C(alpha)-H...O hydrogen bonds that connect two of its three TMHs but there are no intersubunit C(alpha)-H...O hydrogen bonds between the three PsaL . Of the three intersubunit C(alpha)-H...O hydrogen bonds, two are formed between PsaA and PsaB and one between PsaB and PsaM . The large number of 75 C(alpha)-H...O hydrogen bonds contrasts the 49 conventional hydrogen bonds, indicating that the former and van der Waals contacts determine association and orientation of TMHs in PSI.

Biosens Bioelectron, 2003 May, 18(5-6), 565 - 9
Fluorescence based rRNA sensor systems for detection of whole cells of Saccharomonospora spp . and Thermoactinomyces spp; Neef A et al.; Airborne thermophilic actinomycetes (TPAs) are a growing hygienic challenge in different occupational situations e.g . large scale composting . This study describes first results of a new approach for highly specific and rapid detection of organisms of this group using fluorescently labelled oligonucleotide probes as sensors for whole cells . Three genus-specific 16S rRNA-targeted probes, two for Saccharomonospora spp . and one for Thermoactinomyces spp . were developed and evaluated in a fluorescence in situ hybridisation (FISH) format with agar-grown whole cells . For optimal sensitivity and specificity of FISH, conditions for cell wall permeabilisation and hybridisation stringency were evaluated independently for both genera . Performing specified pretreatment protocols, all three probes yielded strong fluorescence signals . However, the relative fraction of detectable cells or spores clearly depended on the single bacterial species . The probes can serve as cell sensors for direct detection of TPAs in natural samples.

Microb Ecol, 2003 May, 45(4), 373 - 83 Epub 2003 Apr 22.
The use of neutral lipid fatty acids to indicate the physiological conditions of soil fungi; Baath E; The usefulness of measuring neutral lipid fatty acids (NLFAs) and phospholipid fatty acids (PLFAs) separately in order to interpret perturbation effects on soil and compost microorganisms has been studied . Initially the NLFA/PLFA ratios were studied in different soils . Low ratios were found for fatty acids common in bacteria, especially for cyclopropane fatty acids . Higher ratios were found for fatty acids common in eukaryotic organisms such as fungi (18:1omega9 and 18:2omega6,9) or in saturated fatty acids, common to many types of organisms . Adding glucose to a forest soil increased the amounts of the fungal NLFAs 18:1omega9 and 18:2 omega6,9 up to 60 and 10 times, respectively, after 10 days, followed by a gradual decrease . After 3 months incubation, higher levels of these NLFAs were still found compared with the control samples . Adding glucose together with nitrogen (N) and phosphorus (P) resulted in no increase in NLFAs but a 10-fold increase in the PLFAs 18:1omega9 and 18:2omega6,9 . Thus, the NLFA/PLFA ratios for these fatty acids were lower than in the no-addition control when glucose was added together with N and P, but higher when glucose was added alone, even 3 months after the addition . Adding N+P without glucose did not affect the NLFA/PLFA ratio for any fatty acid . Increasing NLFA/PLFA ratios for the fungal fatty acids were also found with time after the thermophilic phase in a compost, indicating increased availability of easily available carbon.

Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5160 - 3 Epub 2003 Apr 17.
A defined protein-detergent-lipid complex for crystallization of integral membrane proteins: The cytochrome b6f complex of oxygenic photosynthesis; Zhang H et al.; The paucity of integral membrane protein structures creates a major bioinformatics gap, whose origin is the difficulty of crystallizing these detergent-solubilized proteins . The problem is particularly formidable for hetero-oligomeric integral membrane proteins, where crystallization is impeded by the heterogeneity and instability of the protein subunits and the small lateral pressure imposed by the detergent micelle envelope that surrounds the hydrophobic domain . In studies of the hetero (eight subunit)-dimeric 220,000 molecular weight cytochrome b(6)f complex, derived from the thermophilic cyanobacterium, Mastigocladus laminosus, crystals of the complex in an intact state could not be obtained from highly purified delipidated complex despite exhaustive screening . Crystals of proteolyzed complex could be obtained that grew very slowly and diffracted poorly . Addition to the purified lipid-depleted complex of a small amount of synthetic nonnative lipid, dioleolyl-phosphatidylcholine, resulted in a dramatic improvement in crystallization efficiency . Large crystals of the intact complex grew overnight, whose diffraction parameters are as follows: 94% complete at 3.40 A spacing; R(merge) = 8.8% (38.5%), space group, P6(1)22; and unit cell parameters, a = b = 156.3 A, c = 364.0 A, alpha = beta = 90 degrees, gamma = 120 degrees.It is proposed that the methodology of augmentation of a well-defined lipid-depleted integral membrane protein complex with synthetic nonnative lipid, which can provide conformational stability to the protein complex, may be of general use in the crystallization of integral membrane proteins.

Water Sci Technol, 2003, 47(5), 197 - 200
Influence of operational conditions on biofilm specific activity of an anaerobic fluidized bed reactor; Garcia-Morales JL et al.; A key parameter in water and wastewater treatment technology is the biomass activity in terms of substrate removal ability . The effects of organic load rate and percentage of bed expansion on biofilm specific methanogenic activity were determined in an anaerobic fluidized bed reactor treating wine-distillery wastes in the thermophilic range (55 degrees C) . The proposed activity tests are highly reproducible: an experiment with three identical tests has shown that the standard deviation with respect to the mean values is less than 3% . Specific tests are applied to measure the maximum methanogenic activities of the biomass carrier in lab-scale anaerobic biofilm reactors . These tests have been successfully applied for monitoring the support colonization process and the evolution of biofilm activity in reactors, anaerobic filter and fluidized bed, with different operating conditions . The results show a dependence between the percentage of bed expansion and the specific activity of methanogenic microbiote on biofilm . There is a relationship between the percentage of bed expansion, the sheer stress on the biofilm and the hydrodynamic conditions in the system . Initial biofilm detachment can be compensated with the increase of biomass and of its activity due to the reduction of the substrate diffusional limitations to the microorganism growth inside the support pores.

EMBO J, 2003 Apr 15, 22(8), 1898 - 908
Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA; Serganov A et al.; The 16S rRNA-binding ribosomal protein S15 is a key component in the assembly of the small ribosomal subunit in bacteria . We have shown that S15 from the extreme thermophile Thermus thermophilus represses the translation of its own mRNA in vitro, by interacting with the leader segment of its mRNA . The S15 mRNA-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis . S15 binding triggers a conformational rearrangement of its mRNA into a fold that mimics the conserved three-way junction of the S15 rRNA-binding site . This conformational change masks the ribosome entry site, as demonstrated by direct competition between the ribosomal subunit and S15 for mRNA binding . A comparison of the T.thermophilus and Escherichia coli regulation systems reveals that the two regulatory mRNA targets do not share any similarity and that the mechanisms of translational inhibition are different . Our results highlight an astonishing plasticity of mRNA in its ability to adapt to evolutionary constraints, that contrasts with the extreme conservation of the rRNA-binding site.

Biotechnol Bioeng, 2003 Jun 30, 82(7), 791 - 801
Dynamics of the anaerobic process: effects of volatile fatty acids; Pind PF et al.; A complex and fast dynamic response of the anaerobic biogas system was observed when the system was subjected to pulses of volatile fatty acids (VFAs) . It was shown that a pulse of specific VFAs into a well-functioning continuous stirred tank reactor (CSTR) system operating on cow manure affected both CH(4) yield, pH, and gas production and that a unique reaction pattern was seen for the higher VFAs as a result of these pulses . In this study, two thermophilic laboratory reactors were equipped with a novel VFA-sensor for monitoring specific VFAs online . Pulses of VFAs were shown to have a positive effect on process yield and the levels of all VFA were shown to stabilize at a lower level after the biomass had been subjected to several pulses . The response to pulses of propionate or acetate was different from the response to butyrate, iso-butyrate, valerate, or iso-valerate . High concentrations of propionate affected the degradation of all VFAs, while a pulse of acetate affected primarily the degradation of iso-valerate or 2-methylbutyrate . Pulses of n-butyrate, iso-butyrate, and iso-valerate yielded only acetate, while degradation of n-valerate gave both propionate and acetate . Product sensitivity or inhibition was shown for the degradation of all VFAs tested . Based on the results, it was concluded that measurements of all specific VFAs are important for control purposes and increase and decrease in a specific VFA should always be evaluated in close relationship to the conversion of other VFAs and the history of the reactor process . It should be pointed out that the observed dynamics of VFA responses were based on hourly measurements, meaning that the response duration was much lower than the hydraulic retention time, which exceeds several days in anaerobic CSTR systems .

J Ind Microbiol Biotechnol, 2003 Apr, 30(4), 225 - 38 Epub 2003 Apr 17.
Colored moderately thermophilic bacteria in paper-machine biofilms; Kolari M et al.; Biofilms cause several problems in papermaking . This report describes a microbiological survey of colored biofilms in six paper and board machines, including two case studies of outbreaks of colored slimes in which the causative bacteria were found . A total of 95 pink-, red-, orange- or yellow-pigmented strains were isolated . Nearly all (99%) of the strains grew at 52 degrees C, 72% grew at 56 degrees C, but only 30% grew at 28 degrees C, indicating that most of the strains were moderately thermophilic . Biofilm formation potential and biocide susceptibility of the strains were analyzed with a microtiter plate assay . In the presence of 5 ppm of methylene bisthiocyanate or 2,2-dibromo-3-nitrilopropionamide in paper-machine water, 55 strains formed biofims . Moreover, 39 strains increased biofilm production by 5-753% in the presence of biocide, suggesting that biocide concentrations inhibitory to planktonic but not to surface-attached cells may actually promote biofouling . The cells may have inactivated a portion of the biocides, as the cell density in this assay was high, corresponding to the highest cell densities occurring in the circulating waters . Four groups of colored bacteria that were isolated from several mills were identified . Pink-pigmented Deinococcus geothermalis and red-pigmented Meiothermus silvanus occurred as common primary biofilm-formers in paper machines . This report is the first description of the involvement of Meiothermus species in red-slime formation in the paper industry . The third group of bacteria (putative new species related to Roseomonas) contained strains that were not biofilm formers, but which were commonly found in slimes of neutral or alkaline machines . The fourth group contained red-pigmented biofilm-forming strains representing a novel genus of alpha- Proteobacteria related to Rhodobacter . Many colored paper-machine bacteria are species previously known from microbial mats of hot springs . Some characteristics of the bacterial groups are described here in order to facilitate their recognition in future cases of colored-slime outbreaks in the paper industry.

FEMS Microbiol Rev, 2003 Apr, 27(1), 3 - 16
Thermomyces lanuginosus: properties of strains and their hemicellulases; Singh S et al.; The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus . Several strains of this fungus have been reported to produce high levels of cellulase-free beta-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of beta-xylanase production is apparent . Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis . Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50-80 degrees C and over a broad pH range (3-12) . Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1 . The gene encoding the T . lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases . The crystal structure of the xylanase indicates that the enzyme consists of two beta-sheets and one alpha-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted . The presence of an extra disulfide bridge between the beta-strand and the alpha-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability . The ability of T . lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry.

FEMS Microbiol Lett, 2003 Apr 11, 221(1), 137 - 42
Thermophilic biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1; Furuya T et al.; Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts . The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50 degrees C . When WU-F1 was cultivated at 45 degrees C with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs . In addition, when resting cells were incubated at 45 degrees C with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO) . Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization . Therefore, thermophilic M . phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 degrees C, may be a promising biocatalyst for practical biodesulfurization of diesel oil.

J Mol Biol, 2003 Apr 25, 328(2), 463 - 78
Multiple monovalent ion-dependent pathways for the folding of the L-21 Tetrahymena thermophila ribozyme; Uchida T et al.; Synchrotron hydroxyl radical (*OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes time-scales . The Mg(2+)-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200 mM NaCl) and as a function of the initial annealing conditions and substrate . Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4-P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics . For ribozyme annealed in buffer containing 200 mM NaCl and folded by the addition of 10 mM MgCl(2), multiple kinetic phases are observed for *OH protections throughout the ribozyme . The independently folding P4-P6 domain is the first to fold with its protections displaying 50-90% burst phase amplitudes . That the folding of P4-P6 within the ribozyme does not display the 100% burst phase of isolated P4-P6 at 200 mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding . In addition, *OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions . While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity . The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations . Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations . These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions .

J Mol Biol, 2003 Apr 25, 328(2), 419 - 28
Activity, stability and flexibility in glycosidases adapted to extreme thermal environments; Collins T et al.; To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog . Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility . In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility . These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability . Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity . A reduced number of salt-bridges are believed to be responsible for both these effects . Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation .

Bioresour Technol, 2003 Feb, 86(3), 207 - 13
Performance of an intermittent agitation rotating drum type bioreactor for solid-state fermentation of wheat straw; Kalogeris E et al.; A laboratory bioreactor, designed for solid-state fermentation of thermophilic microorganisms, was operated for production of cellulases and hemicellulases by the thermophilic fungus Thermoascus aurantiacus . The suitability of the apparatus for the effective control of important operating variables affecting growth of microbes in solid-state cultivation was determined . Application of the optimum conditions found for the moisture content of the medium, growth temperature and airflow rate produced enzyme yields of 1709 U endoglucanase, 4 U cellobiohydrolase, 79 U beta-glucosidase, 5.5 U FPA, 4490 U xylanase and 45 U beta-xylosidase per g of dry wheat straw . The correlation between microorganism growth and production of enzymes was efficiently described by the Le Duy kinetic model.

Nucleic Acids Res, 2003 Apr 15, 31(8), 2148 - 56
Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures; Droogmans L et al.; N1-methyladenosine (m1A) is found at position 58 in the T-loop of many tRNAs . In yeast, the formation of this modified nucleoside is catalyzed by the essential tRNA (m1A58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p) . In this report we describe the cloning, expression and characterization of a Gcd14p homolog from the hyperthermophilic bacterium Thermus thermophilus . The purified recombinant enzyme behaves as a homotetramer of 150 kDa by gel filtration and catalyzes the site- specific formation of m1A at position 58 of the T-loop of tRNA in the absence of any other complementary protein . S-adenosylmethionine is used as donor of the methyl group . Thus, we propose to name the bacterial enzyme TrmI and accordingly its structural gene trmI . These results provide a key evolutionary link between the functionally characterized two-component eukaryotic enzyme and the recently described crystal structure of an uncharacterized, putative homotetrameric methyltransferase Rv2118c from Mycobacterium tuberculosis . Interest ingly, inactivation of the T.thermophilus trmI gene results in a thermosensitive phenotype (growth defect at 80 degrees C), which suggests a role of the N1-methylation of tRNA adenosine-58 in adaptation of life to extreme temperatures.

Biometals, 2003 Sep, 16(3), 465 - 70
Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role; Nagasaka S et al.; The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs . Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm . Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0 . The electron spin resonance (ESR) spectrum of the intact C . caldarium cells showed an isotropic signal at a g value of 2.00 . Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal . EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA . The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal . Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.

Environ Toxicol Chem, 2003 Apr, 22(4), 706 - 11
Anaerobic degradation of linear alkylbenzene sulfonate; Mogensen AS et al.; Linear alkylbenzene sulfonate (LAS) found in wastewater is removed in the wastewater treatment facilities by sorption and aerobic biodegradation . The anaerobic digestion of sewage sludge has not been shown to contribute to the removal . The concentration of LAS based on dry matter typically increases during anaerobic stabilization due to transformation of easily degradable organic matter . Hence, LAS is regarded as resistant to biodegradation under anaerobic conditions . We present data from a lab-scale semi-continuously stirred tank reactor (CSTR) spiked with linear dodecylbenzene sulfonate (C12 LAS), which show that C12 LAS was biodegradable under methanogenic conditions . Sorption of C12 LAS on sewage sludge was described with a Freundlich isotherm . The C12 LAS sorption was determined with different concentrations of total solids (TS) . In the semi-continuously stirred tank reactor, 18% of the added C12 LAS was bioavailable and 20% was biotransformed when spiking with 100 mg/L of C12 LAS and a TS concentration of 14.2 mg/L . Enhanced bioavailability of C12 LAS was obtained in an upflow anaerobic sludge blanket (UASB) reactor inoculated with granular sludge and sewage sludge . Biodegradation under thermophilic conditions was 37% with LAS as sole carbon source . Benzaldehyde was produced in the UASB reactor during LAS transformation.

J Biol Chem, 2003 Jun 20, 278(25), 22964 - 71 Epub 2003 Apr 08.
Structures of argininosuccinate synthetase in enzyme-ATP substrates and enzyme-AMP product forms: stereochemistry of the catalytic reaction; Goto M et al.; Argininosuccinate synthetase reversibly catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate . The structures of the enzyme from Thermus thermophilus HB8 complexed with intact ATP and substrates (citrulline and aspartate) and with AMP and product (argininosuccinate) have been determined at 2.1- and 2.0-A resolution, respectively . The enzyme does not show the ATP-induced domain rotation observed in the enzyme from Escherichia coli . In the enzyme-substrate complex, the reaction sites of ATP and the bound substrates are adjacent and are sufficiently close for the reaction to proceed without the large conformational change at the domain level . The mobility of the triphosphate group in ATP and the side chain of citrulline play an important role in the catalytic action . The protonated amino group of the bound aspartate interacts with the alpha-phosphate of ATP and the ureido group of citrulline, thus stimulating the adenylation of citrulline . The enzyme-product complex explains how the citrullyl-AMP intermediate is bound to the active site . The stereochemistry of the catalysis of the enzyme is clarified on the basis of the structures of tAsS (argininosuccinate synthetase from T . thermophilus HB8) complexes.

Environ Pollut, 2003, 124(1), 81 - 91
Mineralization of phenanthrene and fluoranthene in yardwaste compost; Carlstrom CJ et al.; The purpose of the study was to evaluate the potential of phenanthrene and fluoranthene biodegradation in yardwaste compost materials . These polynuclear aromatic hydrocarbons were chosen for this work because they are relatively readily biodegradable and ubiquitous in the environment . Compost samples were incubated in biometers with 14C-labeled phenanthrene and the evolution of "4CO2 was assessed as a measure of mineralization . The '4CO2 evolution varied widely among replicate biometers, possibly as the result of (1) uneven and patchy colonization of phenanthrene-degrading microorganisms on compost particles, and (2) non-uniform dispersion of the labeled substrate spike into the yardwaste microenvironment . Mineralization of phenanthrene reached about 40%extent of 14CO2 evolution at best before leveling off, but the maximum varied from sample to sample and could be as low as 1%after three months . Active mineralization occurred at mesophilic and thermophilic temperatures . Methanol extraction was used to recover "4C from biometer samples that were spiked with "4C-labeled phenanthrene . Extraction for 24-48 h yielded 1-14% recovery of 14C, depending on the length of the preceding incubation . The low extraction yield and relatively low maximum mineralization(<40%) indicated that residual phenanthrene was sorbed and bound within the compost matrix in the biometer . Amendment ofbiometers with 0.05% Tween 80 or addition of water did not consistently enhance the mineralization . Variability in mineralization was greatly reduced in liquid samples taken from pre-enriched compost samples . Mineralization of 14C-labeled fluoranthene was negligible in biometers but could be stimulated by pre-enrichment with salicylate or naphthalene . Pre-enrichment also accelerated the mineralization of phenanthrene.

Structure (Camb), 2003 Apr, 11(4), 387 - 97
The 1.5 A crystal structure of a prokaryote serpin: controlling conformational change in a heated environment; Irving JA et al.; Serpins utilize conformational change to inhibit target proteinases; the price paid for this conformational flexibility is that many undergo temperature-induced polymerization . Despite this thermolability, serpins are present in the genomes of thermophilic prokaryotes, and here we characterize the first such serpin, thermopin . Thermopin is a proteinase inhibitor and, in comparison with human alpha(1)-antitrypsin, possesses enhanced stability at 60 degrees C . The 1.5 A crystal structure reveals novel structural features in regions implicated in serpin folding and stability . Thermopin possesses a C-terminal "tail" that interacts with the top of the A beta sheet and plays an important role in the folding/unfolding of the molecule . These data provide evidence as to how this unusual serpin has adapted to fold and function in a heated environment.

Appl Environ Microbiol, 2003 Apr, 69(4), 1944 - 52
Cloning and heterologous expression of a beta-D-mannosidase (EC 3.2.1.25)-encoding gene from Thermobifida fusca TM51; Beki E et al.; Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources . beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms . An expression library of T . fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases . One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated . Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes . The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87 . S . lividans was used as heterologous expression host for the putative beta-mannosidase gene of T . fusca . The purified gene product obtained from the culture filtrate of S . lividans was then subjected to more-detailed biochemical analysis . Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively . Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity . Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM . Glucono-lacton had no effect on the enzyme activity . A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.

Appl Environ Microbiol, 2003 Apr, 69(4), 1936 - 43
Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation; Okibe N et al.; Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45 degrees C . Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate) . The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors . The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined . More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates . The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations . The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.

Bioresour Technol, 2003 Aug, 89(1), 17 - 34
Developments in industrially important thermostable enzymes: a review; Haki GD et al.; Cellular components of thermophilic organisms (enzymes, proteins and nucleic acids) are also thermostable . Apart from high temperature they are also known to withstand denaturants of extremely acidic and alkaline conditions . Thermostable enzymes are highly specific and thus have considerable potential for many industrial applications . The use of such enzymes in maximising reactions accomplished in the food and paper industry, detergents, drugs, toxic wastes removal and drilling for oil is being studied extensively . The enzymes can be produced from the thermophiles through either optimised fermentation of the microorganisms or cloning of fast-growing mesophiles by recombinant DNA technology . In this review, the source microorganisms and properties of thermostable starch hydrolysing amylases, xylanases, cellulases, chitinases, proteases, lipases and DNA polymerases are discussed . The industrial needs for such specific thermostable enzyme and improvements required to maximize their application in the future are also suggested.

Acta Biochim Pol, 2003, 50(1), 81 - 102
Structural and enzymatic properties of the sedolisin family of serine-carboxyl peptidases; Wlodawer A et al.; Sedolisins (serine-carboxyl peptidases) are proteolytic enzymes whose fold resembles that of subtilisin; however, they are considerably larger, with the mature catalytic domains containing approximately 375 amino acids . The defining features of these enzymes are a unique catalytic triad, Ser-Glu-Asp, as well as the presence of an aspartic acid residue in the oxyanion hole . High-resolution crystal structures have now been solved for sedolisin from Pseudomonas sp . 101, as well as for kumamolisin from a thermophilic bacterium, Bacillus novo sp . MN-32 . The availability of these crystal structures enabled us to model the structure of mammalian CLN2, an enzyme which, when mutated in humans, leads to a fatal neurodegenerative disease . This review compares the structural and enzymatic properties of this newly defined MEROPS family of peptidases, S53, and introduces their new nomenclature.

J Biol Chem, 2003 Jun 13, 278(24), 21483 - 92 Epub 2003 Apr 02.
Characterization of a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a polychlorinated biphenyl- and naphthalene-degrading Bacillus sp . JF8; Hatta T et al.; A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp . JF8, and the gene was cloned . The native and recombinant BphC enzyme was purified to homogeneity . The enzyme has a molecular mass of 125 +/- 10 kDa and was composed of four identical subunits (35 kDa) . BphC_JF8 has a temperature optimum of 85 degrees C and a pH optimum of 7.5 . It exhibited a half-life of 30 min at 80 degrees C and 81 min at 75 degrees C, making it the most thermostable extradiol dioxygenase studied . Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0-4.8 manganese atoms per enzyme molecule . The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II) . The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol . The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mm 2,3-dihydroxybiphenyl . A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 microm for 2,3-dihydroxybiphenyl (at 60 degrees C) is among the lowest reported . The kinetic properties and thermal stability of the native and recombinant enzyme were identical . The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases . The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8 . A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme.

