J Bacteriol, 2004 Sep, 186(17), 5819 - 25 Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly; Cameron DM et al.; The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene . The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype . We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase . This mutant shows no growth defects, indicating that T . thermophilus PrmA, like its E . coli homolog, is dispensable . Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T . thermophilus PrmA . MALDI-TOF MS also revealed that T . thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E . coli L11 . Finally, we found that, as with the E . coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes. J Biomol Struct Dyn, 2004 Oct, 22(2), 205 - 14 Investigation on the causes of codon and amino acid usages variation between thermophilic Aquifex aeolicus and mesophilic Bacillus subtilis; Basak S et al.; Base composition, codon usages and amino acid usages have been analyzed by taking 529 orthologous sequences of Aquifex aeolicus and Bacillus subtilis, having different optimal growth temperatures . These two bacteria do not have significant difference in overall GC composition, but GC(1+2) and GC3 levels were found to vary significantly . Significant increments in purine content and GC3 composition have been observed in the coding sequences of Aquifex aeolicus than its Bacillus subtilis counterparts . Correspondence analyses on codon and amino acid usages reveal that variation in base composition actually influences their codon and amino acid usages . Two selection pressures acting on the nucleotide level (GC3 and purine enrichment), causes variation in the amino acid usage differently in different protein secondary structures . Our results suggest that adaptation of amino acid usages in coil structure of Aquifex aeolicus proteins is under the control of both purine increment and GC3 composition, whereas the adaptation of the amino acids in the helical region of thermophilic bacteria is strongly influenced by the purine content . Evolutionary perspectives concerning the temperature adaptation of DNA and protein molecules of these two bacteria have been discussed on the basis of these results. Glycoconj J, 2004, 20(7-8), 435 - 47 Genetic organization of chromosomal S-layer glycan biosynthesis loci of Bacillaceae; Novotny R et al.; S-layer glycoproteins are cell surface glycoconjugates that have been identified in archaea and in bacteria . Usually, S-layer glycoproteins assemble into regular, crystalline arrays covering the entire bacterium . Our research focuses on thermophilic Bacillaceae, which are considered a suitable model system for studying bacterial glycosylation . During the past decade, investigations of S-layer glycoproteins dealt with the elucidation of the highly variable glycan structures by a combination of chemical degradation methods and nuclear magnetic resonance spectroscopy . It was only recently that the molecular characterization of the genes governing the formation of the S-layer glycoprotein glycan chains has been initiated . The S-layer glycosylation (slg) gene clusters of four of the 11 known S-layer glycan structures from members of the Bacillaceae have now been studied . The clusters are approximately 16 to approximately 25 kb in size and transcribed as polycistronic units . They include nucleotide sugar pathway genes that are arranged as operons, sugar transferase genes, glycan processing genes, and transporter genes . So far, the biochemical functions only of the genes required for nucleotide sugar biosynthesis have been demonstrated experimentally . The presence of insertion sequences and the decrease of the G + C content at the slg locus suggest that the investigated organisms have acquired their specific S-layer glycosylation potential by lateral gene transfer . In addition, S-layer protein glycosylation requires the participation of housekeeping genes that map outside the cluster . The gene encoding the respective S-layer target protein is transcribed monocistronically and independently of the slg cluster genes . Its chromosomal location is not necessarily in close vicinity to the slg gene cluster. J Colloid Interface Sci, 2004 Oct 1, 278(1), 251 - 4 Atomic force microscopic corroboration of bond aging for adhesion of Streptococcus thermophilus to solid substrata; Vadillo-Rodriguez V et al.; Initial bacterial adhesion is considered to be reversible, but over time the adhesive bond between a bacterium and a substratum surface may strengthen, turning the process into an irreversible state . Microbial desorption has been studied in situ in controlled flow devices as a function of the organisms resident time on the surface (J . Colloid Interface Sci . 164 (1994) 355) . It appeared that desorption of Streptococcus thermophilus decreased strongly within approximately 50 s after initial adhesion due to bond aging . In this paper, bond aging between the S . thermophilus cell surface and the silicon nitride tip of an AFM (atomic force microscope) is corroborated microscopically and related to the macroscopic, residence time-dependent desorption of the organism under flow . AFM indicated bond strengthening between the tip and the cell surface within 100 s of contact, which is on the same order of magnitude as bond aging inferred from residence time-dependent desorption . Comparison of the interaction energies derived from AFM and macroscopic desorption indicate that bond strengthening arises as a result of multiple attachments of extracellular polymeric substances to a substratum surface. J Mol Biol, 2004 Sep 3, 342(1), 247 - 60 Stability and folding mechanism of mesophilic, thermophilic and hyperthermophilic archael histones: the importance of folding intermediates; Topping TB et al.; The equilibrium stabilities to guanidinium chloride (GdmCl)-induced denaturation and kinetic folding mechanisms have been characterized for three archael histones: hFoB from the mesophile Methanobacterium formicicum; hMfB from the thermophile Methanothermus fervidus; and hPyA1 from the hyperthermophile Pyrococcus strain GB-3a . These histones are homodimers of 67 to 69 residues per monomer . The equilibrium unfolding transitions, as measured by far-UV circular dichroism (CD) are highly reversible, two-state processes . The mesophilic hFoB is very unstable and requires approximately 1 M trimethyl-amine-N-oxide (TMAO) to completely populate the native state . The thermophilic histones are more stable, with deltaG degrees (H2O) values of 14 and 16 kcal mol(-1) for hMfB and hPyA1, respectively . The kinetic folding of hFoB and hPyA1 are two-state processes, with no detectable transient kinetic intermediates . For hMfB, there is significant development of CD signal in the stopped-flow dead time, indicative of the formation of a monomeric intermediate, which then folds/associates in a single, second-order step to form the native dimer . While the equilibrium stability to chemical denaturation correlates very well with host growth temperature, there is no simple relationship between folding rates and stability for the archael histones . In the absence of denaturant, the log of the unfolding rates correlate with equilibrium stability . The folding/association of the moderately stable hMfB is the most rapid, with a rate constant in the absence of GdmCl of 3 x 10(6) M(-1) s(-1), compared to 9 x 10(5) M(-1) s(-1) for the more stable hPyA1 . It appears that the formation of the hMfB burst-phase monomeric ensemble serves to enhance folding efficiency, rather than act as a kinetic trap . The folding mechanism of the archael histones is compared to the folding of other intertwined, segment-swapped, alpha-helical, DNA-binding dimers (ISSADD), including the eukaryotic heterodimeric histones, which fold more rapidly . The importance of monomeric and dimeric kinetic intermediates in accelerating ISSADD folding reactions is discussed. J Ind Microbiol Biotechnol, 2004 Oct, 31(9), 409 - 14 Epub 2004 Oct. Characterization of iron- and sulphide mineral-oxidizing moderately thermophilic acidophilic bacteria from an Indonesian auto-heating copper mine waste heap and a deep South African gold mine; Kinnunen PH et al.; Iron- and chalcopyrite-oxidizing enrichment cultures were obtained at 50 degrees C from acidic, high-temperature, copper/gold mine environments in Indonesia and South Africa . Over 90% copper yield was obtained from chalcopyrite concentrate with the Indonesian enrichment in 3 months with 2% solids concentration, when pH was maintained at around 2 . Neither addition of silver cations nor an enhanced nutrient concentration influenced chalcopyrite leaching . Excision and sequencing of bands from denaturing gradient gel electrophoresis of the amplified partial 16S rRNA gene showed that the enrichment cultures from different environments in South Africa and Indonesia were very simple, and similar . Chalcopyrite concentrate supported a simpler and different community than Fe2+ . The members of the enrichment cultures were closely related to Sulfobacillus yellowstonensis and Sulfobacillus acidophilus. J Biol Chem, 2004 Oct 22, 279(43), 44704 - 12 Epub 2004 Aug 12. Mechanism-based fluorescent labeling of beta-galactosidases . An efficient method in proteomics for glycoside hydrolases; Kurogochi M et al.; (4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S . T., and Landry, D . Glycobiology (1995) 5, 19-28 and Taron, C . H., Benner, J . S., Hornstra, L . J., and Guthrie, E . P . (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R . H., Zhang, X . J., DuBose, R . F., and Matthews, B . W . (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K . (1988) Glycoconj . J . 15, 155-160) for the rapid identification of the catalytic site . Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) . Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases . It was demonstrated by using X . manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group . This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T . (2002) J . Mol . Biol . 322, 79-91) . A catalytic glutamic acid (Glu-537) of E . coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity . Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B . circulans beta-galactosidase, indicating that Glu-259 is one of the nucleophiles in the active site . The present method can be readily extended to other glycosidases and should greatly aid the high throughput proteomics of many glycoside hydrolases showing both retaining- and inverting-type mechanisms. Rocz Panstw Zakl Hig, 2004, 55(1), 83 - 7 {Ability of the strains of the thermophilic fungus Thermomyces lanuginosus to hydrolyze cocoa fat and lard}; Janda K; The purpose of the study was the estimation of the lipolytic activity of the thermophilic fungus Thermomyces lanuginosus on the solid base with the cocoa fat and the lard . The material was 144 strains isolated from biohumus, garden compost, leaf compost, mushroom compost, hazelnuts and raw coffee beans . The study proved, that all species was able to hydrolyze both the cocoa oil and the lard . The index of the lipolytic activity was the same on the medium with cocoa oil and on the medium with the lard. Protist, 2004 Jun, 155(2), 157 - 68 Biochemical characterization and quantitative gene expression analysis of the multi-stress inducible metallothionein from Tetrahymena thermophila; Dondero F et al.; A cadmium-binding protein with biochemical features of a metallothionein (MT) has been isolated and purified to homogeneity from the ciliate Tetrahymena thermophila . N-terminal sequencing revealed the posttranslational cleavage of the first two amino acids and, in general, a high degree of identity with known MTs from other ciliates . Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis of the apothionein revealed a molecular mass (16,763 Da) higher to those of mammals and of other protozoa . Finally, quantitative real-time PCR has been used to investigate the susceptibility of this ciliate MT to gene activation in response to heavy metals and to other stressors . Our data indicate that while zinc is not effective at all and cadmium is the best inducer, other stress factors, such as mercury, copper, heat and hydrogen peroxide, also activated gene transcription . As in vertebrate cells, interleukin-6 (IL-6) that stimulates ciliate growth, was able to enhance MT gene synthesis . This complex of data seems to indicate a general role of this protein in stress response. Appl Biochem Biotechnol, 1999 Jul, 81(1), 53 - 66 Purification and Characterization of Thermostable D-Hydantoinase from Bacillus thermocatenulatus GH-2; Park JH et al.; A thermostable D-hydantoinase was isolated from thermophilic Bacillus thermocatenulatus GH-2 and purified to homogeneity by using immunoaffinity chromatography . The molecular mass of the enzyme was determined to be about 230 kDa, and a value of 56 kDa was obtained as a molecular mass of the subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that oligomeric structure of the enzyme is tetrameric . Isoelectric pH of the enzyme was found to be approx 4.3 . The enzyme required Mn2+ for the activity and exhibited its highest activity with phenylhydantoin as a substrate . The optimal pH and temperature for catalytic activity were about 7.5 and 65 degrees C, respectively . The half-life of the enzyme was estimated to be about 45 min at 80 degrees C. Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 205 - 14 Thermostable phytase production by Thermoascus aurantiacus in submerged fermentation; Nampoothiri KM et al.; Phytases act on phytic acid, an antinutrient factor present in animal feeds, and release inorganic phosphate . We optimized the production parameters for phytase production using Thermoascus aurantiacus (TUB F 43), a thermophilic fungal culture, by submerged fermentation . A semisynthetic medium containing glucose, starch, peptone, and minerals supplemented with 3.75% (w/v) wheat bran particles was found to be the best production medium among the various combinations tried . Further supplementation of this medium with surfactants such as Tween-20 and Tween-80 considerably enhanced the enzyme yield . A maximum phytase activity (468.22 U/mL) was obtained using this production medium containing 2% (v/v) Tween-20 after 72 h of fermentation at 45 degrees C in shake-flask cultures with a rotation of 150 rpm . Herein we present details of a few of the process parameter optimizations . The phytase enzyme was found to be thermostable, and the optimal temperature for phytase activity was found to be 55 degrees C . However, 80% of the activity still remained when the temperature was shifted to 70 degrees C. Appl Biochem Biotechnol, 1999 Spring, 77-79, 671 - 80 Bioconversion of acrylonitrile to acrylamide using a thermostable nitrile hydratase; Padmakumar R et al.; Although providing an attractive route for production of acrylamide from acrylonitrile, utilization of nitrile hydratase enzymes has been limited by the requirement for low temperature bioconversion conditions . This report summarizes a search for thermostable nitrile hydratases from aerobic moderate thermophiles screened for ability to grow on acrylonitrile at concentrations to 1% at elevated temperatures . A new isolate Bacillus sp . BR449 constitutively expresses a thermostable nitrile hydratase with properties including low substrate inhibition and broad temperature range with optimal activity at 55 degrees C . With prolonged exposure, BR449 nitrile hydratase exhibited temperature- dependent inactivation by acrylonitrile, which is attributed to alkylation of nucleophilic sites on the enzyme. Nucleic Acids Res, 2004 Aug 10, 32(14), 4313 - 21 Print 2004. Transcriptional control of RAD51 expression in the ciliate Tetrahymena thermophila; Smith JJ et al.; The expression of Rad51p, a DNA repair protein that mediates homologous recombination, is induced by DNA damage and during both meiosis and exconjugant development in the ciliate Tetrahymena thermophila . To completely investigate the transcriptional regulation of Tetrahymena RAD51 expression, reporter genes consisting of the RAD51 5' non-translated sequence (5' NTS) positioned upstream of either the firefly luciferase or green fluorescent protein coding sequences have been targeted for recombination at the macronuclear btu1-1 (K350M) locus of T . thermophila strain CU522 . Expression from RAD51-luciferase reporter constructs has been directly quantified from transformant whole cell lysates . Luciferase is induced to maximum levels in transformants harboring the full-length RAD51-luciferase reporter gene following exposure to DNA damaging UV irradiation . A series of truncations, deletions, insertions, substitutions and inversions of the RAD51 5' NTS have led to the identification of three distinct transcriptional promoter elements . The first of these sequence elements is required for basal levels of transcription . The second modulates expression in the absence of DNA damage, whereas the third ensures increased RAD51 transcription in response to DNA damage and during meiosis . Tetrahymena RAD51 is tightly regulated through these transcriptional elements to produce the appropriate expression during conjugation, and in response to DNA damage. Water Sci Technol, 2004, 49(11-12), 269 - 75 The fate of legionellae within distribution pipe biofilms: measurement of their persistence, inactivation and detachment; Storey MV et al.; Distribution pipe biofilms present a currently unquantified public health risk to consumers receiving water for domestic potable and non-potable use . The aim of this study was to quantify the numbers of legionellae, used here as model bacterial pathogens, that may accumulate, persist within and detach from distribution pipe biofilms . L . pneumophila recovered by standard culture from an 8 week-old biofilm formed within a novel pilot-scale water distribution system represented 1% of those present in the adjacent bulk water . A combined chlorine concentration exceeding 0.2 mg x L(-1) eliminated culturable sessile legionellae altogether, though the reduction in FISH-positive cells represented just 75+/-25% of the original amount, compared to a 5-log reduction in culturable cells during the same period . Where there was < 0.1 mg x L(-1) combined chlorine, an exponential decay/loss of sessile L . pneumophila was observed (k = 0.37 - 0.41) over the course of a 38-day experimental period . The inoculation of the system with 1 microm fluorescent microspheres and legionellae demonstrated that removal of the latter was dominated by chemical disinfection, with erosion and biological grazing playing lesser roles . Under turbulent (Re approximately 5000) conditions, larger clusters of biofilm become detached from substrata, with more than 90% of sessile legionellae mobilised into the bulk water phase . Interaction with both biofilms and a thermophilic Acanthamoeba isolate reduced the susceptibility of legionellae to thermal inactivation by between one and two orders of magnitude, though it increased their sensitivity to chemical (free and combined chlorine) disinfection. Proc Natl Acad Sci U S A, 2004 Aug 17, 101(33), 12159 - 64 Epub 2004 Aug 09. Thermophilic ATP synthase has a decamer c-ring: indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling; Mitome N et al.; In a rotary motor F(o)F(1)-ATP synthase that couples H(+) transport with ATP synthesis/hydrolysis, it is thought that an F(o)c subunit oligomer ring (c-ring) in the membrane rotates as protons pass through F(o) and a 120 degrees rotation produces one ATP at F(1) . Despite several structural studies, the copy number of F(o)c subunits in the c-ring has not been determined for any functional F(o)F(1) . Here, we have generated and isolated thermophilic Bacillus F(o)F(1), each containing genetically fused 2-mer-14-mer c (c(2)-c(14)) . Among them, F(o)F(1) containing c(2), c(5), or c(10) showed ATP-synthesis and other activities . When F(1) was removed, F(o) containing c(10) worked as an H(+) channel but F(o)s containing c(9), c(11) or c(12) did not . Thus, the c-ring of functional F(o)F(1) of this organism is a decamer . The inevitable consequence of this finding is noninteger ratios of rotation step sizes of F(1)/F(o) (120 degrees /36 degrees ) and of H(+)/ATP (10:3) . This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by H(+) transport at F(o) and elementary events in catalysis at F(1). J Investig Allergol Clin Immunol, 2004, 14(2), 165 - 7 Hypersensitivity pneumonitis due to occupational inhalation of fungi-contaminated corn dust; Moreno-Ancillol A et al.; Hypersensitivity pneumonitis or extrinsic allergic alveolitis can be defined as a lung disease caused by a wide group of antigens that reach the lung by inhalation of organic and/or inorganic dust of various sources . The dust of the stored maize corn has been reported as cause of respiratory symptoms . During the storage process, maize corn dust can be contaminated by moulds and thermophilic actinomycetes, which have not been described until now as the causing antigens of these symptoms . We present a case of occupational hypersensitivity pneumonitis in an agricultural worker who cultured and stored maize corn . Clinical findings, precipitating antibodies, and evolution after having removed him from his work, confirmed the diagnosis . In our case, Aspergillus species contaminating the maize corn dust are probably the antigens that caused the disease. Acta Crystallogr D Biol Crystallogr, 1997 Mar, 53(Pt 2), 195 - 6 Preliminary X-ray diffraction studies on asparaginyl-tRNA synthetase from Thermus thermophilus; Berthet-Colominas C; The recombinant asparginyl-tRNA synthetase from the thermophilic bacterium Thermus thermophilus expressed in Escherichia coli has been crystallized from PEG 6000 solutions . Depending on the PEG concentrations the crystals were in either tetragonal or hexagonal space groups . Although generally smaller, the latter (space group P6(4)22) diffracted better, to a resolution of 2.8 A . Using the coordinates of the yeast aspartyl-tRNA synthetase structure molecular replacement methods were applied to both tetragonal and hexagonal crystals; a solution was found which gave excellent crystal packing in both space groups. Acta Crystallogr D Biol Crystallogr, 1997 Sep, 53(Pt 5), 608 - 11 Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6; Teplitsky A; The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial . The xylanase gene was cloned and sequenced . The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43 808 and pI of 9.0 . Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis . This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme . The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive . The crystals belong to a primitive trigonal crystal system (space group P3(1) or P3(2)) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A . At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A . These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution . The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays . A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation . Two derivatives of XT-6 were recently prepared . In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site . The second derivative was selenomethionyl xylanase which was produced biosynthetically . These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions. Acta Crystallogr D Biol Crystallogr, 1997 Sep, 53(Pt 5), 599 - 604 Crystallization and preliminary X-ray analysis of the major endoglucanase from Thermoascus aurantiacus; Lo Leggio L; The major endoglucanase (35 kDa) from the thermophilic fungus Thermoascus aurantiacus has been purified from culture filtrates using an affinity method and the sequence for 35 N-terminal amino acids determined . This has allowed assignment of the enzyme to subtype A6 of family 5 endoglucanases . The enzyme has been crystallized as thick plates by the hanging-drop method using ammonium sulfate as precipitant . The crystals belong to space group P2(1)2(1)2(1) with cell edges a = 76.4, b = 85.7 and c = 89.5 A, with two molecules in the asymmetric unit, and diffract to 1.62 A resolution using synchrotron radiation . The structure will be solved by isomorphous replacement. Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 840 - 1 Crystallization and preliminary X-ray diffraction studies of arginase from a thermophilic organism Bacillus caldevelox; Smith CA; A thermostable hexameric arginase purified from the extreme thermophile Bacillus caldevelox has been crystallized from Hepes buffer at pH 7.5 in the presence of 12% polyethylene glycol 4000 and 10% 2-propanol, and from cacodylate buffer at pH 7.2 in the presence of 15% 2-propanol and sodium citrate . The latter crystals are more suitable for X-ray diffraction analysis . The crystals are in the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions a = 156.3, b = 148.0 and c = 85.4 A . The asymmetric unit contains one hexamer (approximate molecular mass 183 kDa) and has a solvent content of approximately 54% . The crystals diffract to 2.8 A resolution. Acta Crystallogr D Biol Crystallogr, 1996 Jan, 52(Pt 1), 124 - 8 Structure of a loop-deleted variant of 3-isopropylmalate dehydrogenase from Thermus thermophilus: an internal reprieve tolerance mechanism; Sakurai M; A loop-deleted mutant form of 3-isopropylmalate dehydrogenase from Thermus thermophilus was constructed to investigate the relationship between the flexibility of the structure and the thermostability of the enzyme . The structure of the mutant enzyme was determined by X-ray crystallography and was found to be almost the same as that of the native enzyme with a reduced temperature factor . Although the mutant protein had lost the flexible loop, its function and thermostability had remained unchanged . This phenomenon can be explained by an internal reprieve tolerance mechanism. Acta Crystallogr D Biol Crystallogr, 1996 Jul, 52(Pt 4), 882 - 6 Crystalline alcohol dehydrogenases from the mesophilic bacterium Clostridium beijerinckii and the thermophilic bacterium Thermoanaerobium brockii: preparation, characterization and molecular symmetry; Korkhin Y; Two tetrameric NADP(+)-dependent bacterial secondary alcohol dehydrogenases have been crystallized in the apo- and the holo-enzyme forms . Crystals of the holo-enzyme from the mesophilic Clostridium beijerinckii (NCBAD) belong to space group P2(1)2(1)2(1) with unit-cell dimensions a = 90.5, b = 127.9, c = 151.4 A . Crystals of the apo-enzyme (CBAD) belong to the same space group with unit-cell dimensions a = 80.4, b = 102.3, c = 193.5 A . Crystals of the holo-enzyme from the thermophilic Thermoanaerobium brockii (NTBAD) belong to space group P6(1(5)) (a = b = 80.6, c = 400.7 A) . Crystals of the apo-form of TBAD (point mutant GI98D) belong to space group P2(1) with cell dimensions a = 123.0, b = 84.8, c = 160.4 A beta = 99.5 degrees . Crystals of CBAD, NCBAD and NTBAD contain one tetramer per asymmetric unit . They diffract to 2.0 A resolution at liquid nitrogen temperature . Crystals of TBAD(GI98D) have two tetramers per asymmetric unit and diffract to 2.7 A at 276 K . Self-rotation analysis shows that both enzymes are tetramers of 222 symmetry. Acta Crystallogr D Biol Crystallogr, 1996 Jul, 52(Pt 4), 623 - 30 Cryocrystallography of 3-Isopropylmalate Dehydrogenase from Thermus thermophilus and its Chimeric Enzyme; Nagata C; The crystal structures of thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus (10T) and a chimeric enzyme between T . thermophilus and Bacillus subtilus with one point mutation (cS82R), were determined at both 100 and 150 K . At the cryogenic condition, the volume of the unit cell decreased by 5% as a result of a contraction in the solvent region . Although the overall structures of both enzymes at low temperature were the same as that of 10T at room temperature, interactions between two domains and between two subunits in a functional dimer of cS82R were significantly altered . The decrease in the average temperature factor of 10T at low temperature and no significant decrease for cS82R suggested that the structure of the engineered enzyme (cS82R) may have many conformational substates even at low temperature, while the native enzyme (10T) at low temperature has a more definite conformation than that at room temperature . The location of water molecules around the enzyme molecule and the calculation of the radii of gyration suggested that cS82R had a weaker hydration than 10T. Acta Crystallogr D Biol Crystallogr, 1996 Sep, 52(Pt 5), 1030 - 2 Crystallization and preliminary X-ray analysis of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans; Tsuchiya D; Three crystalline forms of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans were obtained by hanging-drop vapor-diffusion methods . One of them, which had crystallized under slightly milder conditions than the others, was suitable for X-ray analysis . Its asymmetric unit contains one dimeric molecule and the solvent content is higher than in other protein crystals . The crystal structure was solved in a preliminary manner by the molecular-replacement technique. Acta Crystallogr D Biol Crystallogr, 1996 Nov, 52(Pt 6), 1224 - 5 Crystallization and preliminary crystallographic analysis of two beta-mannanase isoforms from Thermomonospora fusca KW3; Hilge M; Three beta-mannanase isoforms were isolated from the supernatant of a thermophilic actinomycete culture from Thermomonospora fusca KW3 . Two of the isoforms (Q1, Q 1.1) were crystallized by the hanging-drop method at room temperature using ammonium sulfate as a precipitant . The isoforms form rod-shaped colorless crystals . Both belong to the orthorhombic space group P2(1)2(1)2(1) . The cell dimensions are a = 46.7, b = 61.1, and c = 128.2 A for isoform Q1, and a = 43.8, b = 46.2, and c = 132.8 A for isoform Q1.1 . The asymmetric unit of either isoform contains one mannanase molecule . Native data have been collected to 2.2 A resolution for Q1 and to 1.65 A resolution for Q1.1 using synchrotron radiation. Acta Crystallogr D Biol Crystallogr, 1996 Nov, 52(Pt 6), 1092 - 7 Approaches to Very Low Resolution Phasing of the Ribosome 50S Particle from Thermus thermophilus by the Few-Atoms-Models and Molecular-Replacement Methods; Urzhumtsev AG; Estimates for the phases of the X-ray diffraction data from the 50S ribosomal particle of Thermus thermophilus has been made to an effective resolution around 80 A using the few-atoms-modes ab initio technique {Lunin, Lunina, Petrova, Vernoslova, Urzhumtsev & Podjarny (1995) . Acta Cryst . D51, 896-903} . This technique models the density with a small number of Gaussian spheres to generate a large number of possible phase sets and then uses clustering algorithms to identify the best ones . Independently, an envelope obtained from electron-micrograph image reconstruction {Yonath, Leonard & Wittmann (1987) . Science, 236, 813-816} was oriented and positioned using the molecular-replacement technique, specially adapted to the very low resolution case {Urzhumtsev & Podjarny (1995) . Acta Cryst . D51, 888-895} . The two methods show similar packing arrangements.The electron density calculated by the few-atoms-models technique without any assumption on the number of molecules in asymmetric unit or on their shape shows recognizable features of the particle. Acta Crystallogr D Biol Crystallogr, 1994 Sep, 50(Pt 5), 744 - 8 Preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus; Tanner J; Crystals have been obtained of glyceraldehyde 3-phosphate dehydrogenase from the extreme thermophile, Thermus aquaticus . This enzyme is stable and active at 363 K, thus its three-dimensional structure should add insight into the structural basis of protein thermostability . Large high-quality crystals were grown using isopropanol and polyethylene glycol at pH 8.4 . They crystallize in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 144.77 (6), b = 148.77 (5), c = 149.50 (7) A, and diffract to beyond 2.8 A . The volume of the unit cell and the packing observed in other GAPDH structures suggest that there are two tetramers per asymmetric unit . With 300 kDa/asymmetric unit expected in this form, its solution represents a challenging molecular replacement problem . A low-resolution data set has been recorded and used to carry out self-rotation, cross-rotation and Patterson-correlation refinement calculations . We found that the Q molecular axes of both tetramers are approximately coincident with the crystallographic a axis, and the non-crystallographic symmetry relating the two tetramers is approximately a rotation of 90 degrees about the a axis. Curr Microbiol, 2004 Jul, 49(1), 28 - 31 Ca2+ dependence and inhibitory effects of trifluoperazine on plasma membrane ATPase of Thermoactinomyces vulgaris; Bhatnagar K et al.; Ca2+ enhanced the plasma membrane Ca(2+)-ATPase (PMCA) specific activities in wild-type strain 1227 and mutant strains 1278, 1286, and 1261 of Thermoactinomyces vulgaris . The Ca(2+)-ATPase specific activities showed marked increase with increasing concentrations of Ca2+ added in the form of CaCl2 in the culture medium and reached the optimum values at 0.6 mM in strains 1227, 1278, and 1286 and at 0.7 mM in strain 1261 of T . vulgaris . Trifluoperazine, a specific blocker of calmodulin, when added in vivo at concentrations of 2 microM and 8 microM along with the respective optimal concentrations of Ca2+, decreased the PMCA-specific activities to a low level in a dose-dependent manner . The results of the present investigation suggest the presence of a Ca(2+)-dependent protein activator (CaDPA) in the microenvironment constituting this enzyme; and such Ca(2+)-modulated protein has been assigned to play an important role in the enhancement of PMCA levels in this aerobic, spore-forming, thermophilic actinomycete. Am J Physiol Gastrointest Liver Physiol, 2005 Jan, 288(1), G151 - 8 Epub 2004 Aug 05. Accelerated intestinal transit in inbred mice with an increased number of interstitial cells of Cajal; Bellier S et al.; The interstitial cells of Cajal (ICC) play an important role in coordinating intestinal motility, and structural alterations in ICC are found in several human digestive diseases . Mouse models with defects in ICC allow a better understanding of their functions . We investigated the pattern of intestinal motility and the distribution of ICC in the PRM/Alf inbred mouse strain, characterized by a selective intestinal lengthening . In PRM/Alf mice, the digestive transit time, evaluated by using thermophilic Bacillus subtilis spores, was normal, indicating accelerated transit . The contractility and slow-wave frequency, recorded on isolated segments from the proximal small intestine, were significantly increased . The number of ICC was also significantly higher along the small intestine and the colon . The concomitant increase of the contractility, the slow-wave frequency, and the number of ICC is consistent with the proposal of a role of ICC number increase in the higher intestinal transit speed . The PRM/Alf model should be useful to further investigate the roles of ICC in the control of digestive motility. Clin Nutr, 2004 Aug, 23(4), 467 - 75 Influence of synbiotic containing Lactobacillus acidophilus La5, Bifidobacterium lactis Bb 12, Streptococcus thermophilus, Lactobacillus bulgaricus and oligofructose on gut barrier function and sepsis in critically ill patients: a randomised controlled trial; Jain PK et al.; BACKGROUND & AIMS: Infective complications are a common cause of mortality and morbidity in critically ill patients . Many factors affect sepsis, one of which is gut barrier function . The aim of this study was to determine whether the oral administration of a synbiotic preparation could alter gut barrier function in critically ill patients and thus reduce sepsis . METHODS: A total of 90 patients admitted to an intensive care unit (ICU) were randomised to receive