Int J Food Microbiol, 2003 May 25, 83(1), 87 - 103
Quantitative risk assessment of human campylobacteriosis associated with thermophilic Campylobacter species in chickens; Rosenquist H et al.; A quantitative risk assessment comprising the elements hazard identification, hazard characterization, exposure assessment, and risk characterization has been prepared to assess the effect of different mitigation strategies on the number of human cases in Denmark associated with thermophilic Campylobacter spp . in chickens . To estimate the human exposure to Campylobacter from a chicken meal and the number of human cases associated with this exposure, a mathematical risk model was developed . The model details the spread and transfer of Campylobacter in chickens from slaughter to consumption and the relationship between ingested dose and the probability of developing campylobacteriosis . Human exposure was estimated in two successive mathematical modules . Module 1 addresses changes in prevalence and numbers of Campylobacter on chicken carcasses throughout the processing steps of a slaughterhouse . Module 2 covers the transfer of Campylobacter during food handling in private kitchens . The age and sex of consumers were included in this module to introduce variable hygiene levels during food preparation and variable sizes and compositions of meals . Finally, the outcome of the exposure assessment modules was integrated with a Beta-Poisson dose-response model to provide a risk estimate . Simulations designed to predict the effect of different mitigation strategies showed that the incidence of campylobacteriosis associated with consumption of chicken meals could be reduced 30 times by introducing a 2 log reduction of the number of Campylobacter on the chicken carcasses . To obtain a similar reduction of the incidence, the flock prevalence should be reduced approximately 30 times or the kitchen hygiene improved approximately 30 times . Cross-contamination from positive to negative flocks during slaughter had almost no effect on the human Campylobacter incidence, which indicates that implementation of logistic slaughter will only have a minor influence on the risk . Finally, the simulations showed that people in the age of 18-29 years had the highest risk of developing campylobacteriosis.

Biochem Biophys Res Commun, 2003 Apr 11, 303(3), 848 - 54
A gene cluster involved in pyrimidine reductive catabolism from Brevibacillus agri NCHU1002; Kao CH et al.; Genes involved in pyrimidine reductive catabolism (pyd) were isolated from a moderate thermophile, Brevibacillus agri NCHU1002, and nine ORFs in an 8.2-kb DNA fragment were identified by DNA sequence analysis . The pyd gene cluster included three closely spaced ORFs, designated pydA, pydB, and pydC, transcribed in the same orientation . Based on their amino acid sequence identity and enzyme activity assay, the gene products were identified as dihydropyrimidine dehydrogenase (PydA), dihydropyrimidinase (PydB), and beta-alanine synthase (PydC) . Northern blot and primer extension analyses revealed that the pydBC genes are induced by dihydrouracil and regulated under the control of sigma(54) recognized promoter at transcriptional level as a polycistronic operon . All results indicate that the pydABC genes participate in the pathway of the pyrimidine reductive catabolism . This is the first bacterial pyd gene cluster to be reported.

Biophys J, 2003 Apr, 84(4), 2216 - 22
Electrostatic contributions to the stability of a thermophilic cold shock protein; Zhou HX et al.; The thermophilic Bacillus caldolyticus cold shock protein (Bc-Csp) differs from the mesophilic Bacillus subtilis cold shock protein B (Bs-CspB) in 11 of the 66 residues . Stability measurements of Schmid and co-workers have implicated contributions of electrostatic interactions to the thermostability . To further elucidate the physical basis of the difference in stability, previously developed theoretical methods that treat electrostatic effects in both the folded and the unfolded states were used in this paper to study the effects of mutations, ionic strength, and temperature . For 27 mutations that narrow the difference in sequence between Bc-Csp and Bs-CspB, calculated changes in unfolding free energy (Delta G) and experimental results have a correlation coefficient of 0.98 . Bc-Csp appears to use destabilization of the unfolded state by unfavorable charge-charge interactions as a mechanism for increasing stability . Accounting for the effects of ionic strength and temperature on the electrostatic free energies in both the folded and the unfolded states, explanations for two important experimental observations are presented . The disparate ionic strength dependences of Delta G for Bc-Csp and Bs-CspB were attributed to the difference in the total charges (-2e and -6e, respectively) . A main contribution to the much higher unfolding entropy of Bs-CspB was found to come from the less favorable electrostatic interactions in the folded state . These results should provide insight for understanding the thermostability of other thermophilic proteins.

J Biotechnol, 2003 Apr 10, 102(1), 33 - 44
Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water-organic solvent mixtures; Topakas E et al.; An extracellular feruloyl esterase (FAE-II) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by SP-Sepharose, t-butyl-HIC and Sephacryl S-200 column chromatography . The protein corresponded to molecular mass and pI values of 27 kDa and 9.9, respectively . The enzyme was optimally active at pH 7 and 45 degrees C . The purified esterase was fully stable at pH 7.0-9.0 and temperature up to 45 degrees C after 1 h incubation . Determination of k(cat)/K(m) revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively . The enzyme was active on substrates containing ferulic acid ester linked to the C-5 but inactive to the C-2 positions of arabinofuranose such as 4-nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside . In the presence of Sporotrichum thermophile xylanase, there was a significant release of ferulic acid from destarched wheat bran by FAE-II, indicating a synergistic interaction between FAE-II and S . thermophile xylanase . FAE-II by itself could release only little ferulic acid from destarched wheat bran . The potential of FAE-II for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsion formed in ternary mixture consisting of n-hexane, 1-propanol and water.

Mol Cell Biol, 2003 Apr, 23(8), 2778 - 89
The nonessential H2A N-terminal tail can function as an essential charge patch on the H2A.Z variant N-terminal tail; Ren Q et al.; Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up approximately 80% of total H2A, and a conserved variant, H2A.Z . We showed previously that acetylation of H2A.Z was essential (Q . Ren and M . A . Gorovsky, Mol . Cell 7:1329-1335, 2001) . Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo . Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein . Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential . Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail . Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

Extremophiles, 2003 Apr, 7(2), 111 - 22 Epub 2002 Dec 12.
High-resolution X-ray structure of the DNA-binding protein HU from the hyper-thermophilic Thermotoga maritima and the determinants of its thermostability; Christodoulou E et al.; The histone-like DNA-binding proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability . We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU protein from the hyper-thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C) . The structural data presented clearly show that the HU Tmar has a fold similar to its thermophilic homologue HU from Bacillus stearothermophilus (HU Bst) . Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar . Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme thermophilic protein HU Tmar to mesophilic, like HU Bsu . In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu . The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type . The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution . A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa . Our analysis points to this as the prime cause of the destabilization compared to the wild type . The three new structures were compared, together with the X-ray structure of a similar protein, HU Bst, with the aim of relating their structural properties and different thermal stability . The presented results show that the HU Tmar protein achieves its stability by employing a dual strategy . On the one hand, we observe local hydrophobic interactions, which stabilize the secondary structure elements, and on the other hand, electrostatic interactions between side chains.

Extremophiles, 2003 Apr, 7(2), 101 - 9 Epub 2002 Nov 23.
Thermococcus atlanticus sp nov., a hyperthermophilic Archaeon isolated from a deep-sea hydrothermal vent in the Mid-Atlantic Ridge {corrected}; Cambon-Bonavita MA et al.; An extremely thermophilic archaeon, strain MA898, was isolated from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge . This strain is a strictly anaerobic coccus of approximately 0.7-1.2 microm in diameter . Optimal temperature, pH, and NaCl concentration for growth are around 85 degrees C, pH 7, and 3%, respectively . Strain MA898 grows preferentially in the presence of elemental sulfur, polysulfur, cystine, or L-cysteine . The microorganism requires rich proteinaceous substrates . BHI-S medium supports rapid growth, with a final concentration of more than 1.2 x 10(9) cells ml(-1), but strain MA898 exhibits poor growth on 2216S medium (yeast/peptone) and poor growth on starch . Growth is inhibited by rifampicin and chloramphenicol at a concentration of 100 microg/ml . The DNA G+C content is 50 mol% . Sequencing of the 16S rRNA gene indicates that strain MA898 belongs to the Thermococcusgenus, and from DNA/DNA hybridization data it is proposed as a new species: Thermococcus atlanticus . The deposition numbers are CIP-107420T and DSM15226.

Appl Microbiol Biotechnol, 2003 Feb, 60(6), 748 - 53 Epub 2002 Dec 19.
The isolation and use of iron-oxidizing, moderately thermophilic acidophiles from the Collie coal mine for the generation of ferric iron leaching solution; Kinnunen PH et al.; Moderately thermophilic, iron-oxidizing acidophiles were enriched from coal collected from an open-cut mine in Collie, Western Australia . Iron-oxidizers were enriched in fluidized-bed reactors (FBR) at 60 degrees C and 70 degrees C; and iron-oxidation rates were determined . Ferrous iron oxidation by the microbiota in the original coal material was inhibited above 63;C . In addition to four iron-oxidizers, closely related to Sulfobacillus spp that had been earlier isolated from the 60 degrees C FBR, one heterotroph closely related to Alicyclobacillus spp was isolated . The Alicyclobacillus sp . isolated from the Collie coal mine tolerated a lower pH than known Alicyclobacillus spp and therefore may represent a new species . The optimum temperature for growth of the iron-oxidizing strains was approximately 50 degrees C and their maximum temperatures were approximately 60 degrees C . The FBR was adjusted to operate at 50 degrees C and was inoculated with all of the isolated iron-oxidizing strains . At 60 degrees C, an iron-oxidation rate of 0.5 g Fe(2+) l(-1) x h(-1) was obtained . At 50 degrees C, the iron-oxidation rate was only 0.3 g Fe(2+) l(-1) x h(-1) . These rates compare favourably with the iron-oxidation rate of Acidianus brierleyi in shake-flasks, but are considerably lower than mesophilic iron-oxidation rates.

Appl Microbiol Biotechnol, 2003 Feb, 60(6), 605 - 11 Epub 2002 Dec 18.
Microbial silica deposition in geothermal hot waters; Inagaki F et al.; A combined use of molecular ecological techniques and geochemical surveys revealed that thermophilic or hyperthermophilic microorganisms living in geothermal environments are likely to be implicated in the formation of biogenic siliceous deposits . Electron microscopic observations indicated that numerous microorganism-like fabrics were preserved in naturally occurring siliceous deposits such as siliceous sinter, geyserite, and silica scale, which suggests microbial contribution to silica precipitation . Molecular phylogenetic analyses suggested that extreme thermophilic bacteria within the genera Thermus and Hydrogenobacter are predominant components among the indigenous microbial community in siliceous deposits formed in pipes and equipment of Japanese geothermal power plants . These bacteria seem to actively contribute to the rapid formation of huge siliceous deposits . Additionally, in vitro examination suggested that Thermus cells induced the precipitation of supersaturated amorphous silica during the exponential growth phase, concomitant with the production of a specific cell envelope protein . Dissolved silica in geothermal hot water may be a significant component in the maintenance of position and survival of microorganisms in limited niches.

Environ Microbiol, 2003 Apr, 5(4), 267 - 77
Related assemblages of sulphate-reducing bacteria associated with ultradeep gold mines of South Africa and deep basalt aquifers of Washington State; Baker BJ et al.; We characterized the diversity of sulphate-reducing bacteria (SRB) associated with South African gold mine boreholes and deep aquifer systems in Washington State, USA . Sterile cartridges filled with crushed country rock were installed on two hydrologically isolated and chemically distinct sites at depths of 3.2 and 2.7 km below the land surface (kmbls) to allow development of biofilms . Enrichments of sulphate-reducing chemolithotrophic (H2) and organotrophic (lactate) bacteria were established from each site under both meso- and thermophilic conditions . Dissimilatory sulphite reductase (Dsr) and 16S ribosomal RNA (rRNA) genes amplified from DNA extracted from the cartridges were most closely related to the Gram-positive species Desulfotomaculum thermosapovorans and Desulfotomaculum geothermicum, or affiliated with a novel deeply branching clade . The dsr sequences recovered from the Washington State deep aquifer systems affiliated closely with the South African sequences, suggesting that Gram-positive sulphate-reducing bacteria are widely distributed in the deep subsurface.

EMBO J, 2003 Apr 1, 22(7), 1632 - 43
Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain; Charron C et al.; In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs) . In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs . While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNA(Asp) and tRNA(Asn) . The structure at 2.3 A resolution of AspRS-2, the first of a non-discriminating synthetase, was solved . It differs from that of AspRS-1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis . The protein presents non-conventional features in its OB-fold anticodon-binding domain, namely the absence of a helix inserted between two beta-strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNA(Asp) identity determinant C36 in conventional AspRSs . In AspRS-2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline . In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS-2 or AsnRS . Its particular status is demonstrated by a loop-exchange experiment that renders the Pyrococcus AspRS non-discriminating.

Bioorg Khim, 2003 Jan-Feb, 29(1), 75 - 82
{Comparative study of the efficacy of modifying DNA polymerase and DNA matrix by different photoactive groups at the 3'-end of the DNA primer}; Dezhurov SV et al.; The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoactivatable group and the length of the linker that joins the group with the heterocyclic base of the primer 3'-terminal nucleotide was studied . The primers that contained the photoreactive groups at their 3'-termini were obtained using the rat DNA polymerase beta or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue . After irradiating the reaction mixture with UV light and separating the modification products, the level of covalent binding of the {5'-32P}primer to DNA polymerases and template was determined . The primers containing 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 765 - 8 Epub 2003 Mar 25.
Structure of 20K endoglucanase from Melanocarpus albomyces at 1.8 A resolution; Valjakka J et al.; The crystal structure of the 20K endoglucanase from the thermophilic fungus Melanocarpus albomyces (Ma20k) has been determined . The structure was refined to 1.8 A resolution using data obtained at 120 K . Ma20k belongs to glycoside hydrolase family 45 . The three-dimensional structures of endoglucanase V (EGV) from the fungus Humicola insolens and of an endoglucanase from H . grisea var . thermoidea have previously been determined . The overall structure of Ma20k consists of a six-stranded beta-barrel domain similar to that found previously in family 45 endoglucanases . The flexible loop between strands V and VI, which was disordered in the uncomplexed structures of the Humicola endoglucanases but was ordered in complexed structures of EGV, is found to be well ordered in the native structure of Ma20k . The structure of Ma20k allows comparison between thermophilic and mesophilic proteins of family 45 and different principles for thermostability are discussed.

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 734 - 6 Epub 2003 Mar 25.
Purification, crystallization and preliminary X-ray analysis of ferredoxin isolated from thermophilic cyanobacterium Mastigocladus laminosus; Fish A et al.; Ferredoxins are soluble iron-sulfur proteins that are involved in numerous electron-transfer reactions . Plant-type ferredoxins, which carry a single {2Fe-2S} cluster, serve as the electron acceptors of Photosystem I . The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus has unique thermostable properties . The isolated protein is active at temperatures of higher than 338 K . The gene encoding the ferredoxin from M . laminosus was subcloned into an expression vector and overproduced in Escherichia coli . The recombinant protein was purified to near-homogeneity and crystallized using the hanging-drop method . Thin needle-like crystals were grown in several crystallization conditions but were unsuitable for X-ray analysis owing to poor scattering . In order to obtain better diffracting crystals, ferredoxin was purified directly from M . laminosus cells . Single crystals were obtained using 30% PEG 4000, 0.32 M Mg(NO(3))(2), 20 mM Tris-HCl pH 8.2 . These crystals diffracted to 1.25 A resolution using synchrotron radiation and were found to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 28.45, b = 50.93, c = 110.91 A . The asymmetric unit was found to contain two ferredoxin molecules, with a corresponding V(M) of 1.9 A(3) Da(-1) and a solvent content of 34% . The present study indicates that overcoming the poor diffraction of crystals obtained from recombinant protein can be achieved by producing crystals from protein purified from the native host.

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 721 - 3 Epub 2003 Mar 25.
Crystallization and preliminary X-ray data of the alpha2epsilon2 subcomponent of the acetyl-CoA decarbonylase/synthase multienzyme complex from Methanosarcina thermophila; Balbo P et al.; The alpha(2) epsilon (2) subcomponent (218.6 kDa) of the 1.99 MDa acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex is an Ni/Fe-S enzyme that catalyzes reversible CO(2)/CO reduction in the context of acetyl-CoA synthesis . The ACDS complex is required for methanogenesis from acetate in methanogenic archaea . The alpha(2)epsilon(2) subcomponent from Methanosarcina thermophila, grown on acetate, was purified and crystallized . The crystals were mounted in a capillary and diffracted to 4.0 A resolution at room temperature . Different flash-cooling approaches were attempted, all of which resulted in poor diffraction.

Int J Immunopathol Pharmacol, 2000 Sep, 13(3), 141 - 150
Intestinal pathway of internalisation of lactic acid bacteria and gut mucosal immunostimulation; Perdigon G et al.; The induction of the gut mucosal immune response is dependent on the antigen interacting with the M cells of Peyer's patches and with the immune cells associated with this lymphoid organ . In previous studies we showed that the mucosal immunostimulation by LAB varied depending upon the strain being studied . Some of them increased the inflammatory immune response and others enhanced the level of secretory antibody (S-IgA) . Our aim was to determine the pathway of the internalisation of LAB strains in the intestine, to provide a basis for understing the different behaviour exhibited by them . The presence of LAB on Peyer's patches or in the immune cells associated with the villi of small intestine was determined using fluorescein-labelled bacteria and by transmission electron microscopy . Mice were dosed orally by intubation, with 10<sup>1</sup> cells of labelled single strains of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus thermophilus . For electron microscopy studies unlabelled bacteria were used . Histological preparations of the small and large intestine were performed 1 hour after administration of the bacteria . Fluorescent bacteria of L . casei and S . thermophilus were found only in Peyer's patches while L . delbrueckii ssp . bulgaricus and L . acidophilus were observed in Peyer's patches and in the small intestine . L . acidophilus was also found in the large intestine . We confirmed these findings by electron microscopy . We determined that for L . casei the pathway of internalisation was via the M and FAE cells of Peyer's patches, while for S . thermophilus, L . acidophilus and L . delbrueckii ssp . bulgaricus the interaction with the immune cells of Peyer's patches was through the follicle associated epithelium (FAE) . L . delbrueckii ssp . bulgaricus and L . acidophilus also interacted with the epithelial cells of the small intestine and L . acidophilus with epithelial cells of the large intestine . These results suggest that the different effects of LAB on the mucosal immunostimulation, are related to the different pathways of gut internalisation used to take contact with the immune cells associated with the lamina propria intestinal.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 269 - 73
Streptomyces mexicanus sp . nov., a xylanolytic micro-organism isolated from soil; Petrosyan P et al.; The taxonomic position of a thermophilic actinomycete strain isolated from soil was examined using a polyphasic approach . The strain, designated CH-M-1035T, was assigned to the genus Streptomyces on the basis of chemical and morphological criteria . It formed Rectiflexibiles aerial hyphae that carried long chains of rounded, smooth spores . The almost complete nucleotide sequence of the 16S rRNA gene of strain CH-M-1035T was determined and its comparison with the 16S rDNA sequences of previously studied streptomycetes confirmed the assignment of the novel strain to the genus Streptomyces . Strain CH-M-1035T clustered with species belonging to the Streptomyces thermodiastaticus clade in the 1 6S-rDNA-based phylogenetic tree . However, the phenotypic properties of strain CH-M-1035T differed from those of the recognized species within this clade . Therefore, it is proposed that strain CH-M-1035T be classified as a novel species within the genus Streptomyces, as Streptomyces mexicanus (type strain CH-M-1035T =DSM 41796T =BM-B-384T =NRRL B-24196T).

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 59 - 65
Marinithermus hydrothermalis gen . nov., sp . nov., a strictly aerobic, thermophilic bacterium from a deep-sea hydrothermal vent chimney; Sako Y et al.; A novel thermophilic marine bacterium, designated strain T1T, was isolated from a deep-sea hydrothermal vent chimney sample collected from the Suiyo Seamount in the Izu-Bonin Arc, Japan, at a depth of 1,385 m . Cells of strain T1T were rod-shaped, occurring in pairs or filamentous, and stained Gram-negative . Growth was observed between 50.0 and 72.5 degrees C (optimum 67.5 degrees C; 30 min doubling time) and at pH 6.25-7.75 (optimum pH 7.00) . The isolate absolutely required NaCl, at a concentration of 0.5-4.5% (optimum 30%) . It was a strictly aerobic heterotroph capable of growing solely on complex organic substrates such as yeast extract, tryptone and Casamino acids, utilizing glutamate, proline, serine, cellobiose, trehalose, sucrose, acetate and pyruvate as complementary substrates . The G+C content of the genomic DNA was 68.6 mol% . The 16S rRNA gene sequence of the isolate was most similar to those from members of the genus Thermus, but the isolate was distantly related to them at the genus level (<90%) . In addition, phylogenetic analysis indicated that the isolate was on a novel lineage, deeply branched prior to divergence of the genus Thermus . On the basis of phylogenetic analysis and physiological traits of the isolate, it should be described as a member of a novel genus distinct from the previously described genus Thermus . The name Marinithermus gen . nov . is proposed, with Marinithermus hydrothermalis gen . nov., sp . nov . as the type species . The type strain of M . hydrothermalis gen . nov., sp . nov . is strain T1T (=JCM 11576T =DSM 14884T).

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 35 - 41
Porphyrobacter cryptus sp . nov., a novel slightly thermophilic, aerobic, bacteriochlorophyll a-containing species; Rainey FA et al.; Two strains of a novel aerobic, bacteriochlorophyll a-containing species of the alpha-4 subclass of the Proteobacteria were isolated from the hot spring at Alcafache in central Portugal . 16S rRNA gene sequence-based phylogenetic analyses showed the two novel isolates to be phylogenetically related to members of the genera Erythrobacter, Erythromicrobium and Porphyrobacter . The strains produce reddish-orange-pigmented colonies, have an optimum growth temperature of about 50 degrees C and could be distinguished from the species Porphyrobacter tepidarius, which also has a high growth temperature, primarily on the basis of the fatty acid composition . The novel species does not grow anaerobically in the presence or absence of a light source . The strains of the novel species utilize several single carbon sources for growth, most of which are also used by P . tepidarius . The species status of strains ALC-2T and ALC-3 was confirmed by low reassociation values of the DNA with species of the genera Erythrobacter, Erythromicrobium and Porphyrobacter . Phenotypic characteristics and 16S rRNA gene sequence analyses also show that strains ALC-2T (=DSM 12079T =ATCC BAA-386T) and ALC-3 (=DSM 12080) represent a novel species, for which the name Porphyrobacter cryptus sp . nov . is proposed.

Nucleic Acids Res, 2003 Apr 1, 31(7), 1913 - 20
Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus; Laging M et al.; Hydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that-if left unrepaired-leads to a C-->T transition mutation in half of the progeny . In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5' to the mismatched T . We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region . Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity . Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches . The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco.

J Biol Chem, 2003 Jul 11, 278(28), 25417 - 27 Epub 2003 Mar 21.
Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea; Johnsen U et al.; Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties . PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits . The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+ . Temperature optima for activity were 85 degrees C (A . fulgidus) and above 98 degrees C (A . pernix and P . aerophilum) . The PKs were highly thermostable up to 110 degrees C (A . pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A . fulgidus) or above 98 degrees C (A . pernix and P . aerophilum) . All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates . Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g . fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation . PK from the bacterium T . maritima is also a homotetramer of 50-kDa subunits . The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C . The enzyme showed cooperative response to PEP and ADP . In contrast to its archaeal counterparts, the T . maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP . Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya . Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T . maritima.

Eur J Biochem, 2003 Apr, 270(7), 1399 - 412
Three-dimensional structures of thermophilic beta-1,4-xylanases from Chaetomium thermophilum and Nonomuraea flexuosa . Comparison of twelve xylanases in relation to their thermal stability; Hakulinen N et al.; The crystal structures of thermophilic xylanases from Chaetomium thermophilum and Nonomuraea flexuosa were determined at 1.75 and 2.1 A resolution, respectively . Both enzymes have the overall fold typical to family 11 xylanases with two highly twisted beta-sheets forming a large cleft . The comparison of 12 crystal structures of family 11 xylanases from both mesophilic and thermophilic organisms showed that the structures of different xylanases are very similar . The sequence identity differences correlated well with the structural differences . Several minor modifications appeared to be responsible for the increased thermal stability of family 11 xylanases: (a) higher Thr : Ser ratio (b) increased number of charged residues, especially Arg, resulting in enhanced polar interactions, and (c) improved stabilization of secondary structures involved the higher number of residues in the beta-strands and stabilization of the alpha-helix region . Some members of family 11 xylanases have a unique strategy to improve their stability, such as a higher number of ion pairs or aromatic residues on protein surface, a more compact structure, a tighter packing, and insertions at some regions resulting in enhanced interactions.

Bioresour Technol, 2003 Jan, 86(2), 123 - 9
Semi-dry thermophilic anaerobic digestion of the organic fraction of municipal solid waste: focusing on the start-up phase; Bolzonella D et al.; The paper concerns the results of a pilot-scale study of the simulation of the start-up phase of the thermophilic semi-dry anaerobic digestion of the organic fraction of municipal solid wastes . The aim of the study was to aid and shorten the start-up phase of the full-scale plant (500 t/d) in Verona--Ca' del Bue, where the semi-dry anaerobic digestion process is being used . The substrate used in the experimentation was the mechanically sorted organic fraction of municipal solid waste (MS-OFMSW) enriched with the putrescent fraction from the source sorted OFMSW in order to simulate the substrate which is dealt with in the Verona plant . The results of the pilot scale study agreed with literature data and previous work of the authors: it showed a specific gas production of 0.23 m3/kg TVSfeed and a gas production rate of 2.1 m3/m3 d when operating at a specific organic loading rate of 0.135 kgTVSfeed/kgTVSreacter d . No problems regarding process stability were encountered in the gradual acclimation of the biomass . The design organic loading rate of 9 kg TVSfeed/m3reactor d was reached in about 30 days, during which the total solids content in the feedwas increased . Only a partial comparison with the full scale start-up, which is now in progress, is possible: this shows an initial general concordance with the results found in previous work.

Protein Expr Purif, 2003 Mar, 28(1), 69 - 77
Construction and purification of his6-Thermus thermophilus MutS protein; Stanislawska-Sachadyn A et al.; The mutS gene from the thermophilic bacterium Thermus thermophilus was PCR amplified, cloned, and expressed in Escherichia coli . The recombinant MutS protein containing an oligohistidine domain at the N-terminus was purified in a single step by Ni(2+) affinity chromatography to apparent homogeneity . The mismatch recognition properties of the his(6)-tagged MutS protein were confirmed by DNA protection against exonuclease digestion and retardation assays . The results of analytical gel filtration indicate that the predominant form of T . thermophilus MutS at micromolar concentrations is a tetramer.

Plant Mol Biol, 2003 Mar, 51(4), 493 - 507
Expression and import of an active cellulase from a thermophilic bacterium into the chloroplast both in vitro and in vivo; Jin R et al.; A bacterial thermostable cellulase, the endo-1,4-beta-D-glucanase E1 from Acidothermus cellulolyticus, was imported into chloroplasts, and an active enzyme was recovered both in vitro and in vivo . Precursor fusion proteins were synthesized with E1 or its catalytic domain, CD, fused to the transit peptide of ferredoxin or ribulose-bisphosphate carboxylase activase for stromal targeting . A spacer region of 1, 5 or 15 amino acids was included carboxy to the transit peptide . The efficiency of import and processing by the stromal processing peptidase depended on the nature of the transit peptide and the passenger protein, and increased with the length of the spacer between them . Besides finding E1 or CD in the stroma, protein was arrested in the envelope during import showing that structural features of E1 and CD, along with their proximity to the transit peptide, influence translocation . The cellulose binding domain and/or serine/proline/threoline-rich linker of E1 may impede efficient import . Significantly, most precursors for E1 and CD synthesized by in vitro translation possessed endoglucanse activity that was temperature-dependent, and required the residues AGGGY at the N-terminus of E1 and CD . Furthermore, activity was detected upon import into chloroplasts . Based on the in vitro analyses, five precursor fusion proteins were selected to determine if E1 and CD would be successfully targeted to chloroplasts in vivo . In transgenic tobacco plants, E1 and CD accumulated in both the stromal and membrane fractions and, importantly, chloroplast extracts showed endoglucanase activity.

Science, 2003 Mar 21, 299(5614), 1892 - 5
Identifying kinetic barriers to mechanical unfolding of the T . thermophila ribozyme; Onoa B et al.; Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons . Barriers are magnesium dependent and correspond to known intra- and interdomain interactions . Several barrier structures are "brittle," breakage requiring high forces but small (1 to 3 nanometers) deformations . Barrier crossing is stochastic, leading to variable unfolding paths . The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

J Dairy Sci, 2003 Feb, 86(2), 407 - 23
Biochemistry, genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review; Broadbent JR et al.; Many strains of Streptococcus thermophilus synthesize extracellular polysaccharides . These molecules may be produced as capsules that are tightly associated with the cell, or they may be liberated into the medium as a loose slime (i.e., "ropy" polysaccharide) . Although the presence of exopolysaccharide does not confer any obvious advantage to growth or survival of S . thermophilus in milk, in situ production by this species or other dairy lactic acid bacteria typically imparts a desirable "ropy" or viscous texture to fermented milk products . Recent work has also shown that exopolysaccharide-producing S . thermophilus can enhance the functional properties of Mozzarella cheese, but they are not phage-proof . As our understanding of the genetics, physiology, and functionality of bacterial exopolysaccharides continues to improve, novel applications for polysaccharides and polysaccharide-producing cultures are likely to emerge inside and outside the dairy industry . This article provides an overview of biochemistry, genetics, and applications of exopolysaccharide production in S . thermophilus.

Oecologia, 2003 Feb, 134(3), 439 - 44 Epub 2002 Dec 20.
Seasonal variation in the niche, habitat availability and population fluctuations of a bivoltine thermophilous insect near its range margin; Roy DB et al.; We investigated the niche requirements of the summer and autumn/spring generations of the bivoltine butterfly, Polyommatus bellargusRott., and their implications for population dynamics at sites occurring near its northern range margin . The larvae of this species are sedentary, and the turf height and shelter of Hippocrepis comosa foodplants selected for egg-laying accurately predict larval distributions within United Kingdom (UK) sites . We found a significant shift between the plants used for egg-laying in each generation, with the niche occupied by summer-feeding larvae being broader and different to the autumn one . Measurements of soil temperature confirmed that the short, sheltered foodplants selected by ovipositing females in autumn placed the autumn/spring-feeding generation of larvae in the warmest available microclimates within sites . In late spring, egg-laying females avoided the hottest spots but extended egg-laying into taller, less sheltered (relatively cool) turf where the microclimate was similar to that experienced by autumn/spring-feeding larvae . Using each generations' definition of niche requirement, we analysed surveys of foodplant populations available on 24 UK sites for P . bellargus, and estimated that nearly twice as many plants were available to the summer-feeding larvae compared to those feeding in the autumn . Annual adult population counts match these seasonal differences in site carrying capacity; first generation counts (from autumn-laid eggs) were generally half as abundant as in the second generation, and more variable . These results suggest that the seasonal cycle of niche switches represents an annual (autumn-spring) bottleneck for populations of this butterfly at its northern range margin . Under climate warming we predict that the inter-generational difference in niche availability, carrying capacity and population size will be reduced . We recommend revised management requirements for this threatened species under current and predicted climates in northern Europe.

J Bacteriol, 2003 Apr, 185(7), 2161 - 8
Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi; Xu Y et al.; The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified . OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25 degrees C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes . In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state . The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof . OTCase(Mab) displays higher K(m) values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO) . OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K(m) values and the lower sensitivity to PALO . The K(m) for ornithine, however, is substantially lower at low temperatures . A survey of the catalytic efficiencies (k(cat)/K(m)) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K(m) value for ornithine observed when the temperature is brought from 20 to 5 degrees C . OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.

J Theor Biol, 2003 Apr 7, 221(3), 425 - 36
The universal ancestor was a thermophile or a hyperthermophile: tests and further evidence; Di Giulio M; The existence of a correlation between the optimal growth temperature of various organisms and a thermophily index (based on the propensity of amino acids to enter more frequently into the proteins of thermophiles/hyperthermophiles) allows inferences to be made on the mesophilic or thermophilic nature of the last universal common ancestor (LUCA) . By reconstructing the ancestral sequences of the various ancestors using methods based on maximum likelihood and maximum parsimony, these sequences can be attributed to the mesophiles or (hyper)thermophiles and the following conclusions can be drawn . (1) There is no evidence that the LUCA might have been a mesophile and observations seem to imply that the LUCA was a thermophile or a hyperthermophile; (2) The ancestors of the Archaea and Bacteria domains seem to be (hyper)thermophiles while that of the Eukarya domain turns out to be a mesophile . These conclusions are independent of both (i) where the root is located on the topology of the universal tree (based on that of the small subunit ribosomal RNA) and (ii) the presence of hyperthermophile bacteria near the node of the Bacteria domain ancestor . These conclusions are easier to interpret in the light of the hypotheses that see the origin of life taking place at a high temperature .

Environ Technol, 2003 Jan, 24(1), 23 - 30
Effect of organic waste amendments on degradation of PAHs in soil using thermophillic composting; Wan CK et al.; The feasibility of using various types of organic wastes including pig manure, sewage sludge, and soybean refuse for remediation of soil spiked with phenanthrene, anthracene and pyrene (PAHs) was evaluated through batch-scale composting reactors . The most active degradation of PAHs occurred between day 4 to 30 and maximum removal at the end of composting accounted for 90% of the initial concentrations of the three PAH compounds . Among the three PAHs, degradation of pyrene in the composting mass was relatively slow as indicated by a longer lag period than that of phenanthrene and anthracene . This corresponded well with the high molecular weight and log K(ow) values of pyrene . The organic amendments were effective in enhancing the degradation of PAHs, and pig manure amendment exhibited a slightly higher removal efficiency than sewage sludge and soybean refuse . A decrease in total organic matter in all treatments indicated that the decomposition process occurred . Toxicity test with cress seed germination was evaluated and no phytotoxicity was noted after 21 days of composting . This preliminary study positively supports that pig manure is an effective organic additive for bioremediation of PAH-contaminated soil using composting as a treatment technology.

Folia Parasitol (Praha), 2002, 49(4), 299 - 303
Moravecnema segonzaci gen . et sp . n . (Nematoda: Cystidicolidae) from Pachycara thermophilum (Zoarcidae), a deep-sea hydrothermal vent fish from the Mid-Atlantic Ridge; Justine JL et al.; A new cystidicolid nematode, Moravecnema segonzaci gen . et sp . n . is described from the intestine of the teleost fish Pachycara thermophilum Geistdoerfer (Zoarcidae) from the hydrothermal sites Logatchev and Snake Pit-Moose of the Mid-Atlantic Ridge, at depths of 3,008; 3,492, and 3,510 m . The new genus Moravecnema is characterised by a dorsoventrally elongated oral opening, rudimentary pseudolabia, and four pairs of precloacal and six pairs of postcloacal caudal papillae in the male . The species has two spicules of unequal length, about 330 and 80 microm long . Males are about 5 mm and females about 5-10 mm long . Eggs have long thin polar filaments . This is the first species of parasitic nematode described from a fish endemic to hydrothermal deep-sea vents.

Aerosp Am, 2003 Mar, 41(3), 24 - 5
Geysers spawn hot new nanotechnology; Flinn ED; NASA: The article reports on efforts by NASA scientists to build nanostructures from thermophilic cyanobacteria living in hot springs . Heat shock protein 60 (HSP60) is purified and engineered to serve as a substrate for quantum dots that are about 1-10 nm across, much smaller than the typical 130 nm found in standard computer chips .

Water Sci Technol, 2003, 47(3), 151 - 6
Efficiency of autothermal thermophilic aerobic digestion and thermophilic anaerobic digestion of municipal wastewater sludge in removing Salmonella spp . and indicator bacteria; Zabranska J et al.; The study is focused on the comparison of autothermal thermophilic aerobic digestion, thermophilic and mesophilic anaerobic digestion, based on long-term monitoring of all processes in full-scale wastewater treatment plants, with an emphasis on the efficiency in destroying pathogens . The hygienisation effect was evaluated as a removal of counts of indicator bacteria, thermotolerant coliforms and enterococci as CFU/g total sludge solids and a frequency of a positive Salmonella spp . detection . Both thermophilic technologies of municipal wastewater sludge stabilisation had the capability of producing sludge A biosolids suitable for agricultural land application when all operational parameters (mainly temperature, mixing and retention time) were stable and maintained at an appropriate level.

Arch Microbiol, 2003 May, 179(5), 315 - 20 Epub 2003 Mar 14.
Methanol utilization by a novel thermophilic homoacetogenic bacterium, Moorella mulderi sp . nov., isolated from a bioreactor; Balk M et al.; A thermophilic, anaerobic, spore-forming bacterium (strain TMS) was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as the energy source . Cells were gram-positive straight rods, 0.4-0.6 microm x 2-8 microm, growing as single cells or in pairs . The temperature range for growth was 40-70 degrees C with an optimum at 65 degrees C . Growth was observed from pH 5.5 to 8.5, and the optimum pH was around 7 . The salinity range for growth was 0-45 g NaCl l(-1 )with an optimum at 10 g l(-1) . The isolate was able to grow on methanol, H(2)-CO(2 )(80/20%, v/v), formate, lactate, pyruvate, glucose, fructose, cellobiose and pectin . The bacterium reduced thiosulfate to sulfide . The G+C content of the DNA was 53 mol% . Comparison of 16S rRNA genes revealed that strain TMS is related to Moorella glycerini (96%, sequence similarity), Moorella thermoacetica (92%) and Moorella thermoautotrophica (92%) . On the basis of physiological and phylogenetic differences, strain TMS is proposed as a new species within the genus Moorella, Moorella mulderi sp . nov . (=DSM 14980, =ATCC BAA-608).

Biochim Biophys Acta, 2003 Mar 21, 1646(1-2), 136 - 44
Chimeric sensory kinases containing O2 sensor domain of FixL and histidine kinase domain from thermophile; Kumita H et al.; To explore the functional mechanism of inter-domain interaction in a sensor histidine kinase, five chimeric sensory kinases were constructed . In each of these chimeric proteins (CskA254, CskA264, CskA274, CskA284, and CskA294), the sensor domain of heme-based O(2) sensor FixL, obtained from Sinorhizobium meliloti, was fused with the histidine kinase domain from a hyperthermophile, Thermotoga maritima, each at a systematically different position . The UV-visible (UV-vis), resonance Raman (RR), and circular dichroism (CD) spectral characteristics of the CskAs indicated that the secondary and heme environmental structures of all five CskAs examined are identical to those of FixL . In spite of these structural similarities, all CskAs did not exhibit O(2)-dependent regulation of autophosphorylation activity . Furthermore, their functional properties were much different from those of FixL: The O(2) binding affinity and the autophosphorylation activity for CskA254, CskA264, and CskA274 were similar to those of the truncated sensor and histidine kinase domain, whereas CskA284 and CskA294 display extremely low O(2) affinity and low autophosphorylation activity, as compared with each truncated domain . These observations indicated that the interdomain interaction was presented in those CskAs, and that interaction could be related to the O(2)-dependent regulatory interaction of FixL . In the present study, we demonstrated that the interaction in the physiological sensor histidine kinase would be strictly and finely controlled to mediate the signal ligation-dependent autophosphorylation activity in its histidine kinase domain.

Microbiology, 2003 Mar, 149(Pt 3), 807 - 13
16S-23S rRNA intergenic spacer region sequence variation in Streptococcus thermophilus and related dairy streptococci and development of a multiplex ITS-SSCP analysis for their identification; Mora D et al.; The 16S-23S rRNA internal transcribed spacer (ITS) region of several Streptococcus thermophilus strains and some related dairy streptococci, S . macedonicus, S . salivarius and S . bovis, was analysed by sequence analysis . All the Streptococcus species were easily discriminated on the basis of sequence variations principally located upstream and downstream of the region encompassing the double-stranded processing sites and the tRNA(Ala) gene . Comparison between tRNA(Ala) gene sequences highlighted a high level of sequence conservation among the Streptococcus species investigated despite their belonging to separated phylogenetic clusters, i.e . the S . salivarius and S . bovis rRNA groups . A low but significant degree of variability was detected among the S . thermophilus strains, allowing the identification of four different ITS sequences . Similarity analysis of the ITS sequences showed that the Streptococcus species were clustered in two main branches, one containing S . macedonicus and S . bovis strains, and one containing S . thermophilus and S . salivarius strains . With the aim of developing a rapid tool for the identification of the dairy streptococci species a multiplex ITS-SSCP analysis of two discrete regions within the ITS locus was carried out.

J Mol Biol, 2003 Mar 28, 327(3), 745 - 57
Protein dynamics in a family of laboratory evolved thermophilic enzymes; Wintrode PL et al.; Molecular dynamics simulations were employed to study how protein solution structure and dynamics are affected by adaptation to high temperature . Simulations were carried out on a para-nitrobenzyl esterase (484 residues) and two thermostable variants that were generated by laboratory evolution . Although these variants display much higher melting temperatures than wild-type (up to 18 degrees C higher) they are both >97% identical in sequence to the wild-type . In simulations at 300 K the thermostable variants remain closer to their crystal structures than wild-type . However, they also display increased fluctuations about their time-averaged structures . Additionally, both variants show a small but significant increase in radius of gyration relative to wild-type . The vibrational density of states was calculated for each of the esterases . While the density of states profiles are similar overall, both thermostable mutants show increased populations of the very lowest frequency modes (<10 cm(-1)), with the more stable mutant showing the larger increase . This indicates that the thermally stable variants experience increased concerted motions relative to wild-type . Taken together, these data suggest that adaptation for high temperature stability has resulted in a restriction of large deviations from the native state and a corresponding increase in smaller scale fluctuations about the native state . These fluctuations contribute to entropy and hence to the stability of the native state . The largest changes in localized dynamics occur in surface loops, while other regions, particularly the active site residues, remain essentially unchanged . Several mutations, most notably L313F and H322Y in variant 8G8, are in the region showing the largest increase in fluctuations, suggesting that these mutations confer more flexibility to the loops . As a validation of our simulations, the fluctuations of Trp102 were examined in detail, and compared with Trp102 phosphorescence lifetimes that were previously measured . Consistent with expectations from the theory of phosphorescence, an inverse correlation between out-of-plane fluctuations on the picosecond time scale and phosphorescence lifetime was observed.

J Environ Radioact, 2003, 67(1), 1 - 14
Ecological effects of various toxic agents on the aquatic microcosm in comparison with acute ionizing radiation; Fuma S et al.; The purpose of this study was an evaluation of the effect levels of various toxic agents compared with acute doses of ionizing radiation for the experimental model ecosystem, i.e., microcosm mimicking aquatic microbial communities . For this purpose, the authors used the microcosm consisting of populations of the flagellate alga Euglena gracilis as a producer, the ciliate protozoan Tetrahymena thermophila as a consumer and the bacterium Escherichia coli as a decomposer . Effects of aluminum and copper on the microcosm were investigated in this study, while effects of gamma-rays, ultraviolet radiation, acidification, manganese, nickel and gadolinium were reported in previous studies . The microcosm could detect not only the direct effects of these agents but also the community-level effects due to the interspecies interactions or the interactions between organisms and toxic agents . The authors evaluated doses or concentrations of each toxic agent which had the following effects on the microcosm: (1) no effects; (2) recognizable effects, i.e., decrease or increase in the cell densities of at least one species; (3) severe effects, i.e., extinction of one or two species; and (4) destructive effects, i.e., extinction of all species . The resulting effects data will contribute to an ecological risk assessment of the toxic agents compared with acute doses of ionizing radiation.

Folia Microbiol (Praha), 2002, 47(6), 685 - 90
Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus; de Palma-Fernandez ER et al.; beta-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps--ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized . After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3) . Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix . Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively . The temperature optimum of both glucosidases was at 75-80 degrees C . The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability . Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively . Using 4-nitrophenyl beta-D-glucopyranoside as substrate, Km values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively . Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Biochemistry, 2003 Mar 18, 42(10), 2857 - 65
Crystal structure of ribosomal protein L30e from the extreme thermophile Thermococcus celer: thermal stability and RNA binding; Chen YW et al.; We report here the high-resolution crystal structure of the ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer determined at cryo-temperature . When it is compared with its mesophilic homologue, L30e from yeast, a number of structural features that can enhance thermostability are revealed . Disordered residues corresponding to a large RNA-binding loop in yeast L30e are well structured in the T . celer protein . The overall charge of T . celer L30e is near neutral, whereas that of the yeast homologue is highly positive . This is the result of an increase in the number of acidic residues at the expense of polar residues, Asn, Ser, and Thr . Extensive ion pair networks are found on the molecular surface . Exposed nonpolar surface areas are reduced in the T . celer protein . Its side chain atoms preferably form hydrogen bonds with main chain atoms . Taken together, these factors contribute to high protein stability . The roles of well-conserved L30e residues are studied and found to be important in defining a very compact overall structure and in maintaining the structure of the RNA binding site . By comparing it with the yeast homologue, we also identified the residues that are responsible for RNA binding and built a model to illustrate how L30e binds to an RNA kink turn motif.

Biochemistry, 2003 Mar 18, 42(10), 2781 - 9
Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase: insertion of valine C gamma into the substrate access channel; Hearn AS et al.; Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion . We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal . Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD . These effects are not attributed to the redox potentials, which are similar for all of these variants . An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type . The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site . This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished . Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.

Nucleic Acids Res, 2003 Mar 15, 31(6), 1673 - 82
Complete sequence of the mitochondrial genome of Tetrahymena thermophila and comparative methods for identifying highly divergent genes; Brunk CF et al.; The complete sequence of the mitochondrial genome of Tetrahymena thermophila has been determined and compared with the mitochondrial genome of Tetrahymena pyriformis . The sequence similarity clearly indicates homology of the entire T.thermophila and T.pyriformis mitochondrial genomes . The T.thermophila genome is very compact, most of the intergenic regions are short (only three are longer than 63 bp) and comprise only 3.8% of the genome . The nad9 gene is tandemly duplicated in T.thermophila . Long terminal inverted repeats and the nad9 genes are undergoing concerted evolution . There are 55 putative genes: three ribosomal RNA genes, eight transfer RNA genes, 22 proteins with putatively assigned functions and 22 additional open reading frames of unknown function . In order to extend indications of homology beyond amino acid sequence similarity we have examined a number of physico-chemical properties of the mitochondrial proteins, including theoretical pI, molecular weight and particularly the predicted transmembrane spanning regions . This approach has allowed us to identify homologs to ymf58 (nad4L), ymf62 (nad6) and ymf60 (rpl6).

Rocz Panstw Zakl Hig, 2002, 53(3), 277 - 82
{The evaluation of thermophilic fungi in raw coffee beans.}; Falkowski J et al.; The purpose of the study was the attempt of the isolation of the thermophilic fungi from raw coffee beans . The material constituted of 24 coffee beans samples came from 12 countries . The isolation and the identification of the thermophilic fungi was conducted according to Bilaj {2}, Bilaj and Zacharczenko {3} . The study proved, that raw coffee beans were the rich source of the thermophilic mycoflora . From all tested samples 270 species were isolated . The most refused sample came from Ecuador--81% coffee beans were infected . The most of species (90% from among isolated) were species belonged to the Thermomyces lanuginosus.

Appl Environ Microbiol, 2003 Mar, 69(3), 1680 - 6
Biogeochemical evidence that thermophilic archaea mediate the anaerobic oxidation of methane; Schouten S et al.; Distributions and isotopic analyses of lipids from sediment cores at a hydrothermally active site in the Guaymas Basin with a steep sedimentary temperature gradient revealed the presence of archaea that oxidize methane anaerobically . The presence of strongly (13)C-depleted lipids at greater depths in the sediments suggests that microbes involved in anaerobic oxidation of methane are present and presumably active at environmental temperatures of >30 degrees C, indicating that this process can occur not only at cold seeps but also at hydrothermal sites . The distribution of the membrane tetraether lipids of the methanotrophic archaea shows that these organisms have adapted their membrane composition to these high environmental temperatures.

Appl Environ Microbiol, 2003 Mar, 69(3), 1417 - 27
Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric, and ferulic acids; Peng X et al.; Thirty-four thermophilic Bacillus sp . strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions . It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids . The metabolic pathways for the degradation of these aromatic acids at 60 degrees C were examined by using one of the isolates, strain B1 . Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively . The beta-oxidative mechanism was proposed to be responsible for these conversions . The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission . It is likely that a non-beta-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-beta-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates . Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.

Appl Environ Microbiol, 2003 Mar, 69(3), 1377 - 82
New thermosensitive delivery vector and its use to enable nisin-controlled gene expression in Lactobacillus gasseri; Neu T et al.; Derivatives of a cryptic plasmid from Lactobacillus curvatus showed temperature-sensitive replication in thermophilic lactobacilli . The thermosensitive replicon was used to construct the new delivery vector pTN1, which allows site-specific replacement of chromosomal DNA sequences . pTN1 carries an erythromycin resistance marker suitable for selection of single-copy integrants and replicates readily at 35 degrees C, whereas replication is efficiently shut down at 42 degrees C . To demonstrate the functionality of pTN1, the signal transduction genes (nisRK) of the nisin-controlled expression system were integrated downstream of the pepN gene into the chromosome of Lactobacillus gasseri . In the resulting strain, UKLbg1, expression of nisRK was likely driven by cotranscription with pepN and enabled nisin-dependent induction of a fusion of a reporter gene (pepI) to the nisA promoter . The induction rates were correlated with the amount of nisin used, and maximum pepI expression was achieved with nisin concentrations (above 25 ng/ml) at which growth of the bacteria was already inhibited.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 297 - 304
Methanogenic population structure in a variety of anaerobic bioreactors; McHugh S et al.; The methanogenic community structures of six anaerobic sludges were examined using culture-independent techniques . The sludges were obtained from full-scale and laboratory-scale bioreactors, treating a variety of low- and high-strength, simple and complex wastewaters at psychrophilic (10-14 degrees C), mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures . Amplified rDNA restriction analysis identified 18 methanogenic operational taxonomic units in the six samples . 16S rRNA gene sequencing and phylogenetic reconstruction demonstrated that five separate groups of methanogens were represented with Methanosaeta-like species dominant in all sludges, but particularly in samples from a psychrophilic bioreactor treating low-strength synthetic sewage (75% of all clones detected).