either synbiotic or placebo preparations (45 into each group) . The synbiotic preparation consisted of Lactobacillus acidophilus La5, Bifidobacterium lactis Bb 12, Streptococcus thermophilus and Lactobacillus bulgaricus (probiotics) with oligofructose (prebiotic) . Gut barrier function was assessed by measurement of intestinal permeability (lactulose/rhamnose test) and culture of nasogastric aspirate on days 1 and 8 . All septic complications and mortality were recorded . RESULTS: There were no differences between the groups in terms of age, sex, APACHE II or POSSUM scores . After 1 week of therapy, patients in the synbiotic group had a significantly lower incidence of potentially pathogenic bacteria (43% versus 75%, P = 0.05) and multiple organisms (39% versus 75%, P = 0.01) in their nasogastric aspirates than controls . There were no significant differences between the groups in terms of intestinal permeability, septic complications or mortality . CONCLUSIONS: The administration of synbiotic in critically ill patients favourably altered the microbial composition of the upper gastrointestinal tract but had no effect on intestinal permeability and was not associated with measurable clinical benefit . Structure (Camb), 2004 Aug, 12(8), 1481 - 8 Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1; Bartlam M et al.; Acylpeptide hydrolases (APH; also known as acylamino acid releasing enzyme) catalyze the removal of an N-acylated amino acid from blocked peptides . The crystal structure of an APH from the thermophilic archaeon Aeropyrum pernix K1 to 2.1 A resolution confirms it to be a member of the prolyl oligopeptidase family of serine proteases . The structure of apAPH is a symmetric homodimer with each subunit comprised of two domains . The N-terminal domain is a regular seven-bladed beta-propeller, while the C-terminal domain has a canonical alpha/beta hydrolase fold and includes the active site and a conserved Ser445-Asp524-His556 catalytic triad . The complex structure of apAPH with an organophosphorus substrate, p-nitrophenyl phosphate, has also been determined . The complex structure unambiguously maps out the substrate binding pocket and provides a basis for substrate recognition by apAPH . A conserved mechanism for protein degradation from archaea to mammals is suggested by the structural features of apAPH. Structure (Camb), 2004 Aug, 12(8), 1471 - 80 Crystal structure of the native chaperonin complex from Thermus thermophilus revealed unexpected asymmetry at the cis-cavity; Shimamura T et al.; The chaperonins GroEL and GroES are essential mediators of protein folding . GroEL binds nonnative protein, ATP, and GroES, generating a ternary complex in which protein folding occurs within the cavity capped by GroES (cis-cavity) . We determined the crystal structure of the native GroEL-GroES-ADP homolog from Thermus thermophilus, with substrate proteins in the cis-cavity, at 2.8 A resolution . Twenty-four in vivo substrate proteins within the cis-cavity were identified from the crystals . The structure around the cis-cavity, which encapsulates substrate proteins, shows significant differences from that observed for the substrate-free Escherichia coli GroEL-GroES complex . The apical domain around the cis-cavity of the Thermus GroEL-GroES complex exhibits a large deviation from the 7-fold symmetry . As a result, the GroEL-GroES interface differs considerably from the previously reported E . coli GroEL-GroES complex, including a previously unknown contact between GroEL and GroES. Structure (Camb), 2004 Aug, 12(8), 1413 - 23 Crystal structures of CTP synthetase reveal ATP, UTP, and glutamine binding sites; Goto M et al.; CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP . Glutamine hydrolysis is active in the presence of ATP and UTP and is stimulated by the addition of GTP . We report the crystal structures of Thermus thermophilus HB8 CTPs alone, CTPs with 3SO4(2-), and CTPs with glutamine . The enzyme is folded into a homotetramer with a cross-shaped structure . Based on the binding mode of sulfate anions to the synthetase site, ATP and UTP are computer modeled into CTPs with a geometry favorable for the reaction . Glutamine bound to the glutaminase domain is situated next to the triad of Glu-His-Cys as a catalyst and a water molecule . Structural information provides an insight into the conformational changes associated with the binding of ATP and UTP and the formation of the GTP binding site. Appl Environ Microbiol, 2004 Aug, 70(8), 5041 - 6 Temporal transcription map of the virulent Streptococcus thermophilus bacteriophage Sfi19; Ventura M et al.; A transcription map was developed for the virulent Streptococcus thermophilus phage Sfi19 on the basis of systematic Northern blot hybridizations . All deduced 5' ends were confirmed by primer extension experiments . Three classes of transcripts were detected based on the different times of appearance . Early transcripts were identified in three genome regions; middle transcripts covered cro-like, DNA replication, and transcriptional regulation genes; and late genes consisted of structural and lysis genes . Chloramphenicol treatment suppressed the translation of a putative transcriptional factor necessary for the production of late transcripts and shifted middle transcripts to early transcription times. Appl Environ Microbiol, 2004 Aug, 70(8), 4702 - 10 Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity; Bruno-Barcena JM et al.; In living organisms, exposure to oxygen provokes oxidative stress . A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases . Generally, these enzymes are not present in Lactobacillus spp . In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli . Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli . A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs . The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932 . Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli . The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L . gasseri, L . johnsonii, L . acidophilus, and L . reuteri, respectively . The expression of S . thermophilus MnSOD in L . gasseri and L . acidophilus provided protection against hydrogen peroxide stress . The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron . To our best knowledge, this is the first report of a sodA from S . thermophilus being expressed in other lactic acid bacteria. Appl Environ Microbiol, 2004 Aug, 70(8), 4642 - 7 Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag; Moldes C et al.; The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin . The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography . Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag . Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix . The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins . Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper . The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications. Appl Environ Microbiol, 2004 Aug, 70(8), 4596 - 603 Characterization of a galactokinase-positive recombinant strain of Streptococcus thermophilus; Vaillancourt K et al.; The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid . Unlike the phylogenetically related species Streptococcus salivarius, S . thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose . This metabolic property may result from the inability of S . thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate . In this work, we report the complementation of Gal(-) strain S . thermophilus SMQ-301 with S . salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01 . The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S . salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose . Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S . salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons . Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05% . Thus, unlike S . salivarius, the ability to metabolize galactose was not sufficient for S . thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose . Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal(+) strain grew and produced acid more rapidly than the Gal(-) wild-type strain. Mol Cell Probes, 2004 Oct, 18(5), 321 - 7 A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter; Perelle S et al.; In a previous study, we reported the performance of a PCR assay amplifying 287-bp of the 16S rRNA gene of thermo-tolerant Campylobacter (C . jejuni, C . lari, C . coli) through an international ring-trial involving 12 participating laboratories . Based on the validated set of primers, a LightCycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed . The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications . The specificity study involving 39 Campylobacter and nine strains of other species indicated that the LC-PCR test was highly specific, giving cross-reactivity with only one strain of C . upsaliensis (CCUG19559) . The sensitivity of the LC-PCR assay, evaluated in 32 spiked poultry-rinse or pork carcass-swab samples, was determined at 10CFU/ml carcass-rinse . The prevalence of samples positive for thermo-tolerant Campylobacter was 58.8% in 68 naturally contaminated poultry rinse samples tested by LC-PCR and the data were in good concordance with those of bacteriological method . The Ct values of the three replicates obtained for each sample tested in three different runs demonstrate that the LC-PCR was highly reproducible and afford a powerful tool for rapid detection of the thermo-tolerant Campylobacter strains. Mol Biol Rep, 2004 Jun, 31(2), 139 - 42 Bsu2413I and Bfi2411I, two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification . Thermophilic endonucleases from two Bacillus species; Jutur PP et al.; Two new thermophilic type II restriction endonucleases, which we designated as Bsu2413I and Bfi2411I, have been isolated from gram-positive thermophilic bacteria Bacillus subtilis strain 2413 and Bacillus firmus strain 2411 respectively and partially purified . The restriction endonucleases were extracted from cell extracts and purified using single step purification through phosphocellulose column chromatography . SDS-PAGE profile showed denatured molecular weights of 33 and 67 kDa for the Bsu2413I and 39 and 67 kDa for the Bfi2411I . The partially purified Bsu2413I enzyme restricted pBR322 DNA into two fragments of 3250 and 1100 bp whereas Bfi2411I enzyme restricted pBR322 DNA into two fragments of 3500 and 800 bp . The activity of both endonucleases was assayed at 55 degrees C and they required Mg+2 as cofactor like other type II restriction endonucleases. J Biol Chem, 2004 Oct 29, 279(44), 45369 - 78 Epub 2004 Jul 28. A new type of NADH dehydrogenase specific for nitrate respiration in the extreme thermophile Thermus thermophilus; Cava F et al.; A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor . Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs) . The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank . Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants . A fusion to a thermophilic reporter gene was used for the first time in Thermus spp . to show that expression of nrc required the presence of nitrate and anoxic conditions . Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced . Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen . The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system . Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins . Interactions were also detected between NrcN and NrcF . Inactivation of nrcF produced solubilization of NrcN, but not of NrcD . These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH. J Bacteriol, 2004 Aug, 186(16), 5400 - 9 Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway; Xu H et al.; Three leuA-like protein-coding sequences were identified in Leptospira interrogans . One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-) . The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12) . Expressed in Escherichia coli, the citramalate synthase was purified and characterized . Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate . Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L . interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate . The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction . This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E . coli extracts bearing the corresponding clones and in vivo by complementation of E . coli ilvA, leuC/D, and leuB mutants . Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L . interrogans under physiological conditions was unequivocally proven . Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L . interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium . The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes. Extremophiles . 2004 Jul 30; {Epub ahead of print} A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus; Li DC et al.; A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100 . The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each . The SOD was found to be inhibited by NaN(3), but not by KCN or H(2)O(2), suggesting that the SOD in T . lanuginosus was of the manganese superoxide dismutase type . The SOD exhibited maximal activity at pH 7.5 . The optimum temperature for the activity was 55 degrees C . It was thermostable at 50 and 60 degrees C and retained 55% activity after 60 min at 70 degrees C . The half-life of the SOD at 80 degrees C was approximately 28 min and even retained 20% activity after 20 min at 90 degrees C. Biotechnol Lett, 2004 Aug, 26(15), 1181 - 9 Degradation of microbial polyesters; Tokiwa Y et al.; Microbial polyhydroxyalkanoates (PHAs), one of the largest groups of thermoplastic polyesters are receiving much attention as biodegradable substitutes for non-degradable plastics . Poly(D-3-hydroxybutyrate) (PHB) is the most ubiquitous and most intensively studied PHA . Microorganisms degrading these polyesters are widely distributed in various environments . Although various PHB-degrading microorganisms and PHB depolymerases have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications . Distributions of PHB-degrading microorganisms, factors affecting the biodegradability of PHB, and microbial and enzymatic degradation of PHB are discussed in this review . We also propose an application of a new isolated, thermophilic PHB-degrading microorganism, Streptomyces strain MG, for producing pure monomers of PHA and useful chemicals, including D-3-hydroxycarboxylic acids such as D-3-hydroxybutyric acid, by enzymatic degradation of PHB. Bioresour Technol, 2004 Dec, 95(3), 319 - 26 Performance characteristics of three aeration systems in the swine manure composting; Zhu N et al.; Pilot composting of swine manure mixed with rice straw was carried out to evaluate performance characteristics of three aeration systems: forced aeration, passive aeration and natural aeration . It was expected to provide academic basis for farmers to select an advisable aeration system . The results showed that the thermophilic durations were long enough to satisfy the sanitary standard, and swine manure could reach maturity . The indexes of the composting, including physical changes, pH value, TOC, OM, TKN, WSC, WSN, solid C/N ratio, water-soluble C/N ratio, TOM, NH4+-N, (NO3(-) + NO2(-))-N, and GI had no significant difference among the treatments (P > 0.05) except the average temperature profiles (P12 = 0.001, P13 = 0.036) . Economic analysis showed that a passive aeration system was suitable for a small-scale swine farm, and forced aeration system should be considered to apply in the middle and large-scale swine farms with a high extent of industrialization . But, in order to avoid too high temperature occurring during composting, an active aeration control system needed to be developed. Bioresour Technol, 2004 Dec, 95(3), 245 - 54 Aerobic thermophilic treatment of farm slurry and food wastes; Mohaibes M et al.; The review discusses the aerobic treatments for farm slurry and food wastes and concentrates in particular on the thermophilic aerobic treatments . Methods are discussed under the heading of chemical, physical and other treatments . From those methods considered, the most suitable physical-microbiological treatment are aerobic thermophilic treatments . The main problem faced in aerobic thermophilic treatments could be the foaming formation during the process, and this could be solved by using different methods, mainly mechanical control method . Aerobic thermophilic treatments are also simple, economical and environmentally accepted . This method is known to have effects, and could be used to assist decontaminations on farms, as such technologies are already used in routine slurry treatment in many farms. Bioresour Technol, 2004 Dec, 95(3), 235 - 44 Greenhouse gas emission during storage of pig manure on a pilot scale; Wolter M et al.; The greenhouse gas emissions (CO2, CH4, N2O) from a 2 ton (4.4 m3) deep litter pig manure pile (storage time 113 days during winter season) were quantified by using a tent, which covered the whole pile during the measuring periods only . The emissions were calculated in CO2 equivalents per kilogram dry matter by . Additionally the retention time (use of tracer gas SF6) and the concentrations of the gases in different parts of the pile were determined . The average retention time of the gases in the pile was less than 2 h . CH4 is assumed to have been generated only in the centre of the pile, whereas CO2 was assumed to have been generated in a wider zone . The emissions of CH4, CO2 and N2O were at the highest in the beginning when nearly the whole pile had temperatures in the range of thermophilic microorganisms . This leads to the assumption that mainly thermophilic microorganisms formed the gases . The most important gas for global warming was found to be nitrous oxide. J Appl Microbiol, 2004, 97(3), 512 - 9 Exopolysaccharides produced by mixed culture of yeast Rhodotorula rubra GED10 and yogurt bacteria (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11); Simova ED et al.; AIMs: The studies of the production of exopolysaccharides by lactose-negative yeast and a yogurt starter co-cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high-efficiency synthesis of biopolymers by mixed cultivation . METHODS AND RESULTS: The mixed culture Rhodotorula rubra GED10 + (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11) was cultivated in cheese whey ultrafiltrate (WU) (44.0 g lactose l(-1)) at initial pH 6.0, 28 degrees C, under intensive aeration (air-flow rate 1.0 l l(-1) min(-1), agitation 220 rev min(-1)) in a MBR AG fermentor . The mixed culture manifested the highest activity for synthesis of exopolysaccharides (19.3 g l(-1)) and cell mass (21.0 g l(-1)) at the 84th hour . The yogurt starter synthesized neutral exopolysaccharides, while the mixed culture yeast + yogurt starter produced acidic exopolysaccharides containing uronic acid (6%) . The neutral sugar composition was identified as mannose, glucose, galactose, xylose and arabinose . Mannose dominated in the polymer composition (83%) that was produced only by the yeast (97%) . CONCLUSIONS: Lactose in the WU can be effectively utilized by a co-culture of lactose-negative yeast-yogurt starter for synthesis of exopolysaccharides . SIGNIFICANCE AND IMPACT OF THE STUDY: The present findings propose an alternative use of WU as a cost-effective carbohydrate substrate, and suggest that the lactose-negative yeast Rhodotorula rubra can have industrial application as producers of exopolysaccharides . Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1239 - 42 Anoxybacillus voinovskiensis sp . nov., a moderately thermophilic bacterium from a hot spring in Kamchatka; Yumoto I et al.; A novel moderately thermophilic bacterium, strain TH13T, was isolated from a hot spring in Kamchatka . It was found to be a Gram-positive, facultative aerobe; the straight, non-motile rods grew at 30-64 degrees C (optimum 54 degrees C) . The isolate was positive for catalase and oxidase tests and reduced nitrate to nitrite, but was negative for H2S production and growth in more than 3% NaCl (w/v) . The isolate grew at pH 7-8, but not at pH values higher than 9 . The DNA G+C content was 43.9 mol% . Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain TH13T was a member of the genus Anoxybacillus . DNA-DNA hybridization revealed a low relatedness (less than 30.2%) between the isolate and its close phylogenetic neighbours Anoxybacillus pushchinoensis and Anoxybacillus flavithermus . On the basis of phenotypic characteristics, phylogenetic data and DNA-DNA hybridization data, it was concluded that the isolate merited classification as a novel species, for which the name Anoxybacillus voinovskiensis sp . nov . is proposed . The type strain of this species is TH13T (=NCIMB 13956T=JCM 12111T). Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1095 - 100 Methanotorris formicicus sp . nov., a novel extremely thermophilic, methane-producing archaeon isolated from a black smoker chimney in the Central Indian Ridge; Takai K et al.; A novel extremely thermophilic, methane-producing archaeon was isolated from a black smoker chimney at the Kairei field in the Central Indian Ridge . Cells of this isolate were irregular cocci with several flagella; motility was not observed . Growth was observed between 55 and 83 degrees C (optimum of 75 degrees C; 30 min doubling time) and between pH 6.0 and 8.5 (optimum of pH 6.7) . The isolate was a strictly anaerobic, methanogenic autotroph capable of using hydrogen and carbon dioxide as sole energy and carbon sources . Formate was utilized as an alternative energy source . The G+C content of the genomic DNA was 33.3 mol% . Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate was most closely related to Methanotorris igneus strain Kol 5T . The isolate, however, could be genetically differentiated from this species by DNA-DNA hybridization analysis and on the basis of its physiological properties . The name Methanotorris formicicus sp . nov . is proposed for this isolate; the type strain is Mc-S-70T (=JCM 11930T=ATCC BAA-687T). Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1063 - 9 Cerasibacillus quisquiliarum gen . nov., sp . nov., isolated from a semi-continuous decomposing system of kitchen refuse; Nakamura K et al.; A moderately thermophilic and alkaliphilic bacillus, which had been reported and designated BLx (Haruta et al., 2002), was isolated from a semi-continuous decomposing system of kitchen refuse . Cells of strain BLxT were strictly aerobic, rod-shaped, motile and spore forming . The optimum temperature and pH for growth were approximately 50 degrees C and pH 8-9 . Strain BLxT was able to grow at NaCl concentrations from 0.5 to 7.5%, with optimum growth at 0.5% NaCl . The predominant menaquinone was MK-7, and the major fatty acid was iso-C(15 : 0) . Phylogenetic analysis showed that strain BLxT was positioned in an independent lineage within the cluster that includes the genera Virgibacillus and Lentibacillus in Bacillus rRNA group 1 . Strain BLxT exhibited 16S rDNA similarity of 92.8-94.8% to Virgibacillus species and 92.3% to Lentibacillus salicampi . Phenotypic, chemotaxonomic and phylogenetic analyses supported the classification of strain BLxT in a novel genus and species . Cerasibacillus quisquiliarum gen . nov., sp . nov . is proposed on the basis of phenotypic, chemotaxonomic and phylogenetic data . The type strain is BLxT (DSM 15825T=IAM15044T=KCTC 3815T). Genetics, 2004 Jul, 167(3), 1507 - 12 Genome-wide patterns of nucleotide substitution reveal stringent functional constraints on the protein sequences of thermophiles; Friedman R et al.; To test the hypothesis that the proteins of thermophilic prokaryotes are subject to unusually stringent functional constraints, we estimated the numbers of synonymous and nonsynonymous nucleotide substitutions per site between 17,957 pairs of orthologous genes from 22 pairs of closely related species of Archaea and Bacteria . The average ratio of nonsynonymous to synonymous substitutions was significantly lower in thermophiles than in nonthermophiles, and this effect was observed in both Archaea and Bacteria . There was no evidence that this difference could be explained by factors such as nucleotide content bias . Rather, the results support the hypothesis that proteins of thermophiles are subject to unusually strong purifying selection, leading to a reduced overall level of amino acid evolution per mutational event . The results show that genome-wide patterns of sequence evolution can be influenced by natural selection exerted by a species' environment and shed light on a previous observation that relatively few of the mutations arising in a thermophilic archaeon were nucleotide substitutions in contrast to indels. Appl Microbiol Biotechnol . 2004 Jul 23; {Epub ahead of print} Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste; Juteau P et al.; The microflora of a self-heating aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste was investigated by a combination of culture and culture-independent techniques . The temperature increased quickly in the first hours of the treatment cycles and values up to 72 degrees C were reached . Denaturing gradient gel electrophoresis of the PCR-amplified V3 region of 16S rDNA (PCR-DGGE) revealed important changes in the bacterial community during 3-day cycles . A clone library was constructed with the near-full-length 16S rDNA amplified from a mixed-liquor sample taken at 60 degrees C . Among the 78 non-chimeric clones analysed, 20 species (here defined as clones showing more than 97% sequence homology) were found . In contrast to other culture-independent bacterial analyses of aerobic thermophilic wastewater treatments, species belonging to the Bacilli class were dominant (64%) with Bacillus thermocloacae being the most abundant species (38%) . The other Bacilli could not be assigned to a known species . Schineria larvae was the second most abundant species (14%) in the clone library . Four species were also found among the 19 strains isolated, cultivated and identified from samples taken at 40 degrees C and 60 degrees C . Ten isolates showed high 16S rDNA sequence homology with the dominant bacterium of a composting process that had not been previously isolated. J Mol Biol, 2004 Aug 6, 341(2), 575 - 88 Modulation of S6 fibrillation by unfolding rates and gatekeeper residues; Pedersen JS et al.; We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus . When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl . Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively . However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured . At 42 degrees C, there is a lag phase of several days after which fibrillation takes place over several hours . Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation . Firstly, there appears to be a contiguous region of "gatekeeper" residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase . This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation . Secondly, longer lag phases correlate with faster rates of unfolding . We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation . This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains. Structure (Camb), 2004 Jun, 12(6), 949 - 59 The first crystal structure of the novel class of fructose-1,6-bisphosphatase present in thermophilic archaea; Nishimasu H et al.; As the first structure of the novel class of fructose-1,6-bisphosphatase (FBPase) present in thermophilic archaea, we solved the crystal structure of the ST0318 gene product (St-Fbp) of Sulfolobus tokodaii strain 7 . The St-Fbp structure comprises a homooctamer of the 422 point-group . The protein folds as a four-layer alpha-beta-beta-alpha sandwich with a novel topology, which is completely different from the sugar phosphatase fold . The structure contains an unhydrolyzed FBP molecule in the open-keto form, as well as four hexacoordinated magnesium ions around the 1-phosphoryl group of FBP . The arrangement of the catalytic side chains and metal ligands is consistent with the three-metal ion assisted catalysis proposed for conventional FBPases . The structure provides an insight into the structural basis of the strict substrate specificity of St-Fbp. Biopolymers, 2004 Aug 15, 74(6), 423 - 31 Ribosome motions modulate electrostatic properties; Trylska J et al.; The electrostatic properties of the 70S ribosome of Thermus thermophilus were studied qualitatively by solving the Poisson-Boltzmann (PB) equation in aqueous solution and with physiological ionic strength . The electrostatic potential was calculated for conformations of the ribosome derived by recent normal mode analysis (Tama, F., et al . Proc Natl Acad Sci USA 2003 100, 9319-9323) of the ratchet-like reorganization that occurs during translocation (Frank, J.; Agrawal, R . K . Nature 2000 406, 318-322) . To solve the PB equation, effective parameters (charges and radii), applicable to a highly charged backbone model of the ribosome, were developed . Regions of positive potential were found at the binding site of the elongation factors G and Tu, as well as where the release factors bind . Large positive potential areas are especially pronounced around the L11 and L6 proteins . The region around the L1 protein is also positively charged, supporting the idea that L1 may interact with the E-site tRNA during its release from the ribosome after translocation . Functional rearrangement of the ribosome leads to electrostatic changes which may help the translocation of the tRNAs during the elongation stage. J Environ Sci (China), 2004, 16(3), 428 - 30 Dissimilatory reduction of FeIII (EDTA) with microorganisms in the system of nitric oxide removal from the flue gas by metal chelate absorption; Ma BY et al.; In the system of nitric oxide removal from the flue gas by metal chelate absorption, it is an obstacle that ferrous absorbents are easily oxidized by oxygen in the flue gas to ferric counterparts, which are not capable of binding NO . By adding iron metal or electrochemical method, FeIII(EDTA) can be reduced to FeII(EDTA) . However, there are various drawbacks associated with these techniques . The dissimilatory reduction of FeIII(EDTA) with microorganisms in the system of nitric oxide removal by metal chelate absorption was investigated . Ammonium salt instead of nitrate was used as the nitrogen source, as nitrates inhibited the reduction of FeIII due to the competition between the two electron acceptors . Supplemental glucose and lactate stimulated the formation of FeII more than ethanol as the carbon sources . The microorganisms cultured at 50 degrees C were not very sensitive to the other experimental temperature, the reduction percentage of FeIII varied little with the temperature range of 30-50 degrees C . Concentrated Na2CO3 solution was added to adjust the solution pH to an optimal pH range of 6-7 . The overall results revealed that the dissimilatory ferric reducing microorganisms present in the mix-culture are probably neutrophilic, moderately thermophilic FeIII reducers. Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1445 - 6 Epub 2004 Jul 21. Crystallization and preliminary X-ray analysis of carboxypeptidase 1 from Thermus thermophilus; Nagata K et al.; Carboxypeptidase 1 from the thermophilic eubacterium Thermus thermophilus (TthCP1, 58 kDa), a member of the M32 family of metallocarboxypeptidases, was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant . The crystals diffracted X-rays to beyond 2.6 A resolution using a synchrotron-radiation source . The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 171.0, b = 231.6, c = 124.9 A . The crystal contains three molecules in an asymmetric unit (VM = 2.11 A3 Da(-1)) and has a solvent content of 61.5%. J Pediatr Gastroenterol Nutr, 2004 Aug, 39(2), 147 - 52 Effects of long-term consumption of a fermented infant formula (with Bifidobacterium breve c50 and Streptococcus thermophilus 065) on acute diarrhea in healthy infants; Thibault H et al.; OBJECTIVE: To determine whether long-term consumption of a fermented infant formula could influence the incidence of acute diarrhea and its severity in healthy infants . METHOD: Nine hundred seventy-one infants, ranging in age from 4 to 6 months, were included in a randomized, double-blind, placebo-controlled trial during a period of 5 months . They consumed daily either a fermented infant formula (FF) (fermentation with Bifidobacterium breve C50 and Streptococcus thermophilus 065) or a standard infant formula (SF) of the same nutritional composition . EVALUATION CRITERIA: Number and duration of acute diarrhea episodes were evaluated . Severity of the episodes was determined by the number of hospital admissions, incidence of dehydration, number of medical consultations, number of oral rehydration solution prescriptions, and number of formula switches . RESULTS: Growth of the infants and acceptability of the formulas were identical in the two groups . Incidence, duration of diarrhea episodes, and number of hospital admissions did not differ significantly between groups . Episodes were less severe in the FF (fermented formula) group . There were fewer cases of dehydration 2.5%versus 6.1% (P = 0.01), fewer medical consultations (46%v 56.6%, P = 0.003), fewer ORS prescriptions 41.9%v 51.9% (P = 0.003) and fewer switches to other formulas (59.5%v 74.9%, P = 0.0001) in FF infants compared to SF . CONCLUSION: A fermented formula may reduce the severity of acute diarrhea among healthy young infants . This outcome may be linked to the bifidogenic effects of fermentation products and their interactions with the intestinal immune system. Eur J Biochem, 2004 Aug, 271(15), 3115 - 26 Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii; Maloney AP et al.; Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps . Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction . Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources . The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes . The m-MDH gene is the first oxidoreductase gene cloned from T . emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote . Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture . The recombinant enzyme behaved as a dimer under nondenaturing conditions . Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag . Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme. J Struct Funct Genomics, 2004, 5(1-2), 133 - 46 A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome; DiDonato M et al.; The wealth of genomic data available for many organisms has set the stage for the next phase of structure-function analysis . High-throughput structural genomics is currently the method of choice for rapid analysis of protein structure-function relationships on a proteome-wide basis . The Joint Center for Structural Genomics (JCSG), established in 2000 under the NIH/NIGMS Protein Structure Initiative, has developed and implemented an integrated high-throughput structure pipeline and applied it in a 2-tiered approach to mining the proteome of the thermophilic bacterium Thermotoga maritima . In the first tier, the successful application of this integrated pipeline has resulted in the cloning and expression of 73% of the T . maritima proteome (1376 out of 1877 predicted genes), and has identified 465 proteins which produced crystal hits . These 465 proteins were compared with existing structural information and a subset of 269 targets were selected to process towards structure determination in a second tier effort . To date, the JCSG pipeline applied to the Thermotoga maritima proteome has resulted in 55 new structures and has identified 6 novel folds and continues to identify structures with novel features. J Biol Chem, 2004 Sep 10, 279(37), 38087 - 90 Epub 2004 Jul 15. Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase; Svetlov V et al.; Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties . While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known . Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose . We utilized the Escherichia coli RNAP model system to assess this prediction . Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension . Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site. J Bacteriol, 2004 Aug, 186(15), 4972 - 7 Circadian rhythms in the thermophilic cyanobacterium Thermosynechococcus elongatus: compensation of period length over a wide temperature range; Onai K et al.; Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies . We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium T . elongatus BP-1 that uses a bacterial luciferase gene set (Xl luxAB) derived from Xenorhabdus luminescens as a bioluminescence reporter gene . A promoter region of the psbA1 gene of T . elongatus was fused to the Xl luxAB gene set and inserted into a specific targeting site in the genome of T . elongatus . The bioluminescence from the cells of the psbA1-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes . The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature . The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60 degrees C) . Theses results indicate that T . elongatus has the circadian clock that is widely temperature compensated. Biochim Biophys Acta, 2004 Aug 2, 1700(2), 161 - 70 Proteolytic cleavage of the hyperthermophilic topoisomerase I from Thermotoga maritima does not impair its enzymatic properties; Cossard R et al.; Using limited proteolysis, we show that the hyperthermophilic topoisomerase I from Thermotoga maritima exhibits a unique hot spot susceptible to proteolytic attack with a variety of proteases . The remaining of the protein is resistant to further proteolysis, which suggests a compact folding of the thermophilic topoisomerase, when compared to its mesophilic Escherichia coli homologue . We further show that a truncated version of the T . maritima enzyme, lacking the last C-terminal 93 amino acids is more susceptible to proteolysis, which suggests that the C-terminal region of the topoisomerase may be important to maintain the compact folding of the enzyme . The hot spot of cleavage is located around amino acids 326-330 and probably corresponds to an exposed loop of the protein, near the active site tyrosine in charge of DNA cleavage and religation . Location of this protease sensitive region in the vicinity of bound DNA is consistent with the partial protection observed in the presence of different DNA substrates . Unexpectedly, although proteolysis splits the enzyme in two halves, each containing part of the motifs involved in catalysis, trypsin-digested topoisomerase I retains full DNA binding, cleavage, and relaxation activities, full thermostability and also the same hydrodynamic and spectral properties as undigested samples . This supports the idea that the two fragments which are generated by proteolysis remain correctly folded and tightly associated after proteolytic cleavage. Ecotoxicol Environ Saf, 2004 Sep, 59(1), 116 - 22 A test battery for the ecotoxicological evaluation of the agri-chemical Environ; Davoren M et al.; The ecotoxicological effects of the agri-chemical Environ were evaluated using a test battery comprising organisms representing three trophic levels of the aquatic ecosystem . The sensitivities of the test species to Environ were as follows: Microtox > Daphnia magna > Pseudokirchneriella subcapitata = Thamnocephalus platyurus = Oncorhynchus mykiss > Artemia salina > Tetrahymena thermophilia . An order of magnitude sensitivity between the test species was observed which emphasizes the importance of a test battery approach in the assessment of possible ecological consequences of agri-chemicals . In addition, the aquatic bioassays were found to be more sensitive (e.g., greater than three orders of magnitude for D . magna) than previously reported mammalian toxicity data for Environ . Toxicity of Environ was also investigated using fish (RTG-2) and human fibroblast cell lines (HepG2 cells) and juvenile O . mykiss . Environ was shown to have greater toxicity in the acute lethality test than with the fish cell line . However, in vivo/in vitro comparisons in this instance we feel would be premature and imprecise owing to valid concerns regarding fish loading rates for the in vivo test, and exposure duration with the in vitro test . Carbohydr Res, 2004 Aug 2, 339(11), 1857 - 71 Enzyme-catalysed synthesis of galactosylated 1d- and 1l-chiro-inositol, 1d-pinitol, myo-inositol and selected derivatives using the beta-galactosidase from the thermophile Thermoanaerobacter sp . strain TP6-B1; Hart JB et al.; The products from the enzymatic beta-d-galactopyranosylation of 1d-chiro-inositol, 1d-pinitol, 1d-3-O-allyl-4-O-methyl-chiro-inositol, 1d-3,4-di-O-methyl-chiro-inositol, 1l-chiro-inositol and myo-inositol in combined yields ranging from 46% to 64% have been obtained using the beta-galactosidase isolated from an anaerobic extreme thermophile, Thermoanaerobacter sp . strain TP6-B1 and p-nitrophenyl beta-d-galactopyranoside as the donor . Analysis of the products from these reactions reveals information about the acceptor preferences of the enzyme. Cell Biol Int, 2004, 28(7), 503 - 9 Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA; Iwamoto M et al.; Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation . The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division . On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h . This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium . These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1 . Starved cells that had finished division soon formed mating pairs with cells of the opposite type . These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T . thermophila. Water Sci Technol, 2004, 49(10), 163 - 9 Digestion of sludge and organic waste in the sustainability concept for Malmö, Sweden; la Cour Jansen J et al.; Anaerobic digestion of sludge has been part of the treatment plant in Malmo for many years and several projects on optimisation of the digestion process have been undertaken in full scale as well as in pilot scale . In order to facilitate a more sustainable solution in the future for waste management, solid waste organic waste is sorted out from households for anaerobic treatment in a newly built city district . The system for treatment of the waste is integrated in a centralised solution located at the existing wastewater treatment plant . A new extension of the digester capacity enables separate as well as co-digestion of sludge together with urban organic waste from households, industry, restaurants, big kitchens, food stores, supermarkets, green markets etc . for biogas production and production of fertiliser . Collection and pre-treatment of different types of waste are in progress together with examination of biogas potential for different types of organic waste . Collection of household waste as well as anaerobic digestion in laboratory and pilot scale has been performed during the last year . It is demonstrated that organic household waste can be digested separately or in combination with sludge . In the latter case a higher biogas yield is found than should be expected from digestion of the two materials separately . Household waste from a system based on collection of organic waste from grinders could be digested at mesophilic conditions whereas digestion failed at thermophilic conditions. Water Sci Technol, 2004, 49(10), 139 - 46 Full scale validation of helminth ova (Ascaris suum) inactivation by different sludge treatment processes; Paulsrud B et al.; The Norwegian sewage sludge regulation requires disinfection (hygienisation) of all sludges for land application, and one of the criteria is that disinfected sludge should not contain viable helminth ova . All disinfection processes have to be designed and operated in order to comply with this criterion, and four processes employed in Norway (thermophilic aerobic pre-treatment, pre-pasteurisation, thermal vacuum drying in membrane filter presses and lime treatment) have been tested in full scale by inserting semipermeable bags of Ascaris suum eggs into the processes for certain times . For lime treatment supplementary laboratory tests have been conducted . The paper presents the results of the experiments, and it could be concluded that all processes, except lime treatment, could be operated at less stringent time-temperature regimes than commonly experienced at Norwegian plants today. Water Sci Technol, 2004, 49(10), 89 - 96 Improvement of anaerobic digestion of sludge; Dohanyos M et al.; Anaerobic digestion improvement can be accomplished by different methods . Besides optimization of the process conditions, pretreatment of input sludge and increase of process temperature is frequently used . The thermophilic process brings a higher solids reduction and biogas production, a high resistance to foaming, no problems with odour, better pathogens destruction and an improvement of the energy balance of the whole treatment plant . Disintegration of excess activated sludge in a lysate centrifuge was proved to cause increase of biogas production in full-scale conditions . The rapid thermal conditioning of digested sludge is an acceptable method of particulate matter disintegration and solubilization. Extremophiles, 2004 Oct, 8(5), 411 - 9 Epub 2004 Jul 16. Tetraether-linked membrane monolayers in Ferroplasma spp: a key to survival in acid; Macalady JL et al.; Ferroplasma acidarmanus thrives in hot, extremely low pH, metal-rich solutions associated with dissolving metal sulfide ore deposits . Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin layer chromatography analyses of F . acidarmanus membranes indicate that tetraether lipids predominate, with at least three core lipid structures . NMR measurements indicate that the cytoplasmic pH of F . acidarmanus is approximately 5.6 . The optimal growth pH is approximately 1.2, and the lowest growth pH is approximately 0.0 . Thus, these organisms maintain pH gradients across their membranes that approach 5 pH units . Tetraether lipids were originally thought to be specifically associated with thermophiles but are now known to be widely distributed within the archaeal domain . Our data, in combination with recently published results for thermophilic and mesothermophilic acidophilic archaea, indicate that there may be a stronger association between tetraether lipids and tolerance to acid and/or large metal ion gradients. Proc Natl Acad Sci U S A, 2004 Jul 27, 101(30), 11111 - 6 Epub 2004 Jul 16. Membrane lipid patterns typify distinct anaerobic methanotrophic consortia; Blumenberg M et al.; The anaerobic oxidation of methane (AOM) is one of the major sinks of this substantial greenhouse gas in marine environments . Recent investigations have shown that diverse communities of anaerobic archaea and sulfate-reducing bacteria are involved in AOM . Most of the relevant archaea are assigned to two distinct phylogenetic clusters, ANME-1 and ANME-2 . A suite of specific (13)C-depleted lipids demonstrating the presence of consortia mediating AOM in fossil and recent environments has been established . Here we report on substantial differences in the lipid composition of microbial consortia sampled from distinct compartments of AOM-driven carbonate reefs growing in the northwestern Black Sea . Communities in which the dominant archaea are from the ANME-1 cluster yield internally cyclized tetraether lipids typical of thermophiles . Those in which ANME-2 archaea are dominant yield sn-2-hydroxyarchaeol accompanied by crocetane and crocetenes . The bacterial lipids from these communities are also distinct even though the sulfate-reducing bacteria all belong to the Desulfosarcina/Desulfococcus group . Nonisoprenoidal glycerol diethers are predominantly associated with ANME-1-dominated communities . Communities with ANME-2 yield mainly conventional, ester-linked