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 183 - 5
A novel cellulolytic, anaerobic, and thermophilic bacterium, Moorella sp . strain F21; Karita S et al.; A cellulolytic and thermophilic anaerobe was isolated from soil . This bacterium made a halo on a roll-tube culture containing Avicel . Analysis of the PCR-based 16S rRNA gene sequence showed that the bacterium was closely related to Moorella thermoacetica . Scanning electron microscopy showed the bacterium is a rod and has no protuberant structure on the surface of cells growing on cellulose, suggesting that this strain is a non-cellulosomal cellulolytic bacterium . Carboxymethyl cellulase and xylanase activities were detected in the culture broth . A major fermentation product from ball-milled cellulose was acetate . This strain has a potential to convert cellulosic biomass to acetate, directly.

Bioresour Technol, 2003 Jul, 88(3), 207 - 14
Comparison of laboratory-scale thermophilic biofilm and activated sludge processes integrated with a mesophilic activated sludge process; Suvilampi J et al.; A combined thermophilic-mesophilic wastewater treatment was studied using a laboratory-scale thermophilic activated sludge process (ASP) followed by mesophilic ASP or a thermophilic suspended carrier biofilm process (SCBP) followed by mesophilic ASP, both systems treating diluted molasses (dilution factor 1:500 corresponding GF/A-filtered COD (COD(filt)) of 1900+/-190 mgl(-1)) . With hydraulic retention times (HRTs) of 12-18 h the thermophilic ASP and thermophilic SCBP removed 60+/-13% and 62+/-7% of COD(filt), respectively, with HRT of 8 h the removals were 48+/-1% and 69+/-4% . The sludge volume index (SVI) was notably lower in the thermophilic SCBP (measured from suspended sludge) than in the thermophilic ASP . Under the lowest HRT the mesophilic ASP gave better performance (as SVI, COD(filt), and COD(tot) removals) after the thermophilic SCBP than after the thermophilic ASP . Measured sludge yields were low (less than 0.1 kg suspended solids (SS) kg COD(filt removed)(-1)) in all processes . Both thermophilic treatments removed 80-85% of soluble COD (COD(sol)) whereas suspended COD (COD(susp)) and colloidal COD (COD(col)) were increased . Both mesophilic post-treatments removed all COD(col) and most of the COD(susp) from the thermophilic effluents . In conclusion, combined thermophilic-mesophilic treatment appeared to be easily operable and produced high effluent quality.

J Struct Biol, 2003 Feb, 141(2), 149 - 55
The 1.45 A three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus; Nield J et al.; The conversion of solar radiation to chemical energy by photosynthetic organisms provides the primary driving force for life on earth . Light energy is captured by a variety of pigments, usually bound to proteins, which vary with different types of organisms . We report here the 1.45 A resolution three-dimensional structure of one such pigment protein, C-phycocyanin, from Synechococcus elongatus . The structure is at the highest resolution achieved for any such phycobiliprotein . This level of resolution was made possible by implementing a novel crystallization method whereby nucleation is decoupled from subsequent growth, by incubating crystallizing drops for 7h under nucleation conditions and then transferring them to metastable conditions for growth . This is done without touching the crystallization drops throughout the process.

Aquat Toxicol, 2003 Mar 17, 63(1), 27 - 41
Ecotoxicological study of Lithuanian and Estonian wastewaters: selection of the biotests, and correspondence between toxicity and chemical-based indices; Manusadzianas L et al.; The toxicity of industrial and urban wastewater (WW) samples collected in Lithuania and Estonia was evaluated by using a suite of biological tests comprising the Algaltoxkit F with Selenastrum capricornutum, the Charatox with Nitellopsis obtusa, Daphtoxkit F with Daphnia magna, Thamnotoxkit F with Thamnocephalus platyurus, Protoxkit F with Tetrahymena thermophila and the Microtox with Vibrio fischeri . The Charatox and Thamnotoxkit F tests showed highest relative sensitivity, responding to 80-90% of samples, respectively, and both expressed good discrimination capacity between samples . Principal Component and pairwise correlation analysis allowed to select test-battery consisting of Charatox, Thamnotoxkit and Microtox . The WW toxicity was evaluated by means of cumulative indices such as average toxicity (AvTx) and two indices derived from the PEEP-index (Environ . Toxicol . Water Qual . 8 (1993) 115) . In addition to these integrated evaluations of test-battery response, WW toxicity was evaluated according to the most sensitive test (MST) in the battery . The linear regression analysis between cumulative toxicity indices and chemical-based indices (derived from comparison of WW chemical concentrations and their respective maximum allowable concentration) revealed positive linear relationships (r(2)=0.7-0.8), while toxicity evaluation based on the MST was less positively related with chemical analysis data (r(2)=0.5-0.6) . Although better coincidence between the toxicity and chemical-based assessments was achieved when information from all tests in the battery was assembled, the prediction of toxicity from chemical data was still limited . In search of suitable test-battery for the screening of certain type of WWs, a preliminary study comprising excessive suite of tests might be useful.

Biochim Biophys Acta, 2003 Mar 6, 1557(1-3), 13 - 9
The respiratory chain of the thermophilic archaeon Sulfolobus metallicus: studies on the type-II NADH dehydrogenase; Bandeiras TM et al.; The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O(2) min(-1) mg(-1), which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase . Following this observation, the enzyme was purified from solubilised membranes and characterised . The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type . It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases . Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein . The average reduction potential of the FMN group was determined to be +160 mV, at 25 degrees C and pH 6.5, by redox titrations monitored by visible spectroscopy . Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone . Maximal turnover rates of 195 micromol NADH oxidized min(-1) mg(-1) at 60 degrees C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.

J Ind Microbiol Biotechnol, 2003 Feb, 30(2), 95 - 101 Epub 2003 Jan 15.
Production of exopolysaccharides from a thermophilic microorganism isolated from a marine hot spring in flegrean areas; Schiano Moriello V et al.; A thermophilic strain isolated from sea sand at Maronti, near Sant' Angelo (Ischia), is described . The organism grows well at an optimal temperature of 60 degrees C at pH 7.0 . The thermophilic bacterium, named strain 4004, produces an exocellular polysaccharide (EPS) in yields of 90 mg/l . The EPS fraction was produced with all substrates tested, although a higher yield was obtained with sucrose or trehalose as sole carbon source . During growth, the EPS content was proportional to the biomass . Three fractions (EPS1, EPS2, EPS3) were obtained after purification . Quantitative monosaccharide analysis of the EPSs revealed the presence of mannose:glucose:galactose in a relative ratio of 0.5:1.0:0.3 in EPS1, mannose:glucose:galactose in a relative ratio of 1.0:0.3:trace in EPS2, and galactose:mannose:glucosamine:arabinose in a relative ratio of 1.0:0.8:0.4:0.2 in EPS3 . The average molecular mass of EPS3 was determined to be 1x10(6) Da . From comparison of the chemical shift values in (1)H and (13)C spectra, we conclude that EPS3 presents a pentasaccharide repeating unit.

J Ind Microbiol Biotechnol, 2003 Feb, 30(2), 81 - 8 Epub 2003 Jan 15.
Kinetic comparisons of mesophilic and thermophilic aerobic biomass; Vogelaar JC et al.; Kinetic parameters describing growth and decay of mesophilic (30 degrees C) and thermophilic (55 degrees C) aerobic biomass were determined in continuous and batch experiments by using oxygen uptake rate measurements . Biomass was cultivated on a single soluble substrate (acetate) in a mineral medium . The intrinsic maximum growth rate ( micro (max)) at 55 degrees C was 0.71+/-0.09 h(-1), which is 1.5 times higher than the micro (max) at 30 degrees C (0.48+/-0.11 h(-1)) . The biomass decay rates increased from 0.004 h(-1) at 30 degrees C to 0.017 h(-1) at 55 degrees C . Monod constants were very low for both types of biomass: 9+/-2 mg chemical oxygen demand (COD) l(-1)at 30 degrees C and 3+/-2 mg COD l(-1)at 55 degrees C . Theoretical biomass yields were similar at 30 and 55 degrees C: 0.5 g biomass COD (g acetate COD)(-1) . The observed biomass yields decreased under both temperature conditions as a function of the cell residence time . Under thermophilic conditions, this effect was more pronounced due to the higher decay rates, resulting in lower biomass production at 55 degrees C compared to 30 degrees C.

Arch Microbiol, 2003 Mar, 179(3), 160 - 73 Epub 2003 Feb 12.
Autotrophic CO2 fixation pathways in archaea (Crenarchaeota); Hugler M et al.; Representative autotrophic and thermophilic archaeal species of different families of Crenarchaeota were examined for key enzymes of the known autotrophic CO(2) fixation pathways . Pyrobaculum islandicum ( Thermoproteaceae) contained key enzymes of the reductive citric acid cycle . This finding is consistent with the operation of this pathway in the related Thermoproteus neutrophilus . Pyrodictium abyssi and Pyrodictium occultum ( Pyrodictiaceae) contained ribulose 1,5-bisphosphate carboxylase, which was active in boiling water . Yet, phosphoribulokinase activity was not detectable . Operation of the Calvin cycle remains to be demonstrated . Ignicoccus islandicus and Ignicoccus pacificus ( Desulfurococcaceae) contained pyruvate oxidoreductase as potential carboxylating enzyme, but apparently lacked key enzymes of known pathways; their mode of autotrophic CO(2) fixation is at issue . Metallosphaera sedula, Acidianus ambivalens and Sulfolobus sp . strain VE6 ( Sulfolobaceae) contained key enzymes of a 3-hydroxypropionate cycle . This finding is in line with the demonstration of acetyl-coenzyme A (CoA) and propionyl-CoA carboxylase activities in the related Acidianus brierleyi and Sulfolobus metallicus . Enzymes of central carbon metabolism in Metallosphaera sedula were studied in more detail . Enzyme activities of the 3-hydroxypropionate cycle were strongly up-regulated during autotrophic growth, supporting their role in CO(2) fixation . However, formation of acetyl-CoA from succinyl-CoA could not be demonstrated, suggesting a modified pathway of acetyl-CoA regeneration . We conclude that Crenarchaeota exhibit a mosaic of three or possibly four autotrophic pathways . The distribution of the pathways so far correlates with the 16S-rRNA-based taxa of the Crenarchaeota.

Arch Microbiol, 2003 Mar, 179(3), 145 - 50 Epub 2003 Feb 07.
Laccases and their occurrence in prokaryotes; Claus H; Laccases are copper-containing proteins that require O(2) to oxidize phenols, polyphenols, aromatic amines, and different non-phenolic substrates by one-electron transfer, resulting in the formation of reactive radicals . Although their specific physiological functions are not completely understood, there are several indications that laccases are involved in the morphogenesis of microorganisms (e.g., fungal spore development, melanization) and in the formation and/or degradation of complex organic substances such as lignin or humic matter . Owing to their high relative non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications . To date, laccases have been found only in eukaryotes (fungi, plants); however, databank searches and experimental data now provide evidence for their distribution in prokaryotes . This survey shows that laccase-like enzymes occur in many gram-negative and gram-positive bacteria . Corresponding genes have been found in prokaryotes that are thought to have branched off early during evolution, e.g., the extremely thermophilic Aquifex aeolicus and the archaeon Pyrobaculum aerophilum . Phylogenetically, the enzymes are members of the multi-copper protein family that have developed from small-sized prokaryotic azurins to eukaryotic plasma proteins.

FEBS Lett, 2003 Feb 27, 537(1-3), 53 - 7
Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers; Fox JD et al.; Escherichia coli maltose-binding protein (MBP) is frequently used as an affinity tag to facilitate the purification of recombinant proteins . An important additional attribute of MBP is its remarkable ability to enhance the solubility of its fusion partners . MBPs are present in a wide variety of microorganisms including both mesophilic and thermophilic bacteria and archaea . In the present study, we compared the ability of MBPs from six diverse microorganisms (E . coli, Pyrococcus furiosus, Thermococcus litoralis, Vibrio cholerae, Thermotoga maritima, and Yersinia pestis) to promote the solubility of eight different aggregation-prone proteins in E . coli . In contrast to glutathione S-transferase (GST), all of these MBPs proved to be effective solubility enhancers and some of them were even more potent solubilizing agents than E . coli MBP.

Eur J Biochem, 2003 Jan, 270(2), 190 - 205
Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit; Yamaguchi K et al.; Six ribosomal proteins are specific to higher plant chloroplast ribosomes {Subramanian, A.R . (1993) Trends Biochem . Sci.18, 177-180} . Three of them have been fully characterized {Yamaguchi, K., von Knoblauch, K . & Subramanian, A . R . (2000) J . Biol . Chem . 275, 28455-28465; Yamaguchi, K . & Subramanian, A . R . (2000) J . Biol . Chem . 275, 28466-28482} . The remaining three plastid-specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N-terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs) . PSRP-3 exists in two forms (alpha/beta, N-terminus free and blocked by post-translational modification), whereas PSRP-2 and PSRP-4 appear, from MS data, to be unmodified . PSRP-2 contains two RNA-binding domains which occur in mRNA processing/stabilizing proteins (e.g . U1A snRNP, poly(A)-binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis . PSRP-3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga . The ycf65 gene is absent from the chloroplast genomes of higher plants . Therefore, we suggest that Psrp-3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle-to-nucleus gene transfer in chloroplast evolution . PSRP-4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis . We would therefore suggest that PSRP-4 is an example of gene capture (via horizontal gene transfer) during chloro-ribosome emergence . Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database . All six PSRPs are nucleus-encoded . The cytosolic precursors of PSRP-2, PSRP-3, and PSRP-4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively . Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.

Arch Microbiol, 2003 Apr, 179(4), 250 - 7 Epub 2003 Feb 19.
Characterization and phylogenetic analysis of a thermostable N-carbamoyl- l-amino acid amidohydrolase from Bacillus kaustophilus CCRC11223; Hu HY et al.; A thermostable N-carbamoyl- l-amino acid amidohydrolase ( l-N-carbamoylase) gene composed of an 1,230-bp ORF encoding a 44.3-kDa protein was cloned from the thermophile Bacillus kaustophilus CCRC11223 . This l-N-carbamoylase contained six cysteine residues that form three disulfide bridges . The purified l-N-carbamoylase was stringently l-specific and exhibited high activity in the hydrolysis of N-carbamoyl- l-homophenylalanine . N-carbamoyl derivatives of beta-alanine, beta-aminoisobutyric acids, l-tryptophan, and d-specific amino acids were not recognized as substrates . The l-N-carbamoylase required the divalent metal ions Mn(2+), Co(2+), and Ni(2+) for increasing activity . The pH and temperature optima of the enzyme were pH 7.4 and 70 degrees C, respectively . This enzyme was completely thermostable at 50 degrees C for 36 days in the presence of d- and/or l-specific substrates . Phylogenetic analysis of the available amino acid sequences of N-carbamoyl and N-acyl amino acid amidohydrolases from the three main kingdoms of life showed that they can be divided into four distinct families . The B . kaustophilus enzyme could be classified into the family of l-N-carbamoylases and some beta-ureidopropionases, but did not hydrolyze beta-ureidopropionates.

J Biol Chem, 2003 May 9, 278(19), 17198 - 202 Epub 2003 Feb 24.
Genomic correlates of hyperthermostability, an update; Suhre K et al.; It has been shown (Cambillau, C., and Claverie, J . M . (2000) J . Biol . Chem . 275, 32383-32386) that a large difference between the proportions of charged versus polar (non-charged) amino acids (CvP-bias) was an adequate, if empirical, signature of the proteome of hyperthermophilic organisms (T(growth) >80 degrees C) . Since that study, the number of available microbial genomes has more than doubled, raising the possibility that the simple CvP-bias rule might no longer hold . Taking advantage of the new sequence data, we re-analyzed the genomes of 9 fully sequenced thermophiles, 9 hyperthermophiles, and 53 mesothermophile microorganisms to identify the genomic correlates of hyperthermostability on a wider data set . Our new results confirm that the CvP-bias previously identified on a much smaller data set still holds . Moreover, we show that it is an optimal criterion, in the sense that it corresponds to the most discriminating factor between hyperthermophilic and mesothermophilic microorganisms in a principal component analysis . In parallel, we evaluated two other recently proposed correlates of hyperthermostability, the proteome average pI and the dinucleotide statistical index (Kawashima, T., Amano, N., Koike, H., Makino, S., Higuchi, S., Kawashima-Ohya, Y., Watanabe, K., Yamazaki, M., Kanehori, K., Kawamoto, T., Nunoshiba, T., Yamamoto, Y., Aramaki, H., Makino, K., and Suzuki, M . (2000) Proc . Natl . Acad . Sci . 97, 14257-14262) . We show that the CvP-bias is the sole criterion that is able to clearly discriminate hyperthermophile from mesothermophile microorganisms on a global genomic basis.

Water Res, 2003 Apr, 37(7), 1628 - 36
Performance of temperature-phased anaerobic digestion (TPAD) system treating dairy cattle wastes; Sung S et al.; The performance of temperature-phased anaerobic digestion (TPAD) system in the stabilization of dairy cattle wastes at high solids concentrations has never been evaluated, though the process has been established as a feasible alternative to conventional mesophilic processes for the treatment of municipal wastewater sludges . In this study, the TPAD system operating at a retention time of 14 days was subjected to varying total solids (TS) concentrations (3.46-14.54%) of dairy cattle wastes . At TS concentrations lower than 12.20%, corresponding to system volatile solids (VS) loadings in the range of 1.87-5.82 g VS/L/day, the system achieved an average VS removal of 40.2% . The maximum VS destruction of 42.6% was achieved at a TS concentration of 10.35% . Methane recovery from the wastes was consistently within 0.21-0.22 L/g VS fed . There was a drop in the system performance with respect to VS removal and methane recovery at TS concentrations higher than 10.35% . volatile fatty acid/alkalinity ratios less than 0.35 in the thermophilic reactor and 0.10 in the mesophilic reactor were found favorable for stable operation of the system . For the entire range of TS concentrations, the indicator organism counts in the biosolids were within the limits specified by USEPA in 40 CFR Part 503 regulations for Class A designation . After digestion, nearly 80-85% of total phosphorus was associated with the biosolids .

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2312 - 5 Epub 2003 Feb 21.
Evidence for rotation of V1-ATPase; Imamura H et al.; V(o)V(1)-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria . From the similarity to F(o)F(1)-ATP synthase, V(o)V(1)-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this . Here we visualized the rotation of single molecules of V(1)-ATPase, a catalytic subcomplex of V(o)V(1)-ATPase . V(1)-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage . In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side . Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations . This study provides experimental evidence that V(o)V(1)-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

J Chromatogr A, 2003 Feb 7, 986(2), 275 - 90
Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent; Arvidsson P et al.; A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates . The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N,N'-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization) . After thawing, the column contains a continuous matrix having interconnected pores of 10-100 microm size . Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His)6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli . The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column . No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column . A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties . Mechanically the cryogel adsorbent is very stable . The continuous matrix could easily be removed from the column, dried at 70 degrees C and kept in a dry state . After rehydration and reinsertion of the matrix into an empty column, (His)6-LDH was purified as efficiently as on the newly prepared column . The procedure of manufacturing the supermacroporous continuous cryogel is technically simple . Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions . Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts.

Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 576 - 9 Epub 2003 Feb 21.
Crystallization of a member of the recFOR DNA repair pathway, RecO, with and without bound oligonucleotide; Aono S et al.; RecFOR proteins are important for DNA repair by homologous recombination in bacteria . The RecO protein from Thermus thermophilus was cloned and purified, and its binding to oligonucleotides was characterized . The protein was crystallized alone and in complex with a 14-mer oligonucleotide . Both crystal forms grow under different crystallization conditions in the same space group, P3(1)21 or P3(2)21, with almost identical unit-cell parameters . Complete data sets were collected to 2.8 and 2.5 A for RecO alone and for the RecO-oligonucleotide complex, respectively . Visual comparison of the diffraction patterns between the two crystal forms and calculation of an R(merge) of 33.9% on F indicate that one of the crystal forms is indeed a complex of RecO with bound oligonucleotide.

Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 554 - 7 Epub 2003 Feb 21.
Coexpression, purification, crystallization and preliminary X-ray characterization of glycine decarboxylase (P-protein) of the glycine-cleavage system from Thermus thermophilus HB8; Nakai T et al.; Thermus thermophilus (Tth) HB8 glycine decarboxylase (P-protein) is an alpha(2)beta(2) tetrameric enzyme with a total molecular mass of 200 kDa . The alpha- and beta-subunits of the Tth P-protein have been coexpressed in Escherichia coli and purified as a stable complex . Dynamic light-scattering measurements indicated the recombinant protein to be monodisperse and its size to be consistent with an alpha(2)beta(2) tetrameric composition . Crystals of the protein have been grown in polyethylene glycol 3350 using the vapour-diffusion method at 291 K . Synchrotron radiation from BL45XU at SPring-8 was used to measure a complete native data set to 2.4 A resolution . The crystals belong to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 89.5, c = 371.0 A . Estimation of the crystal packing (V(M) = 2.15 A(3) Da(-1)) and self-rotation function analysis suggest the presence of one alpha(2)beta(2) tetramer per asymmetric unit, with the molecules related by non-crystallographic twofold symmetry.

Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 551 - 3 Epub 2003 Feb 21.
Expression, purification and preliminary X-ray characterization of CTP synthetase from Thermus thermophilus HB8; Goto M et al.; A recombinant form of the CTP synthetase from Thermus thermophilus HB8 (tCTPs) was grown as colourless crystals by the hanging-drop vapour-diffusion technique using ammonium sulfate or sodium citrate as a precipitating agent . The crystals belong to space group I222, with unit-cell parameters a = 88.2, b = 118.9, c = 142.7 A, alpha = beta = gamma = 90 degrees, and are most likely to contain a monomer in the asymmetric unit with a V(M) value of 3.07 A(3) Da(-1) . The crystals obtained from ammonium sulfate and sodium citrate solutions diffract X-rays to a resolution of 2.25 A using synchrotron X-ray sources and to a resolution of 2.35 A using Cu Kalpha X-rays from a rotating-anode generator.

J Biol Chem, 2003 Apr 25, 278(17), 14893 - 6 Epub 2003 Feb 19.
Oxygen-linked equilibrium CuB-CO species in cytochrome ba3 oxidase from thermus thermophilus . Implications for an oxygen channel ar the CuB site; Koutsoupakis K et al.; We report the first study of O(2) migration in the putative O(2) channel of cytochrome ba(3) and its effect to the properties of the binuclear heme a(3)-Cu(B) center of cytochrome ba(3) from Thermus thermophilus . The Fourier transform infrared spectra of the ba(3)-CO complex demonstrate that in the presence of 60-80 micro m O(2), the nu(C-O) of Cu(B)1+-C-O at 2053 cm(-1) (complex A) shifts to 2045 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and in the pH 6.5-9.0 range . The frequencies but not the intensities of the C-O stretching modes of heme a(3)-CO (complex B), however, remain unchanged . The change in the nu(C-O) of complex A results in an increase of k(-2), and thus in a higher affinity of Cu(B) for exogenous ligands . The time-resolved step-scan Fourier transform infrared difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex at pH 6.5 is 30.4 s(-1) and 28.3 s(-1) in the presence of O(2) . Similarly, the rebinding to heme a(3) is slightly affected and occurs with k(2) = 26.3 s(-1) and 24.6 s(-1) in the presence of O(2) . These results provide solid evidence that in cytochrome ba(3), the ligand delivery channel is located at the Cu(B) site, which is the ligand entry to the heme a(3) pocket . We suggest that the properties of the O(2) channel are not limited to facilitating ligand diffusion to the active site but are extended in controlling the dynamics and reactivity of the reactions of ba(3) with O(2) and NO.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3689 - 94 Epub 2003 Feb 14.
Functional copper at the acetyl-CoA synthase active site; Seravalli J et al.; The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation . A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a {4Fe-4S} cluster bridged to a binuclear Cu-Ni site . Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M . thermoacetica ACS active site is essential . Depletion of copper correlates with reduction in ACS activity and in intensity of the "NiFeC" EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH . In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity . Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila . XAS studies are consistent with a distorted Cu(I)-S(3) site in the fully active enzyme in solution . Cu extended x-ray absorption fine structure analysis indicates an average Cu-S bond length of 2.25 A and a metal neighbor at 2.65 A, consistent with the Cu-Ni distance observed in the crystal structure . XAS experiments in the presence of seleno-CoA reveal a Cu-S(3)Se environment with a 2.4-A Se-Cu bond, strongly implicating a Cu-SCoA intermediate in the mechanism of acetyl-CoA synthesis . These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms.

J Appl Microbiol, 2003, 94(3), 495 - 500
Antimicrobial resistance and genomic screening of clinical isolates of thermophilic Campylobacter spp . from south-east Queensland, Australia; Alfredson DA et al.; AIM: To screen 90 clinical isolates of thermophilic Campylobacter species for putative resistance to ampicillin, erythromycin and tetracycline and perform numerical analysis to determine isolate relatedness . METHODS AND RESULTS: Disc diffusion, E-test MIC and agar dilution methods were performed . Disc diffusion testing showed 87 (97%) isolates appeared resistant to ampicillin at 10 microg; 14 (16%) resistant to tetracycline at 30 microg; and three (3.4%) resistant to erythromycin at 15 microg . E-test MICs showed a range of 0.5 to >256 mg l(-1) for ampicillin; 16 to >256 mg l(-1) for tetracycline; and >256 mg l(-1) for erythromycin . E-test showed 68% correlation (+/-1 log2 dilution) with agar dilution for ampicillin, 100% for erythromycin and 64% for tetracycline . Disc diffusion testing showed 100% correlation with agar dilution for erythromycin and tetracycline, and 77% for ampicillin . Numerical analyses of restriction endonuclease (RE) fragment profiles suggested a high level of isolate variation . CONCLUSION: The incidence of resistance of thermophilic Campylobacter spp . to erythromycin and tetracycline is low in south-east Queensland . SIGNIFICANCE AND IMPACT OF THE STUDY: Disc diffusion susceptibility testing may be used to screen thermophilic Campylobacter spp . for putative resistance to erythromycin and tetracycline . Agar dilution should be used to determine ampicillin susceptibility.

Environ Microbiol, 2003 Mar, 5(3), 152 - 62
The impact of grassland management on archaeal community structure in upland pasture rhizosphere soil; Nicol GW et al.; The community structure of rhizosphere soil Archaea from three grassland types, associated with different management practices, was examined at a site in the Borders region of Scotland, by analysis of 16S rRNA gene fragments amplified from 16S rDNA and from rRNA . Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of amplified products indicated high relative abundance within the archaeal community of two distinct lineages of non-thermophilic (group 1) Crenarchaeota . Grassland management practices influenced archaeal community structure, as characterized by both 16S rRNA- and 16S rDNA-derived DGGE profiles . One band dominated DGGE profiles in all three grassland types examined, and reproducible differences in the presence and intensity of bands were observed between profiles from managed and natural grassland sites . Analysis of 16S rRNA-derived amplicons from managed and natural grasslands at sites in the north of England and the north of Wales also indicated high relative abundance of non-thermophilic crenarchaeotes within the archaeal community . The band dominating the Scottish grassland site also dominated DGGE profiles from the English and Welsh sites, and similar differences were seen between profiles derived from soils subjected to different management regimes . The study indicates that grassland archaeal communities are dominated by Crenarchaeota, with closely related members of this lineage ubiquitous in distribution in UK upland pasture, and indicate that management practices influence the nature of the crenarchaeotal community.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 359 - 64
Evidence for limited species diversity of bacteriochlorophyll b-containing purple nonsulfur anoxygenic phototrophs in freshwater habitats; Hoogewerf GJ et al.; Thirteen new isolates of bacteriochlorophyll b-containing purple nonsulfur bacteria were isolated from four freshwater habitats using specific enrichment methods including the use of long wavelength filters and extincting dilution of the inoculum . The new isolates were compared with the type strain of Blastochloris viridis, strain DSM 133(T), as regards pigments, morphology, carbon nutrition, and phylogeny . All new isolates were budding bacteria, and phototrophic mass cultures were green, brown, or brown-green in color . The pattern of carbon sources photocatabolized were similar in all strains; however, sugars, both mono- and disaccharides, were widely used by the new isolates while they did not support growth of strain DSM 133(T) . Phylogenetic analysis showed all new strains to cluster tightly with the type strain with the exception of one brown-colored strain and a mildly thermophilic strain . The results suggest that in contrast to purple nonsulfur bacteria containing bacteriochlorophyll a, those containing bacteriochlorophyll b may not be morphologically or phylogenetically diverse, and group into a tight phylogenetic clade distinct from all other anoxygenic phototrophs.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 285 - 90
Cloning, expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain; Masullo M et al.; The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli . The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S . solfataricus strain MT4 (optimum growth temperature 87 degrees C) . Only one amino acid change (Val15-->Ile) was found . Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity . Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed . Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.

Plasmid, 2003 Jan, 49(1), 2 - 8
Development of a gene expression vector for Thermus thermophilus based on the promoter of the respiratory nitrate reductase; Moreno R et al.; A specific expression system for Thermus spp . is described . Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8 . Two genes, encoding a thermophilic beta-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively . The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T . thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation . Overexpressions in the range of approximately 200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to approximately 20-fold, with respect to their intrinsic respective activities .

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 167 - 74
Isolation and phylogenetic diversity of members of previously uncultivated epsilon-Proteobacteria in deep-sea hydrothermal fields; Takai K et al.; We report the successful cultivation and partial characterization of novel members of epsilon-Proteobacteria, which have long been recognized solely as genetic signatures of small subunit ribosomal RNA genes (rDNA) from a variety of habitats occurring in deep-sea hydrothermal fields . A newly designed microhabitat designated 'in situ colonization system' was used for enrichment . Based on phylogenetic analysis of the rDNA of the isolates, most of these represent the first cultivated members harboring previously uncultivated phylotypes classified into the Uncultivated epsilon-Proteobacteria Groups A, B, F and G, as well as some novel members of Group D . Preliminary characterization of the isolates indicates that all are mesophilic or thermophilic chemolithoautotrophs using H(2) or reduced sulfur compounds (elemental sulfur or thiosulfate) as an electron donor and O(2), nitrate or elemental sulfur as an electron acceptor . The successful cultivation will enable the subsequent characterization of physiological properties and ecological impacts of a diversity of epsilon-Proteobacteria in the deep-sea hydrothermal environments.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 115 - 20
Development of a genetic system for hyperthermophilic Archaea: expression of a moderate thermophilic bacterial alcohol dehydrogenase gene in Sulfolobus solfataricus; Contursi P et al.; The Escherichia coli/Sulfolobus solfataricus shuttle vector pEXSs was used as a cloning vehicle for the gene transfer and expression of two bacterial genes in Sulfolobus solfataricus . The alcohol dehydrogenase (adh) from the moderate thermophilic Bacillus stearothermophilus (strain LLDR) and a mutagenised version encoding a less thermostable ADH enzyme were the selected genes . S . solfataricus adh promoter and aspartate aminotransferase terminator were used to drive the heterologous gene expression and to guarantee the correct termination of the transcripts, respectively . The constructed vectors were found to be able to carry these 'passenger' genes without undergoing any rearrangements . The active transcription of bacillar mRNAs was ascertained in vivo by RT-PCR . Transformed S . solfataricus expressed functional exogenous ADHs that showed unaffected kinetic and chemical-physical features.

Syst Appl Microbiol, 2002 Dec, 25(4), 618 - 26
Enumeration of thermophilic Bacillus species in composts and identification with a Random Amplification Polymorphic DNA (RAPD) protocol; Zhang YC et al.; The thermophilic microbial flora of general garden and domestic wastes composts, derived from thermogenic, post-thermogenic and maturation phases, was analysed using spore and total plate counts in combination with an optimised RAPD protocol . A total of 459 isolates were recovered obtained at 55 degrees C, and another 56 at 70 degrees C using tryptic soy-starch agar plates, with near-equal numbers being derived from total plate counts or spore preparations . The isolates were obtained from 11 compost samples and were assigned to eighteen different RAPD fingerprint types, with 76.1% of these ultimately being positively assigned by their RAPD profiles to just 2 species including Bacillus thermodenitrificans and B . licheniformis . Viable cell numbers ranged from 1.4 to 150 x 10(6) colony forming units per gram compost (wet weight), with the highest two counts being from 2 week and 4 week old compost samples with temperatures of 70 degrees C and 55 degrees C, respectively . B . thermodenitrificans was a dominant isolate (representing more than 50% of isolates from total plate counts) in 7 of the 11 individual compost total plate count samples between 30 degrees C to 73 degrees C, and accounted for 68.9% of all isolates overall . Another relatively common Bacillus species that was identified with RAPDs in significant numbers included B . licheniformis (7.2% of all isolates and dominant isolate in 1 sample) . Three other relatively common RAPD profiles could not be identified by comparison with known species in a RAPD profile database but were tentatively identified using 16S rDNA sequence comparisons . These were B . sporothermodurans (4.9% of all isolates and dominant in 1 sample), B . thermosphaericus (7.4% and dominant in 1 sample) and Terrabacter tumescens (5.0%) . Overall, based on the vegetative and spore count results and the subsequent RAPD-based identification, the data strongly support a significant role for B . thermodenitrificans in the composting process, and casts doubt on the notion that B . stearothermophilus sensu strictu (DSMZ 22) is a prominent member within compost ecology.

Eur J Biochem, 2003 Feb, 270(4), 736 - 44
Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula . Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation; Hugler M et al.; Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation . In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions . These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA . The carboxylase was purified and characterized and the genes were cloned and sequenced . In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M . sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa . The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta) . These subunits probably form an (alpha beta gamma)4 holoenzyme . It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5 . The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate . Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes . Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.

Extremophiles, 2003 Feb, 7(1), 43 - 53 Epub 2002 Oct 18.
Purification and characterization of a new hyperthermostable, allosamidin-insensitive and denaturation-resistant chitinase from the hyperthermophilic archaeon Thermococcus chitonophagus; Andronopoulou E et al.; A new chitinase (1,4-beta-D-N-acetyl-glucosaminidase, EC 3.2.1.14) was detected and purified to homogeneity in its native form from the chitinolytic enzyme system of the extremely thermophilic archaeon Thermococcus chitonophagus . This is the first nonrecombinant chitinase purified and characterized from archaea and also constitutes the first case of a membrane-associated chitinase isolated from archaea . The enzyme is a monomer with an apparent molecular weight of 70 kDa {therefore named chitinase 70 (Chi70)} and pI of 5.9; it is hydrophobic and appears to be associated with the outer side of the cell membrane . Chi70 is optimally active at 70 degrees C and pH 7.0 and exhibits remarkable thermostability, maintaining 50% activity even after 1 h at 120 degrees C, and therefore the enzyme is the most thermostable chitinase so far isolated . The enzyme was not inhibited by allosamidin, the natural inhibitor of chitinolytic activity, and was also resistant to denaturation by urea and SDS . On the other hand, guanidine hydrochloride significantly reduced enzymatic activity, indicating that, apart from the hydrophobic interactions, ion pairs located on the surface of the protein could be playing an important role in maintaining the protein's fold and enzyme activity . Chi70 showed broad substrate specificity for several chitinous substrates and derivatives . The lowest K(m) and highest K(cat) values were found for pNP(NAG)(2) as substrate and were determined to be 0.14 mM and 23 min(-1), respectively . The hydrolysis pattern was similar for oligomers and polymers, with N, N'-diacetylchitobiose {(NAG)(2)} being the final, major hydrolysis product . Chi70 was classified as an endochitinase due to its ability to release chitobiose from colloidal chitin . Additionally, the enzyme presented considerable cellulolytic activity . Analysis of the NH(2)-terminal amino acid sequence showed no detectable homology with other known sequences, suggesting that Chi70 is a new protein.

Extremophiles, 2003 Feb, 7(1), 35 - 41 Epub 2002 Oct 15.
Purification and characterization of Thermus thermophilus UvrD; Collins R et al.; The DNA helicase UvrD (helicase II) protein plays an important role in nucleotide excision repair, mismatch repair, rolling circular plasmid replication, and in DNA replication . A homologue of the Escherichia coli uvrD gene was previously identified in Thermus thermophilus; however, to date, a UvrD helicase has not been purified and characterized from a thermophile . Here we report the purification and characterization of a UvrD protein from Thermus thermophilus HB8 . The purified UvrD has a temperature range from 10 degrees to >65 degrees C, with an optimum of 50 degrees C, within the temperature limits of the assay . The enzyme had a requirement for divalent metal ions and nucleoside triphosphates which related to enzyme activity in the order ATP > dATP > dGTP > GTP >> CTP > dCTP >> UTP . A simple real-time helicase assay was developed that should facilitate detailed kinetic studies of the enzyme . Evaluation of helicase substrates using this assay showed that the enzyme was highly active on a double-stranded DNA with 5' recessed ends in comparison with substrates with 3' recessed or blunt ends, and supports enzyme translocation in a 3'-5' direction relative to the strand bound by the enzyme.

Genetika, 2002 Dec, 38(12), 1614 - 20
{Identification and cloning of peptide synthetase genes of thermostable bacilli using the polymerase chain reaction}; Sokolov SL et al.; Fourteen thermophilic and thermostable strains of the genus Bacillus were studied . Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction . Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined . Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes . On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites . Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B . licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical . The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1 . Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B . subtilis peptide synthetase genes dhbB and dhbF . Strains VK2, VK21, and VK2101 were shown to synthesize siderophores . A method for screening bacteria with peptide synthetase genes has been developed.

Appl Environ Microbiol, 2003 Feb, 69(2), 1287 - 9
Impaired temperature stress response of a Streptococcus thermophilus deoD mutant; Varcamonti M et al.; An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20 degrees C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation . We report evidence that ppGpp is implicated in this dual response.

Appl Environ Microbiol, 2003 Feb, 69(2), 987 - 95
Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution; Bulter T et al.; Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae . Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter) . Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold . Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations . The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host . The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold . Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability . Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity . The molecular mass of MtL expressed in S . cerevisiae is 30% higher than that of the same enzyme expressed in M . thermophila (110 kDa versus 85 kDa) . Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase . This S . cerevisiae expression system makes MtL available for functional tailoring by directed evolution.

Appl Environ Microbiol, 2003 Feb, 69(2), 980 - 6
Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1; Baek DH et al.; A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced . The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits . The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides . The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively . The enzyme remained stable within a broad pH range from 7.0 to 10.0 . The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+) . The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).

Appl Environ Microbiol, 2003 Feb, 69(2), 960 - 70
Phylogenetic diversity of nitrogenase (nifH) genes in deep-sea and hydrothermal vent environments of the Juan de Fuca Ridge; Mehta MP et al.; The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid . Dissolved N2 gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid . In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid . A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal . Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers . Other nifH genes unique to the vent samples include proteobacteria and divergent ARCHAEA: All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity) . These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.

J Biol Chem, 2003 Apr 11, 278(15), 12703 - 9 Epub 2003 Feb 04.
Characterization of a functional bacterial homologue of sodium-dependent neurotransmitter transporters; Androutsellis-Theotokis A et al.; The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters . Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon . Transport was Na(+)-dependent and highly selective . The K(m) for tryptophan was approximately 145 nm, and tryptophan transport was unchanged in the presence of 100 microM concentrations of other amino acids . Tryptamine and serotonin were weak inhibitors with K(I) values of 200 and 440 microM, respectively . By using a T7 promoter-based system, TnaT with an N-terminal His(6) tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.

J Biol Chem, 2003 Apr 25, 278(17), 14622 - 31 Epub 2003 Feb 04.
Identification of an archaeal alpha-L-fucosidase encoded by an interrupted gene . Production of a functional enzyme by mutations mimicking programmed -1 frameshifting; Cobucci-Ponzano B et al.; The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-fucosidase . We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S . solfataricus extracts . Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting . Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity . The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases . The presence of an alpha-fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.

Int J Biol Macromol, 2003 Jan 15, 31(4-5), 171 - 5
Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 11 endoxylanases; Christakopoulos P et al.; Acidic oligosaccharides were obtained from birchwood xylan by treatment with a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases . The main difference between the products liberated by xylanases of family 10 and 11 concerned the length of the products containing 4-O-methyl-D-glucuronic acid . The xylanase from T . aurantiacus liberate from glucuronoxylan an aldotetrauronic acid as the shortest acidic fragment in contrast with the enzyme from S . thermophile, which liberated an aldopentauronic acid . Acidic xylooligosaccharides were separated from the hydrolysate by anion-exchange and size-exclusion chromatography (SEC) and the primary structure was determined by 13C NMR spectroscopy . The acidic xylo-oligosaccharides were tested against three Gram-positive and three Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori . Aldopentauronic acid was proved more active against the Gram-positive bacteria and against H . pylori.

Int J Food Microbiol, 2003 Apr 25, 82(2), 153 - 61
Evolution of microbial populations during traditional Feta cheese manufacture and ripening; Manolopoulou E et al.; In three different dairies (A, B and C) located in Peloponess region (Southern Greece), traditional Feta cheese trials took place February to March using mixtures of sheep's and goat's milk . Only small variations in the evolution of microbial groups were observed during the whole ripening period . The main groups, such as thermophilic cocci, mesophilic lactococci, thermophilic lactobacilli, nonstarter lactic acid bacteria (NSLAB), presumptive Leuconostoc, enterococci and micrococci, reached their highest levels during the first 16 days, and then declined approximately 1-2 log units until the end of ripening . The remaining groups investigated, comprising yeasts, coliforms and Escherichia coli, were highest at day 4 . The yeasts remained constant, while coliforms and E . coli decreased sharply and were not detectable after 120 days of ripening . A number of 146 isolates (dairy A) taken from all stages of the manufacturing and ripening process were purified and studied . Lactobacillus plantarum (58/146) and isolates of related species Lactobacillus pentosus and Lactobacillus paraplantarum (16/146) were the most common microorganisms found during cheese ripening . Streptococcus thermophilus (23/146) and Lactobacillus delbrueckii subsp . bulgaricus (20/146) were detected in high levels up to 20 days, and then gradually reduced . Enterococcus faecium (29/146) was found in all manufacturing and ripening stages.

Biochem Biophys Res Commun, 2003 Feb 7, 301(2), 280 - 6
Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product; Murray PG et al.; A gene (cbh2) encoding cellobiohydrolase II was isolated from the fungus Talaromyces emersonii by rapid amplification of cDNA ends techniques and the equivalent genomic sequence was subsequently cloned . This represents the first report of a key component of the cellulase regulon from this organism . DNA sequencing revealed that cbh2 has an open reading frame of 1377 bp, which encodes a putative polypeptide of 459 amino acids, and is interrupted by seven introns . The deduced amino acid sequence revealed that cbh2 has a modular structure with a predicted molecular mass of 47 kDa and consisting of a fungal type carbohydrate binding module separated from a catalytic domain by a proline/serine/threonine rich linker region . The deduced protein is homologous to fungal cellobiohydrolases in Family 6A of the glycosyl hydrolases . Profiles of cbh2 expression in T . emersonii investigated by Northern blot analysis revealed that expression is regulated at the transcriptional level . Expression of the T . emersonii cbh2 gene is induced by cellulose, xylan, xylose, and gentiobiose and clearly repressed by glucose . Putative regulatory element consensus sequences have been identified in the upstream regulatory sequence of the cbh2 gene including the catabolite repressor element and the activator of cellulase expression (Ace) binding sites . High sequence identity (67%) between the catalytic domain of Cel 6A from Trichoderma reesei and the T . emersonii cbh2 gene product allowed structure prediction for the 3D model of the T . emersonii catalytic domain to be a variant of the classical TIM alpha/beta fold.

FEBS Lett, 2003 Jan 30, 535(1-3), 94 - 100
Solution structure of a tmRNA-binding protein, SmpB, from Thermus thermophilus; Someya T et al.; Small protein B (SmpB) is required for trans-translation, binding specifically to tmRNA . We show here the solution structure of SmpB from an extremely thermophilic bacterium, Thermus thermophilus HB8, determined by heteronuclear nuclear magnetic resonance methods . The core of the protein consists of an antiparallel beta-barrel twisted up from eight beta-strands, each end of which is capped with the second or third helix, and the first helix is located beside the barrel . Its C-terminal sequence (20 residues), which is rich in basic residues, shows a poorly structured form, as often seen in isolated ribosomal proteins . The results are discussed in relation to the oligonucleotide binding fold.

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 471 - 7
{Purification and properties of an endocellulase from the thermophilic fungus Chaetomium thermophile}; Lu M et al.; An endocellulase from culture supernatant of a thermophilic fungus Chaetomium thermophile was purifided to homogeneity, by using ammonium sulfate fraction, DEAE-Sepharose Fast-flow chromatography, Phenyl-Sepharose Fast Flow chromatography and Sephacryl S-100 chromatography . The enzyme was a glycoprotein with an apparent molecular weight of 67,800 and 69,800, as determinded by 12.5% SDS-PAGE and gel filtration respectively . The endocellulase was optimally active at pH 4.0-4.5 and 60 degrees C . It was thermostable at 60 degrees C and retained 30% activity after 60 min at 70 degrees C . The half life time of the enzyme at 80 degrees C was 25 min . Different metal ions showed different effects on the endocellulase activity . Na+ enhanced the enzyme activity, whereas Fe2+, Ag+, Cu2+, Ba2+ and Zn2+ cause obvious inhibition . But it didn't work on crystalline cellulose.

Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 138 - 44
{Isolation, identification and phylogenetic analysis of a thermophilic cellulolytic anaerobic bacterium}; Han R et al.; Four strains of thermophilic cellulolytic anaeobic bacteria were isolated from fresh feces, heat compost, cellulolytic mixed culture with a method based on adherence of cellulolytic bacteria to cellulose . The cells of isolates were straight or slightly curved rods that were 0.4 micron-0.6 micron x 3 microns-15 microns, Gram negative, strictly anaerobic, sulfate reduction negative, spore-forming bacteria . Most of the cells had oval terminal spores, while subterminal spores, middle spores, two or more spores also could be observed and spore formation could occurred in any position . The isolates degraded cellulose filter paper, cellulose powder Whatman CF II, microcrystalline cellulose, cellulose powder MN300 and unpretreated maize stem core, sugarcane residue and rice straw . The pH and temperature ranges for growth on cellulose were 6.2-8.9 and 45 degrees C-65 degrees C respectively with the optima, 7.0-7.5 and 55 degrees C-60 degrees C, respectively . The major fermentation products from cellulose were acetic acid, ethanol, CO2, H2 . The isolates could ferment cellobiose, glucose, fructose, maltose, and sorbital . The phylogenetic analysis based on 16S rDNA suggested strain EVA1 was the closest relative of Clostridium thermocellum with 99.8% sequence similarity.

Proteins, 2003 Feb 15, 50(3), 392 - 9
Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches; Savchenko A et al.; Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345) . This property limits genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein . An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis . To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography for 68 pairs of homologous proteins from E . coli and Thermotoga maritima . A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures . Fourteen (eight E . coli and six T . maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E . coli and 16 T . maritima proteins) samples formed crystals . Only three (one E . coli and two T . maritima proteins) samples both crystallized and had excellent NMR properties . The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies .

Eur J Clin Nutr, 2002 Dec, 56 Suppl 4, S56 - 9
Feasibility studies to control acute diarrhoea in children by feeding fermented milk preparations Actimel and Indian Dahi; Agarwal KN et al.; The aim of this work was to study feasibility of diarrhoea control in children (6 months to 5 y of age) by feeding fermented milk preparations . The design used was a randomized controlled clinical trial and the study was carried out at the Delhi University College Hospital providing tertiary care, and a nearby community centre Nand Nagri, a resettlement colony in East Delhi . Children suffering from acute diarrhoea (75 patients from the hospital and 75 from the community) were allocated to three groups by double-blind technique . Group 1 was given a fermented milk, Actimel, containing 10(8) of each Lactobacillus casei DN-114001, Lactobacillus bulgaricus and Streptococcus thermophilus per gram . Group 2 was given Indian Dahi (Lf 40) containing 10(8) of each Lactococcus lactis, Lactococcus lactis cremoris and Leuconostac mesenteroides cremoris per gram . Group 3 was given ultra-heat-treated yoghurt preparation (no live bacteria) . Actimel was also used as a starter to prepare the curd in order to study the preventive effect of diarrhoea in children in a community . In the hospital study Indian Dahi and Actimel administration reduced mean duration of diarrhoea by 0.3 and 0.6 day (P<0.001), respectively . The corresponding figures in the community study were 0.2 and 0.5 day (P<0.05), respectively . The families using Actimel as a starter showed a reduction in diarrhoeal morbidity episodes by 40% of the children tested in a 3 month follow-up . In conclusion, Actimel, fermented milk containing Lactobacillus casei DN-114001, and Indian Dahi can significantly reduce the duration of diarrhoea in children; the former preparation being superior.

Eur J Clin Nutr, 2002 Dec, 56 Suppl 4, S21 - 6
Interaction of lactic acid bacteria with the gut immune system; Perdigon G et al.; Health claims of lactic acid bacteria (LAB) used in functional foods and pharmaceutical preparations are based on the capacity of these microorganisms to stimulate the host immune system . In this study, the antigenic effect of LAB (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus thermophilus) on the gut immune system of BALB/c mice was evaluated . A dose-dependent increase of the Bcl2 protein was observed with all LAB assayed . Furthermore, the analysis of cytokine-producing cells in the lamina propria of gut showed that TNFalpha and INFgamma values, determined in macrophages cultured from Peyer patches, were enhanced for all the LAB assayed . An important increase of interleukins IL-10 and IL-4 was observed mainly in mice fed with Lactobacillus delbrueckii ssp . bulgaricus or Lactobacillus casei, while a significant induction of IL-2 and IL-12 was only observed with L . acidophilus (P<0.01) . These effects were dose dependent . The role of produced cytokines in the balance Th1/Th2 was determined by a systemic antibody response against parenterally injected ovoalbumin . L . casei, L . delbrueckii ssp . bulgaricus and L . acidophilus enhanced the IgG1 response favouring Th2 balance, while L . acidophilus also increased the IgG2a response inducing Th1 balance . S . thermophilus did not influence the balance Th1/Th2 . Our studies showed that lactic acid bacteria induce distinct mucosal cytokine profiles showing different adjuvant capacity among them . Thus, selection of probiotic strain with immunological properties must be well defined to influence cytokine expression that favour the claimed immune response.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 356 - 8 Epub 2003 Jan 23.
Expression, purification and preliminary X-ray characterization of N-acetyl-gamma-glutamyl-phosphate reductase from Thermus thermophilus HB8; Goto M et al.; N-Acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyses the NADPH-dependent reduction of N-acetyl-gamma-glutamyl phosphate to give the N-acetylglutamic semialdehyde . A recombinant form of AGPR from Thermus thermophilus HB8 has been crystallized by the hanging-drop vapour-diffusion technique using PEG 4000 as a precipitating agent . The crystals grew as colourless prisms, with unit-cell parameters a = b = 90.9, c = 139.5 A, alpha = beta = 90, gamma = 120 degrees . The crystals belong to the hexagonal space group P6(2)22 or P6(4)22 and are most likely to contain a monomer in the asymmetric unit, with a V(M) value of 2.19 A(3) Da(-1) . The crystals diffract to a resolution of 2.2 A at beamline BL44B2 of SPring-8.

RNA, 2003 Jan, 9(1), 100 - 11
Mechanism of molecular interactions for tRNA(Val) recognition by valyl-tRNA synthetase; Fukai S et al.; The molecular interactions between valyl-tRNA synthetase (ValRS) and tRNA(Val), with the C34-A35-C36 anticodon, from Thermus thermophilus were studied by crystallographic analysis and structure-based mutagenesis . In the ValRS-bound structure of tRNA(Val), the successive A35-C36 residues (the major identity elements) of tRNA(Val) are base-stacked upon each other, and fit into a pocket on the alpha-helix bundle domain of ValRS . Hydrogen bonds are formed between ValRS and A35-C36 of tRNA(Val) in a base-specific manner . The C-terminal coiled-coil domain of ValRS interacts electrostatically with A20 and hydrophobically with the G19*C56 tertiary base pair . The loss of these interactions by the deletion of the coiled-coil domain of ValRS increased the K(M) value for tRNA(Val) 28-fold and decreased the k(cat) value 19-fold in the aminoacylation . The tRNA(Val) K(M) and k(cat) values were increased 21-fold and decreased 32-fold, respectively, by the disruption of the G18*U55 and G19*C56 tertiary base pairs, which associate the D- and T-loops for the formation of the L-shaped tRNA structure . Therefore, the coiled-coil domain of ValRS is likely to stabilize the L-shaped tRNA structure during the aminoacylation reaction.

EMBO J, 2003 Feb 3, 22(3), 676 - 88
ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding; Sekine S et al.; Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA . A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA . In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes . In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS . ATP.Glu are too far from each other (6.2 A) to react . In contrast, the ATP-binding mode in GluRS.tRNA . ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite) . Therefore, tRNA binding to GluRS switches the ATP-binding mode . The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.

Water Res, 2003 Mar, 37(5), 1033 - 47
Effect of specific gas loading rate on thermophilic (55 degrees C) acidifying (pH 6) and sulfate reducing granular sludge reactors; Lens PN et al.; The effect of the specific gas loading rate on the acidifying, sulfate reducing and sulfur removal capacity of thermophilic (55 degrees C; pH 6.0) granular sludge bed reactors treating partly acidified wastewater was investigated . A comparison was made between a regular UASB reactor and a UASB reactor continuously sparged with N(2) at a specific gas loading rate of 30 m(3)m(-2)d(-1) . Both UASB reactors (upflow velocity 1.0 mh(-1), hydraulic retention time about 5h) were fed a synthetic wastewater containing starch, sucrose, lactate, propionate and acetate and a low sulfate concentration (COD/SO(4)(2-) ratio of 10) at volumetric organic loading rates (OLR) ranging from 4.0 to 49.8 gCODl(-1) reactord(-1) . Immediately after imposing an OLR of 25 gCODl(-1) reactord(-1), the acidification and sulfate reduction efficiency dropped to 80% and 30%, respectively, in the UASB reactor . Both efficiencies recovered slowly to 100% during the course of the experiment . In the N(2) sparged reactor, both the acidification and sulfate reduction efficiency remained 100% following the OLR increase to 25 gCODl(-1) reactord(-1) . However, the sulfate reduction efficiency gradually decreased to about 20% at the end of the experiment . The biogas (CO(2) and CH(4)) production rate in the UASB was very low, i.e . <3l biogasl(-1) reactorday(-1), resulting in negligible amounts (<20%) of H(2)S stripped from the reactor liquid . The total H(2)S concentration of the N(2) sparged UASB reactor effluent was always below 25 mgl(-1), but incomplete sulfate reduction kept the maximal H(2)S stripping efficiency below 70%.

Biochemistry, 2003 Feb 4, 42(4), 1016 - 23
Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus; Sarrou J et al.; In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal {Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V . (1998) Biochemistry 37, 3581-3587} . This state was recently assigned to the S(-)(2) state of the OEC {Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A . W . (2002) Biochemistry 41, 3057-3064} . On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C . An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S . elongatus only after incubation at -30 degrees C . The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models . The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S . elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.

J Med Assoc Thai, 2002 Nov, 85 Suppl 4, S1225 - 31
Effect of bifidobacterium Bb12 with or without Streptococcus thermophilus supplemented formula on nutritional status; Nopchinda S et al.; Acute diarrhea is a common cause of infant morbidity and mortality . Probiotic supplemented infant formula is one of the effective methods for prevention of rotavirus diarrhea . Other benefits of the probiotics supplemented formula were evaluated by monitoring the growth of the children . A double-blind, placebo-controlled trial was done in 148 children aged 6-36 months . They were divided into 3 groups: the Bb12 group, 51 children received infant formula with Bifidobacteria Bb12 supplement; the Bb12+ST group, 54 children received infant formula with Bb12 and Streptococcus thermophilus supplement; and the control group, 43 children received infant formula without supplement . The mean weight Z-score according to WHO reference standard of the Bbl2 group was -1.8 +/- 0.12, the Bb12+ST group was -1.4 +/- 0.11 and the control group was -1.8 +/- 0.13 at entry . The mean weight Z-score of children after 6 month showed that the children in the Bbl2+ST group had the highest increase in weight which was increased from -1.4 +/- 0.11 to -0.9 +/- 0.12 compared to the Z-score of the Bb12 group which had increased from -1.8 +/- 0.12 to -1.2 +/- 0.13 and in the control group from -1.8 +/- 0.13 to -1.7 +/- 0.25 . In terms of the mean height Z-score, the Bb12 group was -2.7 +/- 0.14 to -1.7 +/- 0.16 which was higher than the Bb12+ST group (- 2.2 +/- 0.13 to -1.7 +/- 0.13) but was not different from the control group . However, the mean weight/height Z-score of the Bbl2+ST group had approached the reference standard (Bb12 group -0.1 +/- 0.11 to -0.1 +/- 0.13, Bb12+ST group -0.1 +/- 0.10 to 0.3 +/- 0.17, control group -0.4 +/- 0.12 to -0.1 +/- 0.16) . Data showed that children who received the probiotics supplement formula had better growth during the 6 month period.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 20 - 5
{A comparison of amino acid composition of proteins from thermophiles and mesophiles}; Lu B et al.; The thermophilic feature of thermophiles are due to the high thermostability of their proteins . In order to investigate the mechanism of the high thermostability of thermophilic proteins and compare the difference of amino acid composition between thermophilic and mesophilic proteins, 110 pairs of homologous protein sequences from thermophiles and mesophiles respectively are collected and the amino acid composition, percentage of hydrophobic amino acids, hydropathy index, percentage of charged amino acids of the two protein groups are compared . It reveals that the two groups have evidently different composition in several kinds of amino acids, and thermophilic proteins have higher average hydropathy and charged amino acids composition . A comparison of the aliphatic indices between the two protein groups indicates that the reason that thermophilic proteins have higher aliphatic index is that thermophilic proteins have higher leucine composition, and thus sheds doubt on the validity of aliphatic index . It shows that it is possible to disclose some general rules with regard to the mechanism of protein thermostability by comparing a large amount of protein sequence data.

Wei Sheng Wu Xue Bao, 1998 Apr, 38(2), 137 - 41
{Studies on actinomycetes population from salt lake of Yuncheng in Shanxi}; Liu D et al.; 120 strain of mesophilic and thermophilic of alkalaphilic actinomycetes of alkalic tolerant actinomycetes isolated from Yuncheng salt lake in Shanxi were identified . On the basis of morphological characteristics and some chemotaxonomic properties (composition of cell wall, whole cell sugar), the isolated strains were classified into Nocadiopsis, Micromonospora, Actinomadura and Streptomyces . The Streptomyces strains were classified into 11 groups.

Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 188 - 92
{Production and some properties of a thermophilic protease from Bacillus stearothermophilus WF146}; Tang B et al.; The factors affecting Bacillus stearothermophilus WF146 for thermophilic protease producing have been investigated, more than 600 units of enzyme in 1 mL of fermented culture could be achieved under suitable condition . The protease had a molecular weight around 34 kD estimated by SDS-PAGE, and functioned optimally at pH 8.0 and 80 degrees C, respectively . In addition, the enzyme exhibited high temperature tolerance and was stable at a wide range of pH, and Ca2+ played a key role for the stability of the enzyme . While the protease activity of the enzyme was strongly inhibited by PMSF, DFP and IAA, and was not affected by DTT.

Ecotoxicol Environ Saf, 2003 Jan, 54(1), 87 - 91
The toxicity of cationic surfactants in four bioassays; Nalecz-Jawecki G et al.; The purpose of this study was to investigate the toxicity of 15 quaternary ammonium compounds (QACs) in a battery of four bioassays comprising the bacterium Vibrio fischeri, two ciliated protozoa Spirostomum ambiguum and Tetrahymena thermophia, and the anostracean crustacean Artemia franciscana . The compounds were prepared by Professor Pernak's group at Poznan University of Technology (Poland) . The toxicity of the test compounds was very high, with EC(LC)(50) values varying from 0.11 to 70 micromol/L . Microtox was the most sensitive bioassay, while the crustacean test was the least sensitive . Among the protozoa T . thermophila was 5-30 times less sensitive than S . ambiguum . The toxicity of the QACs depended on their structure, but no simple correlation was found for all the bioassays applied .

Eur J Biochem, 2003 Feb, 270(3), 463 - 75
tmRNA from Thermus thermophilus . Interaction with alanyl-tRNA synthetase and elongation factor Tu; Stepanov VG et al.; The interaction of a Thermus thermophilus tmRNA transcript with alanyl-tRNA synthetase and elongation factor Tu has been studied . The synthetic tmRNA was found to be stable up to 70 degrees C . The thermal optimum of tmRNA alanylation was determined to be around 50 degrees C . At 50 degrees C, tmRNA transcript was aminoacylated by alanyl-tRNA synthetase with 5.9 times lower efficiency (kcat/Km) than tRNAAla, primarily because of the difference in turnover numbers (kcat) . Studies on EF-Tu protection of Ala approximately tmRNA against alkaline hydrolysis revealed the existence of at least two different binding sites for EF-Tu on charged tmRNA . The possible nature of these binding sites is discussed.

Am J Clin Nutr, 2003 Feb, 77(2), 517 - 20
Ingested probiotics reduce nasal colonization with pathogenic bacteria (Staphylococcus aureus, Streptococcus pneumoniae, and beta-hemolytic streptococci); Gluck U et al.; BACKGROUND: As a bacterial reservoir, the nose may harbor potentially pathogenic bacteria (PPB: Staphylococcus aureus, Streptococcus pneumoniae, beta-hemolytic streptococci, and Haemophilus influenzae) . In patients carrying PPB, antiseptic regimens could be crucial for infection control after major operations on or injuries of the head, nasal sinuses, or lungs . Such regimens may also be important for diabetic patients and persons receiving hemodialysis, in intensive care units, or with impaired immunity due to various other causes . OBJECTIVE: We tested a possible effect of the ingestion of probiotics on the bacterial flora of the nose . DESIGN: In an open, prospective trial, 209 volunteers were randomly assigned to consume either a probiotic, fermented milk drink {65 mL with Lactobacillus GG (ATCC 53103), Bifidobacterium sp B420, Lactobacillus acidophilus 145, and Streptococcus thermophilus; n = 108} or standard yogurt (180 g; n = 101) daily for 3 wk . Nasal microbial flora were analyzed on days 1, 21, and 28 . The microbial examination was blinded to the source of the samples . RESULTS: We found a significant reduction (19%; P < 0.001) in the occurrence of nasal PPB in the group who consumed the probiotic drink but not in the group who consumed yogurt . The effect was mainly on gram-positive bacteria, which decreased significantly (P < 0.05) . CONCLUSIONS: The results indicate that regular intake of probiotics can reduce PPB in the upper respiratory tract . The results also indicate a linkage of the lymphoid tissue between the gut and the upper respiratory tract.

J Food Prot, 2003 Jan, 66(1), 25 - 30
Fate of Escherichia coli O157:H7 during composting of bovine manure in a laboratory-scale bioreactor; Jiang X et al.; Inactivation profiles of Escherichia coli O157:H7 in inoculated bovine manure-based compost ingredients were determined by composting these ingredients in a bioreactor under controlled conditions . A 15-liter bioreactor was constructed to determine the fate of E . coli O157:H7 and changes in pH, moisture content, temperature, and aerobic mesophilic and thermophilic bacterial counts during composting . Fresh cow manure, wheat straw, cottonseed meal, and ammonium sulfate were combined to obtain a moisture content of ca . 60% and a carbon/nitrogen ratio of 29:1 . The compost ingredients were held in the bioreactor at a constant external temperature of 21 or 50 degrees C . Self-heating of the ingredients due to microbial activity occurred during composting, with stratified temperatures occurring within the bioreactor . At an external temperature of 21 degrees C, self-heating occurred for 0 to 3 days, depending on the location within the bioreactor . E . coli O157:H7 populations increased by 1 to 2 log10 CFU/g during the initial 24 h of composting and decreased by ca . 3.5 log10 CFU/g near the bottom of the bioreactor and by ca . 2 log10 CFU/g near the middle and at the top during 36 days of composting . At an external temperature of 50 degrees C . E . coli O157:H7 was inactivated rapidly (by ca . 4.9 log10 CFU/g at the top of the bioreactor, by 4.0 log10 CFU/g near the middle, and by 5.9 log10 CFU/g near the bottom) within 24 h of composting . When inoculated at an initial level of ca . 10(7) CFU/g . E . coli O157:H7 survived for 7 days but not for 14 days at all three sampling locations, as indicated by either direct plating or enrichment culture . At the top of the bioreactor a relatively constant moisture content of 60% was maintained, whereas the moisture content near the bottom decreased steadily to 37 to 45% over 14 days of composting . The pH of the composting mixture decreased to ca . 6 within 1 to 3 days and subsequently increased to 8 to 9 . Results obtained in this study indicate that large populations (10(4) to 10(7) CFU/g) of E coli O157:H7 survived for 36 days during composting in a bioreactor at an external temperature of 21 degrees C but were inactivated to undetectable levels after 7 to 14 days when the external temperature of the bioreactor was 50 degrees C . Hence, manure contaminated with large populations (e.g., 10(7) CFU/g) of E . coli O157:H7 should be composted for more than 1 week, and preferably for 2 weeks, when held at a minimum temperature of 50 degrees C.

Appl Microbiol Biotechnol, 2003 Jan, 60(5), 581 - 7 Epub 2002 Nov 06.
Isolation of a novel thermophilic fungus Chaetomium sp . nov . MS-017 and description of its palm-oil mill fiber-decomposing properties; Suyanto et al.; Palm-oil mill fiber (POMF) is a fibrous, natural hard material discharged in enormous amounts from palm-oil mills in tropical plantations; therefore, research to find microorganisms that decompose POMF was conducted . As the result of screening, a new thermophilic fungus, Chaetomium sp . nov . MS-017, exhibiting rapid growth on POMF was isolated from rotted wood . Based on partial characterization of the decomposition of POMF, it was shown that MS-017 preferentially assimilates polysaccharides, especially hemicelluloses such as xylan . A preliminary composting study indicated that MS-017 produced 855 g of decomposed product from 1,000 g of intact POMF in 12 days under optimized solid-culture conditions . The decomposition rate of POMF was 23% (w/w), and the cell yield calculated from consumed POMF was as high as 36% (w/w) . These results indicate that MS-017 has a very high potential to decompose POMF and that it is suitable for economical production of compost to recycle by-product biomass from oil-palm plantations.

J Biol Chem, 2003 Apr 18, 278(16), 14257 - 64 Epub 2003 Jan 17.
Fluorescence resonance energy transfer analysis of protein translocase . SecYE from Thermus thermophilus HB8 forms a constitutive oligomer in membranes; Mori H et al.; SecY and SecE are the two principal translocase subunits that create a channel-like pathway for the transit of preprotein across the bacterial cytoplasmic membrane . Here we report the cloning, expression, and purification of the SecYE complex (TSecYE) from a thermophilic bacterium, Thermus thermophilus HB8 . Purified TSecYE can be reconstituted into proteoliposomes that function in T . thermophilus SecA (TSecA) dependent preprotein translocation . After the mixing of TSecYE derivatives labeled with either a donor or an acceptor fluorophore during reconstitution, fluorescence resonance energy transfer experiments demonstrated that 2 or more units of TSecYE in the lipid bilayer associate to form a largely non-exchangeable oligomeric structure.

DNA Repair (Amst), 2003 Feb 3, 2(2), 199 - 210
Repair of the mutagenic DNA oxidation product, 5-formyluracil; Liu P et al.; The oxidation of the thymine methyl group can generate 5-formyluracil (FoU) . Template FoU residues are known to miscode, generating base substitution mutations . The repair of the FoU lesion is therefore important in minimizing mutations induced by DNA oxidation . We have studied the repair of FoU in synthetic oligonucleotides when paired with A and G . In E . coli cell extract, the repair of FoU is four orders of magnitude lower than the repair of U and is similar for both FoU:A and FoU:G base pairs . In HeLa nuclear extract, the repair of FoU:A is similarly four orders of magnitude lower than the repair of uracil, although the FoU:G lesion is repaired 10 times more efficiently than FoU:A . The FoU:G lesion is shown to be repaired by E . coli mismatch uracil DNA glycosylase (Mug), thermophile mismatch thymine DNA glycosylase (Tdg), mouse mismatch thymine DNA glycosylase (mTDG) and human methyl-CpG-binding thymine DNA glycosylase (MBD4), whereas the FoU:A lesion is repaired only by Mug and mTDG . The repair of FoU relative to the other pyrimidines examined here in human cell extract differs from the substrate preferences of the known glycosylases, suggesting that additional, and as yet unidentified glycosylases exist in human cells to repair the FoU lesion . Indeed, as observed in HeLa nuclear extract, the repair of mispaired FoU derived from misincorporation of dGMP across from template FoU could promote rather than minimize mutagenesis . The pathways by which this important lesion is repaired in human cells are as yet unexplained, and are likely to be complex.

Mol Biol Cell, 2003 Jan, 14(1), 251 - 61
Cloning, localization, and axonemal function of Tetrahymena centrin; Guerra C et al.; Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila . It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron . It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25 . Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin . Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme . The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays . Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity . Antibodies to centrin abrogated this increase . This is the first demonstration of a specific centrin function associated with axonemal dynein . It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.

J Mol Biol, 2003 Jan 31, 325(5), 1031 - 7
Structure based hyperthermostability of archaeal histone HPhA from Pyrococcus horikoshii; Li T et al.; The histone protein HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii, shows hyperthermostability, as required for optimal growth of the organism at 95 degrees C . The structure of recombinant P.horikoshii HPhA has been determined to 2.3A resolution by molecular replacement, and refined to R(work) and R(free) values of 20.7% and 27.3%, respectively . The HPhA monomer structure is characterized by the histone fold and assembles into a homodimer like other archaeal histones . There are four anions found in the dimer structure, giving rise to clues as to where DNA might bind . A detailed comparison of four known structures of archaeal histones, which evolve and exist under different temperatures, shows that the thermophilic archaeal histone HPhA has a larger hydrophobic contact area, an increased number of hydrogen bonds and a reduced solvent-accessible area . We also observe a unique network of tyrosine residues located at the crossover point of the two HPhA monomers, which locks them together and stabilizes the dimer . These factors together account for the increased thermal stability.

Mikrobiologiia, 2002 Nov-Dec, 71(6), 824 - 8
{Characterization of thermophilic spore-forming bacteria from a geothermal spring in Lithuania based on 16S rDNA and 16S-23S rDNA intergenic spacers analyses}; Kuisene N et al.; Forty-two strains of gram-positive, aerobic, heterotrophic, obligately thermophilic, spore-forming bacteria were isolated from a geothermal site near the Baltic Sea in Lithuania . All of the strains were able to hydrolyze collagen and/or casein . Since characteristics of proteolytic activity are correlated with taxonomic positions of bacteria, the strains were grouped on the basis of molecular biological analyses . On the basis of RFLP patterns of 16S rDNA and 16S-23S rDNA ITS-PCR analysis, the strains were subdivided into nine groups.

Mikrobiologiia, 2002 Nov-Dec, 71(6), 819 - 23
{Comparison of genomes in Streptococcus thermophilus strains of different origins}; Botina SG et al.; According to DNA hybridization data, thermophilic streptococci used in Russia as starters in the dairy industry are divided into 6 different genomovars, with a degree of DNA homology not exceeding 20-50% . The analysis of genomes from these genomovars using SmaI restriction endonuclease and pulsed-field gel electrophoresis revealed a wide variability of the genome size . In some strains, the genome size considerably exceeded 2000 kbp . Most of the strains studied contained plasmids about 120 kbp in size.

Proc Natl Acad Sci U S A, 2003 Jan 21, 100(2), 599 - 604 Epub 2003 Jan 10.
Correlated terrestrial and marine evidence for global climate changes before mass extinction at the Cretaceous-Paleogene boundary; Wilf P et al.; Terrestrial climates near the time of the end-Cretaceous mass extinction are poorly known, limiting understanding of environmentally driven changes in biodiversity that occurred before bolide impact . We estimate paleotemperatures for the last approximately 1.1 million years of the Cretaceous ( approximately 66.6-65.5 million years ago, Ma) by using fossil plants from North Dakota and employ paleomagnetic stratigraphy to correlate the results to foraminiferal paleoclimatic data from four middle- and high-latitude sites . Both plants and foraminifera indicate warming near 66.0 Ma, a warming peak from approximately 65.8 to 65.6 Ma, and cooling near 65.6 Ma, suggesting that these were global climate shifts . The warming peak coincides with the immigration of a thermophilic flora, maximum plant diversity, and the poleward range expansion of thermophilic foraminifera . Plant data indicate the continuation of relatively cool temperatures across the Cretaceous-Paleogene boundary; there is no indication of a major warming immediately after the boundary as previously reported . Our temperature proxies correspond well with recent pCO(2) data from paleosol carbonate, suggesting a coupling of pCO(2) and temperature . To the extent that biodiversity is correlated with temperature, estimates of the severity of end-Cretaceous extinctions that are based on occurrence data from the warming peak are probably inflated, as we illustrate for North Dakota plants . However, our analysis of climate and facies considerations shows that the effects of bolide impact should be regarded as the most significant contributor to these plant extinctions.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Jan, 134(1), 45 - 52
Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food; Peng Y et al.; Bacillus amyloliquefaciens DC-4, which produces a strongly fibrinolytic enzyme, was isolated from douchi, a traditional Chinese soybean-fermented food . A fibrinolytic enzyme (subtilisin DFE) was purified from the supernatant of B . amyloliquefaciens DC-4 culture broth and displayed thermophilic, hydrophilic and strong fibrinolytic activity . Subtilisin DFE was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of 28000 Da and a pI of 8.0 . The optimal reaction pH value and temperature were 9.0 and 48 degrees C, respectively . Subtilisin DFE not only hydrolyzed fibrin but also several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA, and phenylmethylsulfony fluoride can completely inhibit its fibrinolytic activity . These results indicated that subtilisin DFE is a subtilisin-family serine protease, similar to nattokinase from Bacillus natto . The first 24 amino acid residues of the N-terminal sequence of subtilisin DFE were AQSVPYGVSQIKAPALHSQGFTGS, which is identical to that of subtilisin K-54, and different from that of NK and CK . Results from subtilisin DFE gene sequence analysis showed that subtilisin DFE is a novel fibrinolytic enzyme.

Biomacromolecules, 2003 Jan-Feb, 4(1), 107 - 13
One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp . strain T2 by using novel heterofunctional chelate-epoxy Sepabeads; Pessela BC et al.; Using the poly-His-tagged-beta-galactosidase from Thermus sp . strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification . The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments . That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates . These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co(2+) chelated in IDA groups and a high density of epoxy groups . This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place . This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme . The enzyme derivative presented a very high activity at 70 degrees C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 degrees C) . In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads.

Biodegradation, 2002, 13(4), 261 - 71
Mesophilic and thermophilic activated sludge post treatment of anaerobic effluent . Sludge and wastewater characterisation using batch experiments; Vogelaar JC et al.; Anaerobic pretreated paper process water was characterized in terms of readily biodegradable, slowly biodegradable, very slowly biodegradable and inert wastewater fractions under mesophilic and thermophilic conditions . The anaerobic pretreated paper process water contained a relatively high amount of slowly biodegradable components and few easily biodegradable components as indicated by the ratio of short-term BOD over the BOD5 . Wastewater readily biodegradable COD, determined as short-term BOD, was almost similar when measured under both temperature conditions . Fractions of slowly biodegradable COD and inert COD of the same wastewater were found to depend on the type of biomass involved in the test . Thermophilic aerobic biomass was not able to degrade the wastewater to the same extent as the mesophilic biomass resulting in higher apparent inert COD levels . Furthermore, wastewater colloidal COD did not flocculate under thermophilic conditions and was thus not removed from the liquid phase.

Am J Clin Pathol, 2003 Jan, 119(1), 95 - 100
Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction; Hu Y et al.; We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines . These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples . HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed . Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method . PCR products were analyzed by direct DNA sequencing . These results indicate that the HCV can replicate in T and B lymphocytes . This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 98 - 103 Epub 2002 Dec 23.
Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-A resolution; Kamiya N et al.; Photosystem II (PSII) is a multisubunit membrane protein complex performing light-induced electron transfer and water-splitting reactions, leading to the formation of molecular oxygen . The first crystal structure of PSII from a thermophilic cyanobacterium Thermosynechococcus elongatus was reported recently {Zouni, A., Witt, H . T., Kern, J., Fromme, P., Krauss, N., Saenger, W . & Orth, P . (2001) Nature 409, 739-743)} at 3.8-A resolution . To analyze the PSII structure in more detail, we have obtained the crystal structure of PSII from another thermophilic cyanobacterium, Thermosynechococcus vulcanus, at 3.7-A resolution . The present structure was built on the basis of the sequences of PSII large subunits D1, D2, CP47, and CP43; extrinsic 33- and 12-kDa proteins and cytochrome c550; and several low molecular mass subunits, among which the structure of the 12-kDa protein was not reported previously . This yielded much information concerning the molecular interactions within this large protein complex . We also show the arrangement of chlorophylls and cofactors, including two beta-carotenes recently identified in a region close to the reaction center, which provided important clues to the secondary electron transfer pathways around the reaction center . Furthermore, possible ligands for the Mn-cluster were determined . In particular, the C terminus of D1 polypeptide was shown to be connected to the Mn cluster directly . The structural information obtained here provides important insights into the mechanism of PSII reactions.

RNA, 2002 Dec, 8(12), 1548 - 57
Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex; Perederina A et al.; The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution . The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA . The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions . Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively . The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module . Comparison of the T . thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding . In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface . This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

Nat Struct Biol, 2003 Feb, 10(2), 104 - 8
Structure of the L1 protuberance in the ribosome; Nikulin A et al.; The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site . The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome . Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus . This structure fills a major gap in current models of the 50S ribosomal subunit . The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T . thermophilus 70S ribosome . Incorporation of the L1-rRNA complex into the structural models of the T . thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.

Plant Cell Physiol, 2002 Dec, 43(12), 1585 - 8
Bipolar localization of putative photoreceptor protein for phototaxis in thermophilic cyanobacterium Synechococcus elongatus; Kondou Y et al.; We identified an open reading frame from a database of the entire genome of Synechococcus elongatus, the product of which was very similar to pixJ1, which was proposed as photoreceptor gene for phototaxis in Synechocystis sp . PCC6803 {Yoshihara et al . (2000) Plant Cell Physiol . 41: 1299} . The mRNA of S . elongatus pixJ (SepixJ) was expressed in vivo as a part of the product of an operon . SePixJ was detected exclusively in the membrane fraction after cell fractionation . Immunogold labeling of SePixJ in ultra-thin sections indicated that it existed only in both ends of the rod-shaped cell; probably bound with the cytoplasmic membrane.

Appl Environ Microbiol, 2003 Jan, 69(1), 162 - 9
Collagenolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain NTAP-1: purification, characterization, gene cloning, and heterologous expression; Tsuruoka N et al.; Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest . Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides . Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA . Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined . On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k(cat), 5.41 s(-1); K(m), 32 micro M) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (k(cat), 351 s(-1); K(m), 214 micro M), where the asterisks denote the scissile bonds . The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da . Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein . Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results . Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.

Science, 2003 Jan 3, 299(5603), 120 - 3
Fluids from aging ocean crust that support microbial life; Cowen JP et al.; Little is known about the potential for life in the vast, low-temperature (<100 degrees C) reservoir of fluids within mid-ocean ridge flank and ocean basin crust . Recently, an overpressured 300-meter-deep borehole was fitted with an experimental seal (CORK) delivering crustal fluids to the sea floor for discrete and large-volume sampling and characterization . Results demonstrate that the 65 degrees C fluids from 3.5-million-year-old ocean crust support microbial growth . Ribosomal RNA gene sequence data indicate the presence of diverse Bacteria and Archaea, including gene clones of varying degrees of relatedness to known nitrate reducers (with ammonia production), thermophilic sulfate reducers, and thermophilic fermentative heterotrophs, all consistent with fluid chemistry.

J Biol Chem, 2003 Mar 7, 278(10), 7891 - 6 Epub 2003 Jan 02.
Activity-stability relationships in extremophilic enzymes; D'Amico S et al.; Psychrophilic, mesophilic, and thermophilic alpha-amylases have been studied as regards their conformational stability, heat inactivation, irreversible unfolding, activation parameters of the reaction, properties of the enzyme in complex with a transition state analog, and structural permeability . These data allowed us to propose an energy landscape for a family of extremophilic enzymes based on the folding funnel model, integrating the main differences in conformational energy, cooperativity of protein unfolding, and temperature dependence of the activity . In particular, the shape of the funnel bottom, which depicts the stability of the native state ensemble, also accounts for the thermodynamic parameters of activation that characterize these extremophilic enzymes, therefore providing a rational basis for stability-activity relationships in protein adaptation to extreme temperatures.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2251 - 5
Geobacillus toebii sp . nov., a novel thermophilic bacterium isolated from hay compost; Sung MH et al.; A thermophilic, spore-forming rod isolated from hay compost in Korea was subjected to a taxonomic study . The micro-organism, designated strain SK-1(T), was identified as being aerobic, Gram-positive, motile and rod-shaped . Growth of the isolate was observed at 45-70 degrees C (optimum 60 degrees C) and pH 6.0-9.0 (optimum pH 7.5) . The G+C content of the genomic DNA was 43.9 mol% . Chemotaxonomic characteristics of the isolate included the presence of mesodiaminopimelic acid in the cell wall and iso-C15:0 and iso-C17:0 as the major cellular fatty acids . The predominant isoprenoid quinone was MK-7 . The chemotaxonomic characteristics of strain SK-1(T) were the same as those of the genus Geobacillus . Phylogenetic analysis based on 16S rDNA sequences showed that strain SK-1(T) is most closely related to Geobacillus thermoglucosidasius . However, the phenotypic properties of strain SK-1(T) were clearly different from those of G . thermoglucosidasius . The level of DNA-DNA relatedness between strain SK-1(T) and the type strain of G . thermoglucosidasius was 27% . On the basis of the phenotypic traits and molecular systematic data, strain SK-1(T) represents a novel species within the genus Geobacillus, for which the name Geobacillus toebii sp . nov . is proposed . The type strain is strain SK-1(T) (= KCTC 0306BP(T) - DSM 14590(T)).

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 1961 - 7
Carboxydocella thermautotrophica gen . nov., sp . nov., a novel anaerobic, CO-utilizing thermophile from a Kamchatkan hot spring; Sokolova TG et al.; A novel anaerobic, thermophilic, CO-utilizing bacterium, strain 41(T), was isolated from a terrestrial hot vent on the Kamchatka Peninsula . Strain 41(T) was found to be a Gram-positive bacterium, its cells being short, straight, motile rods . Chains of three to five cells were often observed . The isolate grew only chemolithoautotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O --> CO2+H2) . Growth was observed in the temperature range 40-68 degrees C, with an optimum at 58 degrees C, and in the pH range 6.5-7.6, with an optimum at pH 7.0 . The generation time under optimal conditions for chemolithotrophic growth was 1.1 h . The DNA G+C content was 46 +/- 1 mol% . Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin . On the basis of the phenotypic and phylogenetic features, it is proposed that this isolate represents a new genus and species, Carboxydocella thermautotrophica gen . nov., sp . nov . (type strain 41(T) = DSM 12356(T) = VKM B-2282(T)).

Bioresour Technol, 2003 May, 87(3), 331 - 6
Respiration profiles in monitoring the composting of by-products from the olive oil agro-industry; Mari I et al.; The composting of olive press cake (OPC) repeatedly mixed either with olive mill wastewater (OPC+OMW) or with tap water (OPC+W) was studied using the thermogradient respirometer, an apparatus that determines the respiration rates from a substrate over a wide range of different temperatures (respiratory profile) . The composting processes took place over a period of five months during which nine moistenings of the OPC were performed with the respective liquids . The composting resulted in detoxification of the materials used in both treatments, as indicated by seed germination tests . However, the repeated applications of OMW resulted in recurring thermophilic phases (following each application) and in greater pH and conductivity increases in the final product, as compared to water applications . Respiration measurements performed at 35 degrees C were good indicators of the mean metabolic potential in the compost piles (the mean respiration derived from the whole respiration profile over a wide range of environmental temperatures) . However, respiration measurements at higher temperatures (48.5 degrees C) were better indicators of the respiration activity occurring in situ . Following the initial thermophilic phase, the respiration potential of the composts at high temperatures (42-63 degrees C) increased drastically compared to their respiration potential at lower temperatures (17-42 degrees C) indicating the establishment of a thermophilic microflora . Subsequently, only the periodic new substrate-C applications in the form of OMW resulted in increased ratios of low temperature-to-high temperature respiration potential . These ratios decreased again following the respective thermophilic phase that each new OMW application had induced.

Hum Immunol, 2003 Jan, 64(1), 130 - 6
Study of MICA alleles in 201 African Americans by multiplexed single nucleotide extension (MSNE) typing; Zhang Y et al.; We have developed a method for major histocompatibility complex class I chain-related gene A (MICA) genotyping using multiplexed single nucleotide extension (MSNE) and flow cytometric analysis of an array of fluorescent microspheres . This technique employs a polymerase chain reaction-derived target DNA containing all the polymorphic sites of MICA, synthetic complementary primers, biotinylated dideoxynucleotide triphosphate, fluorescent reporter molecules (streptavidin-phycoerythrin), and thermophilic DNA polymerase . Genomic DNA was amplified by MICA locus-specific primers and the MSNE reactions were carried out in the presence of 30 MSNE primers used to assay polymorphisms in exons 2, 3, and 4 of the MICA genes . Thirty-two previously typed cell lines were used as reference material . The MICA gene frequencies among 201 African-American unrelated donors were determined . Of 51 previously known alleles, 18 were observed in African-Americans, compared to 16 that were found in North American Caucasians and 9 in South American Indians, suggesting a more diversified allelic distribution in African-Americans . MICA*00201 and MICA*00801 were the two most frequent alleles in African-Americans . We observed a high degree of linkage disequilibrium between certain alleles of MICA and of human leukocyte antigen-B in the African-American population . The methodology described here offers a powerful new approach to DNA typing of the MICA alleles.

J Gen Appl Microbiol, 1997 Aug, 43(4), 225 - 230
Growth-promoting effect of carbon material upon bacterial cells propagating through a distance; Matsuhashi M et al.; Carbon material such as graphite and activated charcoal, but not diamond, causes the promotion of growth of certain bacteria under ordinarily non-permissive stress conditions over a distance of several centimeters . Bacillus carboniphilus under the stress of a high KCl concentration and high temperature responded to this remote effect of carbon material with enhanced growth, and thermophile bacterium Bacillus stearothermophilus responded similarly yet moderately under the stress of low temperature . The remote effect of carbon was caused by its activation with external energy, probably of electromagnetic nature, as this effect was markedly decreased by sheltering the experimental system with an iron or aluminum barrier . Carbon material probably transforms the external oscillatory pulses or radiation into a signal exerting, far-reaching, growth-promoting effect upon cells . The most plausible candidate of signals emitted from carbon was considered to be (ultra)sonic.

J Gen Appl Microbiol, 1997 Oct, 43(5), 295 - 304
Phylogenetic characterization of a new thermoacidophilic bacterium isolated from hot springs in Japan; Hiraishi A et al.; Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods . The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization . A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A . acidocaldarius and A . acidoterrestris as their closest relatives . The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus . The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species . DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus . On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus . The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 152 - 4 Epub 2002 Dec 19.
Preliminary X-ray crystallographic analysis of tRNA pseudouridine 55 synthase from the thermophilic eubacterium Thermotoga maritima; Wouters J et al.; Thermotoga maritima TruB, an enzyme responsible for the formation of pseudouridine in tRNA, has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li(2)SO(4), 20% glycerol, 13% PEG 8000 . Crystals display orthorhombic symmetry, with unit-cell parameters a = 47.39, b = 83.88, c = 98.72 A, and diffract to 2.0 A resolution using synchrotron radiation . A solution was obtained by molecular replacement using part of the recently published crystal structure of Escherichia coli TruB bound to a synthetic RNA.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 23 - 31 Epub 2002 Dec 19.
Structure and catalytic mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis; Kishida H et al.; Precursors for isoprenoid synthesis are essential in all organisms . These compounds are synthesized by one of two known routes: the well characterized mevalonate pathway or a recently discovered non-mevalonate route which is used in many bacteria and human pathogens . Since the second pathway is both vital and unlike any found in humans, enzymes catalysing reactions along this synthetic route are possible drug targets . The structure of one such enzyme from the thermophilic bacterium Thermus thermophilus has been solved to high resolution in the presence of substrate and with a substrate analogue . Enzyme co-crystallized with substrate shows only one product, cytosine monophosphate (CMP), in the active site . At the high resolution of the refinement (1.6 A) the positions and coordination of the magnesium ions in the active site are clearly seen.

Acta Vet Hung, 2002, 50(4), 395 - 411
Effects of a non-starch polysaccharidase enzyme preparation from Thermomyces lanuginosus on energy and protein metabolism and milk yield of dairy cattle; Jurkovich V et al.; Non-starch polysaccharides (NSPs) form an integral part of the cell walls in plants and represent considerable available energy when degraded into absorbable mono-, di-, tri- and oligosaccharides . The ruminal microflora hydrolyses a good part of NSPs, however, recently there have been attempts to enhance the rate of utilisation by using external polysaccharidase enzymes . In the present study the effects of an enzyme preparation (Rumino-Zyme) high in xylanase activity were studied on ruminal volatile fatty acid (VFA) concentration, parameters of energy and protein metabolism, milk yield, feed conversion ratio (FCR) and body condition score of high-yielding dairy cows . A lignolytic enzyme preparation produced by the thermophilic fungus Thermomyces lanuginosus was applied in the present experiment and fed to dairy cows at 34 g/day dosage in the period between calving and the 110th day of lactation . This preparation increased VFA concentration in the rumen from about 32 days after calving and onward . Increased VFA concentration was followed by an about 5 to 10% increase in milk production and an almost 0.1% increase in butterfat production . Increased VFA concentration produced more balanced energy metabolism in the experimental cows as indicated by the lower incidence rate of hyperketonaemia, and lower acetoacetic acid and non-esterified fatty acid (NEFA) concentration in the blood of the experimental cows . Aspartate aminotransferase (AST) activity was tendentiously higher in the control group and the proportion of cows that had AST activity higher than 100 U/l was also higher in the control group . Both control and experimental cows showed balanced protein and acid-base metabolism throughout the experiment . Enhanced VFA concentration contributed to an improvement in energy balance in the experimental cows with a resultant improvement of feed intake and feed utilisation . Due to the more balanced energy metabolism postparturient body condition loss of the treated cows was reduced.

Int Microbiol, 2002 Dec, 5(4), 201 - 7 Epub 2002 Sep 26.
Microbial transformation of elements: the case of arsenic and selenium; Stolz JF et al.; Microbial activity is responsible for the transformation of at least one third of the elements in the periodic table . These transformations are the result of assimilatory, dissimilatory, or detoxification processes and form the cornerstones of many biogeochemical cycles . Arsenic and selenium are two elements whose roles in microbial ecology have only recently been recognized . Known as "essential toxins", they are required in trace amounts for growth and metabolism but are toxic at elevated concentrations . Arsenic is used as an osmolite in some marine organisms while selenium is required as selenocysteine (i.e . the twenty-first amino acid) or as a ligand to metal in some enzymes (e.g . FeNiSe hydrogenase) . Arsenic resistance involves a small-molecular-weight arsenate reductase (ArsC) . The use of arsenic and selenium oxyanions for energy is widespread in prokaryotes with representative organisms from the Crenarchaeota, thermophilic bacteria, low and high G+C gram-positive bacteria, and Proteobacteria . Recent studies have shown that both elements are actively cycled and play a significant role in carbon mineralization in certain environments . The occurrence of multiple mechanisms involving different enzymes for arsenic and selenium transformation indicates several different evolutionary pathways (e.g . convergence and lateral gene transfer) and underscores the environmental significance and selective impact in microbial evolution of these two elements.

J Biol Chem, 2003 Feb 28, 278(9), 7073 - 80 Epub 2002 Dec 20.
Mutational and functional analysis of a segment of the sigma family bacteriophage T4 late promoter recognition protein gp55; Wong K et al.; Bacteriophage T4 late promoters, which consist of a simple 8-base pair TATA box, are recognized by the gene 55 protein (gp55), a small, highly diverged member of the varsigma family proteins that replaces varsigma(70) during the final phase of the T4 multiplication cycle . A 16-amino acid segment of gp55 that is proposed to be homologous to the varsigma(70) region 2.2 has been subjected to alanine scanning and other mutagenesis . The corresponding proteins have been examined in vitro for binding to Escherichia coli RNA polymerase core enzyme and for the ability to generate accurately initiating basal as well as sliding clamp-activated T4 late transcription . Mutations in the amino acid 68-83 segment of gp55 generate a wide range of effects on these functions . The changes are interpreted in terms of the multiple steps of involvement of gp55, like other varsigma proteins, in transcription . Effects of mutations on RNA polymerase core binding are consistent with the previously proposed homology of amino acids 68-82 of gp55 with varsigma(70) region 2.2 and the recently determined structures of the Thermus thermophilus and Thermus aquaticus varsigma(70)-RNA polymerase holoenzymes.

Biophys J, 2002 Dec, 83(6), 3126 - 33
Toward the physical basis of thermophilic proteins: linking of enriched polar interactions and reduced heat capacity of unfolding; Zhou HX; The enrichment of salt bridges and hydrogen bonding in thermophilic proteins has long been recognized . Another tendency, featuring lower heat capacity of unfolding (DeltaC(p)) than found in mesophilic proteins, is emerging from the recent literature . Here we present a simple electrostatic model to illustrate that formation of a salt-bridge or hydrogen-bonding network around an ionized group in the folded state leads to increased folding stability and decreased DeltaC(p) . We thus suggest that the reduced DeltaC(p) of thermophilic proteins could partly be attributed to enriched polar interactions . A reduced DeltaC(p) might serve as an indicator for the contribution of polar interactions to folding stability.

Int J Food Microbiol, 2003 Feb 15, 80(3), 201 - 10
Isolation, characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage; Muyanja CM et al.; One hundred and thirteen strains of lactic acid bacteria (LAB) were selected from 351 isolates from 15 samples of traditionally fermented household bushera from Uganda and also from laboratory-prepared bushera . Isolates were phenotypically characterised by their ability to ferment 49 carbohydrates using API 50 CHL kits and additional biochemical tests . Coliforms, yeasts and LAB were enumerated in bushera . The pH, volatile organic compounds and organic acids were also determined . The LAB counts in household bushera varied between 7.1 and 9.4 log cfu ml(-1) . The coliform counts varied between < 1 and 5.2 log cfu ml(-1) . The pH of bushera ranged from 3.7 to 4.5 . Ethanol (max, 0.27%) was the major volatile organic compound while lactic acid (max, 0.52%) was identified as the dominant organic acid in household bushera . The initial numbers of LAB and coliforms in laboratory-fermented bushera were similar; however, the LAB numbers increased faster during the first 24 h . LAB counts increased from 5.5 to 9.0 log cfu ml(-1) during the laboratory fermentation . Coliform counts increased from 5.9 to 7.8 log cfu ml(-1) at 24 h, but after 48 h, counts were less 4 log cfu ml(-1) . Yeasts increased from 4.3 to 7.7 log cfu ml(-1) at 48 h, but thereafter decreased slightly . The pH declined from 7.0 to around 4.0 . Lactic acid and ethanol increased from zero to 0.75% and 0.20%, respectively . Lactic acid bacteria isolated from household bushera belonged to Lactobacillus, Streptococcus and Enterococcus genera . Tentatively, Lactobacillus isolates were identified as Lactobacillus plantarum, L . paracasei subsp . paracasei, L . fermentum, L . brevis and L . delbrueckii subsp . delbrueckii . Streptococcus thermophilus strains were also identified in household bushera . LAB isolated from bushera produced in the laboratory belonged to five genera (Lactococcus, Leuconostoc, Lactobacillus, Weissella and Enterococcus . Eight isolates were able to produce acid from starch and were identified as Lactococcus lactis subsp . lactis (four strains), Leuconostoc mesenteroides subsp . mesenteroides (one strain), Leuconostoc mesenteroides subsp . dextranicum (one strain), Weissella confusa (one strain) and L . plantarum (one strain) .

J Appl Microbiol, 2003, 94(1), 127 - 37
Microbiological aspects of biowaste during composting in a monitored compost bin; Ryckeboer J et al.; AIMS: To determine the microbial succession of the dominating taxa and functional groups of microorganisms and the total microbial activity during the composting of biowaste in a monitored process . METHODS AND RESULTS: Biowaste (vegetable, fruit and garden waste) was composted in a monitored composting bin system . During the process, taxonomic and functional subpopulations of microorganisms were enumerated, and dominating colonies were isolated and identified . All counts decreased during the thermophilic phase of the composting, but increased again when the temperature declined . Total microbial activity, measured with an enzyme activity assay, decreased during the thermophilic phase, increased substantially thereafter, and decreased again during maturation . Bacteria dominated during the thermophilic phase while fungi, streptomycetes and yeasts were below the detection limit . Different bacterial populations were found in the thermophilic and mesophilic phases . In fresh wastes and during the peak-heating phase, all bacterial isolates were bacilli . During the cooling and maturation phase the bacterial diversity increased, including also other Gram-positive and Gram-negative bacteria . Among the fungi, Aspergillus spp . and Mucor spp . were predominant after the thermophilic phase . CONCLUSIONS: The microbial abundance, composition and activity changed substantially during composting and compost maturity was correlated with high microbial diversity and low activity . SIGNIFICANCE AND IMPACT OF THE STUDY: A more complete overview of the whole composting process of biowaste, based on microbial counts, species diversity and functional groups and abiotic parameters is presented, and the potential of a simple enzyme assay to measure total microbial activity was demonstrated.

Nucleic Acids Res, 2002 Dec 15, 30(24), 5444 - 51
Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu; Ohtsuki T et al.; Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2 . Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria . Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration . Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble . This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes . The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus) . Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu . Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.

J Dairy Sci, 2002 Nov, 85(11), 2750 - 8
Proteolytic specificity of Lactobacillus delbrueckli subsp . bulgaricus influences functional properties of mozzarella cheese; Oommen BS et al.; Low-moisture part-skim Mozzarella cheeses were manufactured from 2% fat milk and aged for 21 d . Treatments included cheeses made with one of three different strains of Lactobacillus delbrueckii ssp . bulgaricus in combination with a single strain of Streptococcus thermophilus . A fourth, control treatment consisted of cheeses made with only S . thermophilus . Although total proteolytic ability of these strains, as indicated by the o-phthaldialdehyde analysis, was similar in each of the three strains of L . bulgaricus, these strains exhibited different proteolytic specificities toward the peptide, alpha(s1)-CN (f 1-23) . On the basis of their alpha(s1)-CN (f 1-23) cleavage patterns and a previously described classification, these strains were assigned to the groups I, III, and V . The objective of this study was to investigate the influence of lactobacilli proteolytic systems, based on specificity toward alpha(s1)-CN (f 1-23), on functionality of part-skim Mozzarella cheese . Moisture, fat, protein, salt-in-moisture, and moisture in nonfat substances content of cheeses made with groups I, III, and V strain were similar . Control cheese had a lower moisture content than did other treatments . Significant differences were observed in functional properties between cheeses manufactured using groups III and V strains . Cheeses made with groups I and III strains were similar in their meltability, hardness, cohesiveness, melt strength, and stretch quality . Meltability and cohesiveness increased with age, while melt strength and stretch quality decreased with age for all cheeses . Additionally, HPLC showed that total peak areas of water-soluble peptides derived from cleavage of alpha(s1)-CN (f 1-23) by different strains of lactobacilli could be highly correlated to meltability and stretch characteristics of cheeses made with those strains.

J Dairy Sci, 2002 Nov, 85(11), 2705 - 10
Binding of free bile acids by cells of yogurt starter culture bacteria; Pigeon RM et al.; Several strains of Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus thermophilus, which produced exocellular polysaccharides (EPS), varied in the amount produced . The streptococci tended to produce the most EPS per milliliter of culture; however, when compared on the basis of amounts per 10(7) cfu, the lactobacilli produced the most . Lactobacillus delbrueckii ssp . bulgaricus strains Lb-18 and Lb-10442 and S . thermophilus St-143 produced significantly larger amounts per 107 cfu than did other strains tested . These three cultures plus two strains of the streptococci that produced the greatest amounts of EPS per ml of culture were tested for the ability to bind bile acids from laboratory media . The two cultures of L . delbrueckii ssp . bulgaricus (Lb-18 and Lb-10442) bound significantly higher amounts of cholic acid than did the three strains of streptococci . These two cultures of lactobacilli bound up to 15.3% of the cholic acid present in laboratory media, up to 452 microg/mg of EPS and 2.9 microg/10(7) cfu . None of the cultures tested in this study were able to bind the conjugated bile acid, glycocholic acid.

Extremophiles, 2002 Dec, 6(6), 485 - 90 Epub 2002 Sep 04.
Thermal stability of aminoacyl-tRNAs in aqueous solutions; Stepanov VG et al.; Life in hot environments poses certain constraints on the metabolism of thermophilic organisms . Many universal metabolic intermediates are quite labile compounds, and without protection will rapidly decompose at elevated temperatures . Among these are aminoacyl-tRNAs that are necessarily formed upon functioning of the translation apparatus . Aminoacyl-tRNAs are known to be hydrolyzed rapidly even at moderate temperatures under mild alkaline conditions . We studied the thermal stability of phenylalanyl- and alanyl-tRNA in aqueous solutions in order to evaluate a potential threat posed by high temperatures to these components of the translation machinery of thermophiles . Specific second-order rate constants of the aminoacyl-tRNA hydrolysis reaction were determined in the range 20 degrees -80 degrees C . The activation energy of phenylalanyl- and alanyl-tRNA hydrolysis was found to be about 42 and 23 kJ/mol, respectively . The calculated half-lives of aminoacyl-tRNAs at sub-80 degrees C temperatures vary from several seconds to several dozens of seconds at near-neutral pH . The possible mechanisms counteracting the observed thermolability of aminoacyl-tRNAs in vivo are discussed.

Extremophiles, 2002 Dec, 6(6), 463 - 8 Epub 2002 Aug 10.
A novel thermophilic fusion enzyme for trehalose production; de Pascale D et al.; In recent years a number of hyperthermophilic micro-organisms of Sulfolobales have been found to produce trehalose from starch and dextrins . In our laboratory genes encoding the trehalosyl dextrin forming enzyme (TDFE) and the trehalose forming enzyme (TFE) of S . solfataricus MT4 have been cloned and expressed in E . coli (Rb791) . Here we report the construction of a new protein obtained by fusion of TFE and TDFE coding sequences which is able to produce trehalose from dextrins at high temperature by sequential enzymatic steps . We demonstrate that the bifunctional fusion enzyme is able to produce trehalose starting from malto-oligosaccharides at 75 degrees C . Furthermore we partially purified the recombinant fusion protein from bacterial cell free extracts and from insoluble fractions in which the fusion protein was also found as aggregate in inclusion bodies.

J Bacteriol, 2003 Jan, 185(1), 381 - 5
Identification of a helix-turn-helix motif of Bacillus thermoglucosidasius HrcA essential for binding to the CIRCE element and thermostability of the HrcA-CIRCE complex, indicating a role as a thermosensor; Hitomi M et al.; In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element . Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006 . These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor . We identified the position of a helix-turn-helix (HTH) motif in B . thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element . Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B . thermoglucosidasius, which grow at vastly different ranges of temperature . The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B . thermoglucosidasius and B . subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.

Biosens Bioelectron, 2003 Mar, 18(2-3), 319 - 25
Amperometric biosensors based on recombinant laccases for phenols determination; Kulys J et al.; Graphite (GE) or printed graphite electrode (PGE) based biosensors containing recombinant fungal laccase Polyporus pinsitus (rPpL), and Myceliophthora thermophila (rMtL) were developed . The enzymes were immobilized using bovine serum albumin and glutaraldehyde . At pH 5.5 and -0.1 V, the calibration graphs of GE based biosensors were hyperbolic if pyrocatechol was used . The concentration of substrate that results in 50% of steady-state response (EC(50)) was 0.7 mM and sensitivity (S) was 3.8 mA/M . The sensitivity increased up to 4 A/M if larger amount of rPpL was used . The sensitivity of biosensors changed little during 9 days of exploitation, but decreased at longer time . The PGE based biosensors were mounted into the flow-through cell and calibrated under kinetic regime . EC(50) of the biosensors containing rPpL varied from 0.6 to 4.0 mM and sensitivity varied from 0.11 to 1.9 mA/M . The response of biosensor containing thermostable laccase rMtL was less, but response saturated at larger pyrocatechol concentration . The sensitivity changed little during 6 days . Both type of biosensors responded also to 1-naphthol, o-phenylenediamine, guaiacol, o-anizidine, benzidine . The experiments demonstrate recombinant laccases application to biosensor engineering and their use to phenol and related compound determination under steady-state and flow-through regimes.

Biochemistry, 2002 Dec 24, 41(51), 15429 - 35
Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class; Tu C et al.; Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide . Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class . We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class) . A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium . Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers . Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class . The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.

J Gen Appl Microbiol, 2001 Aug, 47(4), 187 - 192
Isolation of a new Thermoanaerobacterium thermosaccharolyticum strain (FH1) producing a thermostable dextranase; Hoster F et al.; A Gram-positive spore-forming thermophilic strict anaerobic bacterium, designated FH1, was isolated from enrichments at 65 degrees C with dextran as sole carbon and energy source . A sequence analysis of the 16S rRNA gene revealed 99.2% identity of FH1 to Thermoanaerobacterium thermosaccharolyticum . Furthermore, the substrate spectra of both organisms were similar . It was therefore concluded that FH1 represents a new strain within the species T . thermosaccharolyticum . The optimal growth temperature of strain FH1 was 68 degrees C . The isolated organism produced a thermostable and thermoactive dextranase with a native molecular mass of approximately 200,000 Da . The enzyme was concentrated from the cell-free culture supernatant by ammonium sulfate precipitation . The resulting crude dextranase exhibited optimal activity from 65 to 70 degrees C and a pH optimum of 5.5.

Virology, 2002 Nov 10, 303(1), 100 - 9
DNA-binding activity of the Streptococcus thermophilus phage Sfi21 repressor; Bruttin A et al.; The cloned Streptococcus thermophilus phage Sfi21 repressor open reading frame (orf) 127 gp protects a cell against superinfection with the homologous temperate, but not against virulent phages . As demonstrated by DNase protection assay and gel shift experiments, the repressor binds to a 25-bp operator site located upstream of the repressor gene . A second sequence-related operator was identified 265 bp apart at the 3'-end of orf 75, the topological equivalent of a cro repressor gene . The replacement of a bp at the middle or at the right side of the operator decreased substantially the affinity of the repressor for the operator . In gel shift assays, the 75 gp did not bind DNA from the genetic switch region . However, when increasing amounts of orf 75 gp containing cell extracts were added to orf 127 gp containing cell extracts, the repressor could no longer bind its operator site.

FEBS Lett, 2002 Dec 18, 532(3), 437 - 40
LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis; Altincicek B et al.; Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity . The purified LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a defined in vitro system . The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate . A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein . The maximal specific activity of 6.6+/-0.3 micromol x min(-1) x mg(-1) protein was determined at pH 7.5 and 60 degrees C . The k(cat) value of the LytB protein was 3.7+/-0.2 s(-1) and the K(m) value for HMBPP was 590+/-60 microM.

FEBS Lett, 2002 Dec 18, 532(3), 432 - 6
Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis; Kollas AK et al.; The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway . This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites . Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen-free conditions . The protein was enzymatically active in converting 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant . The maximal specific activity was 0.6 micromol x min(-1) x mg(-1) at pH 7.5 and 55 degrees C . The kcat value was 0.4 s(-1) and the K(m) value for HMBPP 0.42 mM.

Microbiology, 2002 Dec, 148(Pt 12), 3913 - 20
Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome; Rao AR et al.; Ribosome recycling factor (RRF), coded for by the frr locus, is involved in the disassembly of post-termination complexes and recycling of the ribosomes for a fresh round of initiation in bacteria and in eukaryotic organelles . In a cross-species-complementation experiment, it was shown that the Thermus thermophilus RRF protein lacking five amino acids from its C-terminal end (deltaC5TthRRF) but not the full-length protein (TthRRF) complemented Escherichia coli for its frr(ts) phenotype . It was also shown that the Mycobacterium tuberculosis RFF protein (MtuRRF) did not complement E . coli LJ14 for frr(ts) . However, simultaneous expression of elongation factor G (EFG) and RRF from M . tuberculosis resulted in complementation of E . coli LJ14 . Here it is shown that unlike deltaC5TthRRF, an equivalent mutant of MtuRRF lacking six amino acids from its C-terminal end (deltaC6MtuRRF) did not complement E . coli LJ14 . Surprisingly, deltaC6MtuRRF failed to complement the strain even in the presence of homologous EFG (MtuEFG) . The biochemical and biophysical characterization of these proteins suggested that the mutant RRF folded properly . However, ribosome-binding assays showed that the mutant protein was compromised in its binding to E . coli ribosomes . It is suggested that the conserved amino acids at the C-terminal end of the RRFs contribute to their residency on ribosomes and that the specific interactions between RRF and EFG are crucial in the disassembly of the termination complex.

Biochem Biophys Res Commun, 2003 Jan 3, 300(1), 161 - 6
Comparative modeling of the N-terminal domain of the 67kDa laminin-binding protein: implications for putative ribosomal function; Kazmin DA et al.; Laminin-binding protein/p40 (LBP/p40) precursor appears to be involved in two seemingly unrelated activities-cell adhesion and ribosomal biogenesis . Analysis of primary structure revealed a two-domain organization of the LBP/p40 . The N-terminal portion of LBP is similar to the S2 family of prokaryotic ribosomal proteins, while the C-terminus is unique for Metazoa and is involved in extraribosomal functions . To gain insight into putative ribosomal functions of LBP we performed comparative modeling of the N-terminal domain using crystal structures of S2p from Thermus thermophilus . The LBP model assumes an alpha-beta sandwich fold similar to that of S2 . Modeling revealed the loss of a significant portion of ribosomal RNA (rRNA) interaction domain, lack of conservation of many residues involved in interactions with rRNA, and a major shift in surface charge distribution (compared to the S2 protein) . The overall stability of the fold argues against a proposed transmembrane domain in the central part of the protein . Partial overlap in S2 and laminin-binding domains suggests that ribosomal and surface receptor functions would be mutually exclusive . The possible biological role of LBP/p40 bifunctionality is discussed.

Water Sci Technol, 2002, 46(10), 131 - 8
Identification of factors contributing to degradation in autothermal thermophilic sludge digestion; Csikor Z et al.; A laboratory scale ATAD reactor (6 days SRT, 24 h feed cycle) was operated at quasi steady-state conditions with real waste activated sludge feed . Hydrolysis tests of waste activated sludge demonstrated that 10% of feed suspended matter is hydrolysed practically instantaneously and 20-30% of all fed suspended matter is dissolved after 60 minutes in tap water and in cell free reactor liqueur equally . Respirometric, VSS and COD concentration data served as basis for calibration of a simple VSS based kinetic model . The calibrated model provided good fit to two separate sets of measured data . This model was used to evaluate different operation strategies . Modification of cycle length does not affect overall VSS emoval rate, while shorter cycle length or continuous operation helps avoid oxygen limited conditions . Further advantages of shorter feed cycles (reduced cooling effect, greater realizable load) support choosing continuous operation of the ATAD system if the main goal is VSS reduction . While reactor cascades increase efficiency, this advantage diminishes with increasing load . At high load rates increased construction costs are not justified by the expected improvement in efficiency.

Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 125 - 33
Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima: how is it different from that of the mesophilic enzyme; Kuo TH et al.; Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all-trans C40-octaprenyl pyrophosphate . The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP . Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s(-1) but product release limits the steady-state rate at 0.02 s(-1) {Biochim . Biophys . Acta 1594 (2002) 64} . In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined . The enzyme activity at 25 degrees C was 0.005 s(-1) under steady-state condition and was exponentially increased with elevated temperature . In contrast to E . coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction . The product of chain elongation catalyzed by T . maritima OPPs was C40 and the rate of its conversion to C45 was negligible . Under single-turnover condition with 10 microM OPPs-FPP complex and 1 microM IPP, only the C20 was formed rather than C20-C40 observed for E . coli enzyme . Together, our data suggest that the thermophilic OPPs from T . maritima has lower enzyme activity at 25 degrees C, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E . coli.

J Steroid Biochem Mol Biol, 2002 Oct, 82(2-3), 251 - 6
Studies on Bacillus stearothermophilus . Part II . Transformation of progesterone; Al-Awadi S et al.; Bacillus stearothermophilus, a thermophilic bacterium isolated from Kuwaiti desert, when incubated with exogenous progesterone for 10 days at 65 degrees C produced two new dihydroxy isomers of progesterone, and two known compounds, 5 alpha-pregnane-3,6,20-trione and 6-dehydroprogesterone, along with the earlier reported monohydroxylated metabolites and a B-Seco compound . The two new dihydroxy compounds were identified as 6 alpha,20 alpha-dihydroxyprogesterone and 6 beta,20 alpha-dihydroxyprogesterone . These metabolites were purified by TLC and HPLC followed by their identification through 1H, 13C NMR and other spectroscopic data.

Biochemistry, 2002 Dec 17, 41(50), 14969 - 77
Sheared Aanti.Aanti base pairs in a destabilizing 2 x 2 internal loop: the NMR structure of 5'(rGGCAAGCCU)2; Znosko BM et al.; The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs . The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints . Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron {Cate, J . H., Gooding, A . R., Podell, E., Zhou, K., Golden, B . L., Kundrot, C . E., Cech, T . R., and Doudna, J . A . (1996) Science 273, 1678-1685} . The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5 . An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5 . The cross-strand A5 bases are stacked . The Watson-Crick G-C regions are essentially A-form . The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics . Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.

Cell Mol Life Sci, 2002 Oct, 59(10), 1598 - 606
The DnaK/ClpB chaperone system from Thermus thermophilus; Schlee S et al.; Proteins of thermophilic organisms are adapted to remain well structured and functional at elevated temperatures . Nevertheless like their 'cousins' that reside at medium temperatures, they require the assistance of molecular chaperones to fold properly and prevent aggregation . This review compares structural and functional properties of the DnaK/ClpB systems of Thermus thermophilus and, mainly, Escherichia coli (DnaK(Tth) and DnaK(Eco)) . Many elemental properties of these systems remain conserved . However, in addition to a general increase of the thermal stability of its components, the DnaK(Tth) system shows profound differences in its regulation, and genetic as well as oligomeric organization . Whether these differences are unique or represent general strategies of adaptation to life at elevated temperatures remains to be clarified.

Biotechnol Bioeng, 2003 Feb 5, 81(3), 255 - 62
Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus; van Niel EW et al.; Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied . The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model . Hydrogen was the most severe inhibitor when allowed to accumulate in the culture . Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation . The extent of inhibition by hydrogen was dependent on the density of the culture . The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities . Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely . With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate . The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively . Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition . Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH . Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca . 175 mM caused cell lysis, probably due to activation of autolysins .

J Mol Biol, 2003 Jan 3, 325(1), 65 - 83
The lonepair triloop: a new motif in RNA structure; Lee JC et al.; The lonepair triloop (LPTL) is an RNA structural motif that contains a single ("lone") base-pair capped by a hairpin loop containing three nucleotides . The two nucleotides immediately outside of this motif (5' and 3' to the lonepair) are not base-paired to one another, restricting the length of this helix to a single base-pair . Four examples of this motif, along with three tentative examples, were initially identified in the 16S and 23S rRNAs with covariation analysis . An evaluation of the recently determined crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits revealed the authenticity for all of these proposed interactions and identified 16 more LPTLs in the 5S, 16S and 23S rRNAs . This motif is found in the T loop in the tRNA crystal structures . The lonepairs are positioned, in nearly all examples, immediately 3' to a regular secondary structure helix and are stabilized by coaxial stacking onto this flanking helix . In all but two cases, the nucleotides in the triloop are involved in a tertiary interaction with another section of the rRNA, establishing an overall three-dimensional function for this motif . Of these 24 examples, 14 occur in multi-stem loops, seven in hairpin loops and three in internal loops . While the most common lonepair, U:A, occurs in ten of the 24 LPTLs, the remaining 14 LPTLs contain seven different base-pair types . Only a few of these lonepairs adopt the standard Watson-Crick base-pair conformations, while the majority of the base-pairs have non-standard conformations . While the general three-dimensional conformation is similar for all examples of this motif, characteristic differences lead to several subtypes present in different structural environments . At least one triloop nucleotide in 22 of the 24 LPTLs in the rRNAs and tRNAs forms a tertiary interaction with another part of the RNA . When a LPTL containing the GNR or UYR triloop sequence forms a tertiary interaction with the first (and second) triloop nucleotide, it recruits a fourth nucleotide to mediate stacking and mimic the tetraloop conformation . Approximately half of the LPTL motifs are in close association with proteins . The majority of these LPTLs are positioned at sites in rRNAs that are conserved in the three phylogenetic domains; a few of these occur in regions of the rRNA associated with ribosomal function, including the presumed site of peptidyl transferase activity in the 23S rRNA.

Eur J Biochem, 2002 Dec, 269(24), 6250 - 60
Stepwise adaptations of citrate synthase to survival at life's extremes . From psychrophile to hyperthermophile; Bell GS et al.; The crystal structure of citrate synthase from the thermophilic Archaeon Sulfolobus solfataricus (optimum growth temperature = 85 degrees C) has been determined, extending the number of crystal structures of citrate synthase from different organisms to a total of five that span the temperature range over which life exists (from psychrophile to hyperthermophile) . Detailed structural analysis has revealed possible molecular mechanisms that determine the different stabilities of the five proteins . The key to these mechanisms is the precise structural location of the additional interactions . As one ascends the temperature ladder, the subunit interface of this dimeric enzyme and loop regions are reinforced by complex electrostatic interactions, and there is a reduced exposure of hydrophobic surface . These observations reveal a progressive pattern of stabilization through multiple additional interactions at solvent exposed, loop and interfacial regions.

Proteins, 2003 Jan 1, 50(1), 122 - 34
Structural plasticity of thermophilic serine hydroxymethyltransferases; Paiardini A et al.; Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and monocarbonic groups, essential in several biosynthetic pathways . The availability of crystallographic structures of SHMT from mesophilic organisms and information produced by the genomic projects prompted the analysis of the adaptation of SHMT to "extreme" environments, such as high temperatures, by exploitation of structural data from thermophilic organisms . The sequences of 10 thermophilic/hyperthermophilic SHMTs were multiply aligned to 53 mesophilic homologs and analyzed by a comparative approach, examining the amino acid compositions and preferred residue exchanges between mesophiles and extremophiles . The structural basis of the observed exchanges was further investigated through the application of homology modeling to the 10 extremophilic SHMTs . The results of this study indicate that, in SHMT, thermal stability can be achieved mainly through three strategies: (i) increased number of charged residues at the protein surface; (ii) increased hydrophobicity of the protein core; and (iii) substitution of thermolabile residues exposed to the solvent . Additional features of the archaeal SHMTs, for which no structural data are available yet, were also investigated to explain their quaternary assemblage and the interaction with modified folates .

Mol Genet Genomics, 2002 Dec, 268(4), 500 - 9 Epub 2002 Oct 17.
Identification of a phosphofructokinase-encoding gene from Streptococcus thermophilus CNRZ1205--a novel link between carbon metabolism and gene regulation?
Crispie F, Anba J, Renault P, Ehrlich D, Fitzgerald G, van Sinderen D.
In order to isolate genes encoding so-called Two-Component Regulatory Systems from the lactic acid bacterium Streptococcus thermophilus, a cloning strategy was employed based on suppression of the alkaline phosphatase-negative phenotype displayed by the Escherichia coli strain ANCC22 . Several suppressing clones were obtained which were shown to produce alkaline phosphatase activity . Sequence analysis of four of these clones revealed the presence of overlapping DNA inserts representing two ORFs, designated pfkT and pykT, whose deduced protein products exhibit significant similarity to phosphofructokinases and pyruvate kinases, respectively, from a variety of bacteria . A plasmid bearing pfkT was shown to complement a phosphofructokinase-negative mutant of E . coli, showing that this gene indeed specifies phosphofructokinase activity . It was shown that suppression of the alkaline phosphatase-negative phenotype of E . coli ANCC22 due to the presence of pfkT is caused by modulation of the intracellular level of acetyl phosphate.

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