diglycerides . ANME-1 archaea and associated sulfate-reducing bacteria seem to be enabled to use low concentrations of methane and to grow within a broad range of temperatures . Our results offer a tool for the study of recent and especially of fossil methane environments. Plant Cell, 2004 Aug, 16(8), 2192 - 203 Epub 2004 Jul 16. The SENSITIVE TO FREEZING2 gene, required for freezing tolerance in Arabidopsis thaliana, encodes a beta-glucosidase; Thorlby G et al.; The sensitive to freezing2-1 (sfr2-1) mutation causes freezing sensitivity in Arabidopsis thaliana . By mapping, transgenic complementation, and sequencing, sfr2-1 was revealed to be a mutation in gene At3g06510 . A new knockout allele was obtained, and its identical freezing-sensitive phenotype confirmed that the SFR2 gene product is essential for freezing tolerance . Transcription of SFR2 was observed to be constitutive rather than stress inducible and was distributed throughout most aerial tissues . SFR2 encodes a protein homologous to family 1 glycosyl hydrolases (beta-glycosidases), but the predicted AtSFR2 protein is divergent from all other family 1 beta-glycosidases of Arabidopsis, showing closer homology to the sequences of several beta-glycosidases from thermophilic archea and bacteria . After purification from a heterologous expression system, AtSFR2 displayed a specific hydrolytic activity against beta-d-glucosides. Microbiology, 2004 Jul, 150(Pt 7), 2451 - 63 A multisubunit membrane-bound {NiFe} hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis; Soboh B et al.; Thermoanaerobacter tengcongensis is a thermophilic Gram-positive bacterium able to dispose of the reducing equivalents generated during the fermentation of glucose to acetate and CO(2) by reducing H(+) to H(2) . A unique combination of hydrogenases, a ferredoxin-dependent {NiFe} hydrogenase and an NADH-dependent Fe-only hydrogenase, were found to be responsible for H(2) formation in this organism . Both enzymes were purified and characterized . The tightly membrane-bound {NiFe} hydrogenase belongs to a small group of complex-I-related {NiFe} hydrogenases and has highest sequence similarity to energy-converting {NiFe} hydrogenase (Ech) from Methanosarcina barkeri . A ferredoxin isolated from Ta . tengcongensis was identified as the physiological substrate of this enzyme . The heterotetrameric Fe-only hydrogenase was isolated from the soluble fraction . It contained FMN and multiple iron-sulfur clusters, and exhibited a typical H-cluster EPR signal after autooxidation . Sequence analysis predicted and kinetic studies confirmed that the enzyme is an NAD(H)-dependent Fe-only hydrogenase . When H(2) was allowed to accumulate in the culture, the fermentation was partially shifted to ethanol production . In cells grown at high hydrogen partial pressure {p(H(2))} the NADH-dependent hydrogenase activity was fourfold lower than in cells grown at low p(H(2)), whereas aldehyde dehydrogenase and alcohol dehydrogenase activities were higher in cells grown at elevated p(H(2)) . These results indicate a regulation in response to the p(H(2)). Microbiology, 2004 Jul, 150(Pt 7), 2327 - 34 alpha-Aminoadipate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus; Miyazaki T et al.; The extremely thermophilic bacterium Thermus thermophilus HB27 synthesizes lysine through alpha-aminoadipate (AAA) . In this study, a T . thermophilus gene encoding the enzyme that catalyses transamination of AAA was cloned as a mammalian kynurenine/AAA aminotransferase (Kat2) gene homologue . A T . thermophilus mutant with disruption of the Kat2 homologue required a longer lag phase for growth and showed slower growth in minimal medium . Furthermore, addition of AAA or lysine shortened the lag phase and improved the growth rate . The Kat2 homologue was therefore termed lysN . LysN recognizes not only 2-oxoadipate, an intermediate of lysine biosynthesis, but also 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, intermediates of leucine, valine and isoleucine biosyntheses, respectively, along with oxaloacetate, a compound in the TCA cycle, as an amino acceptor . These results suggest multiple roles of LysN in several cellular metabolic pathways including lysine and branched-chain amino acid biosyntheses. Microbiology, 2004 Jul, 150(Pt 7), 2257 - 66 Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes; Adelsberger H et al.; Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B . They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), beta-xyloside (bxlA, bxlB, bglZ) and alpha-arabinofuranoside (arfA, arfB) . The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C . stercorarium . Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced . Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated . The other glycosidases either did not attack or only marginally attacked these oligosaccharides . Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan . This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo . The active enzyme system was reconstituted in vitro from recombinant enzymes. J Bioenerg Biomembr, 2000 Oct, 32(5), 471 - 84 The alpha/beta Interfaces of alpha1beta1, alpha3beta3, and F1: Domain Motions and Elastic Energy Stored during gamma Rotation; Kagawa Y et al.; ATP synthase (F(o)F(1)) consists of F(1) (ATP-driven motor) and F(o) (H(+)-driven motor) . F(1) is a complex of alpha(3)beta(3)gammadeltaepsilon subunits, and gamma is the rotating cam in alpha(3)beta(3) . Thermophilic F(1) (TF(1)) is exceptional in that it can be crystallized as a beta monomer and an alpha(3)beta(3) oligomer, and it is sufficiently stable to allow alphabeta refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified alpha or beta . The nucleotide-dependent open-close conversion of conformation is an inherent property of an isolated beta and energy and signals are transferred through alpha/beta interfaces . The catalytic and noncatalytic interfaces of both mitochondrial F(1) (MF(1)) and TF(1) were analyzed by an atom search within the limits of 0.40 nm across the alphabeta interfaces . Seven (plus thermophilic loop in TF(1)) contact areas are located at both the catalytic and noncatalytic interfaces on the open beta form . The number of contact areas on closed beta increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces . The interfaces in the barrel domain are immobile . The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in beta . The notion of elastic energy in F(o)F(1) has been revised . X-ray crystallography of F(1) is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of F(o)F(1) . The domain motion and elastic energy in F(o)F(1) will be elucidated by time-resolved crystallography. FEMS Microbiol Lett, 2004 Jul 15, 236(2), 267 - 73 Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans; Rolland JL et al.; A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu) . The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized . The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively . The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM . We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor . In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors . The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain. Environ Microbiol, 2004 Aug, 6(8), 861 - 7 Differential response of archaeal and bacterial communities to nitrogen inputs and pH changes in upland pasture rhizosphere soil; Nicol GW et al.; Grassland management regimens influence the structure of archaeal communities in upland pasture soils, which appear to be dominated by as yet uncultivated non-thermophilic Crenarchaeota . In an attempt to determine which grassland management factors select for particular crenarchaeal community structures, soil microcosm experiments were performed examining the effect of increased pH, application of inorganic fertilizer (ammonium nitrate) and sheep urine deposition on both archaeal and bacterial communities in unmanaged grassland soil . As grassland management typically increases pH, a further experiment examined the effect of a reduction in pH, to that typical of unimproved grassland soils, on archaeal and bacterial communities . The RT-PCR amplification of 16S rRNA followed by denaturing gradient gel electrophoresis analysis demonstrated a distinct and reproducible effect on bacterial communities after incubation for 28 or 30 days . In contrast, none of the treatments had a significant effect on the structure of the crenarchaeal community, indicating that these factors are not major drivers of crenarchaeal community structures in grassland soils. Res Microbiol, 2004 Jul-Aug, 155(6), 422 - 36 Physiology of the thermophilic acetogen Moorella thermoacetica; Drake HL et al.; Moorella thermoacetica (originally isolated as Clostridium thermoaceticum) has served as the primary acetogenic bacterium for the resolution of the acetyl coenzyme A (acetyl-CoA) or Wood-Ljungdahl pathway, a metabolic pathway that (i) autotrophically assimilates CO2 and (ii) is centrally important to the turnover of carbon in many habitats . The purpose of this article is to highlight the diverse physiological features of this model acetogen and to examine some of the consequences of its metabolic capabilities. J Biotechnol, 2004 Aug 5, 111(3), 269 - 77 Protein thermostability: structure-based difference of amino acid between thermophilic and mesophilic proteins; Pack SP et al.; Structural distributions of each amino acid were compared between 20 pairs of thermophilic and mesophilic proteins to obtain thermostable factors . Five kinds of residual structure states such as fully-exposed, exposed, partially exposed (or partially buried), buried, well-buried states were considered for analyzing the structural patterns of amino acids . The statistical tests revealed that lower frequency in partially exposed state of SER, lower frequency in exposed state and higher frequency in well-buried state of ALA, higher frequency in buried state of GLU, higher frequency in exposed state of ARG, etc . could be critical factors related with protein thermostability. Bioresour Technol, 2004 Nov, 95(2), 191 - 201 Effect of temperature and temperature fluctuation on thermophilic anaerobic digestion of cattle manure; El-Mashad HM et al.; The influence of temperature, 50 and 60 degrees C, at hydraulic retention times (HRTs) of 20 and 10 days, on the performance of anaerobic digestion of cow manure has been investigated in completely stirred tank reactors (CSTRs) . Furthermore, the effect of both daily downward and daily upward temperature fluctuations has been studied . In the daily downward temperature fluctuation regime the temperatures of each reactor was reduced by 10 degrees C for 10 h while in the daily upward fluctuation regime the temperature of each reactor was increased 10 degrees C for 5 h . The results show that the methane production rate at 60 degrees C is lower than that at 50 degrees C at all experimental conditions of imposed HRT except when downward temperature fluctuations were applied at an HRT of 10 days . It also was found that the free ammonia concentration not only affects the acetate-utilising bacteria but also the hydrolysis and acidification process . The upward temperature fluctuation affects the maximum specific methanogenesis activity more severely as compared to imposed downward temperature fluctuations . The results clearly reveal the possibility of using available solar energy at daytime to heat up the reactor(s) without the need of heat storage during nights, especially at an operational temperature of 50 degrees C and at a 20 days HRT, and without the jeopardising of the overheating. Bioresour Technol, 2004 Nov, 95(2), 145 - 50 Low pH as an inhibiting factor in the transition from mesophilic to thermophilic phase in composting; Sundberg C et al.; During composting of household waste, the acidity of the material affects the process during the initial phase of rising temperature . In this study, the effects of temperature (36-46 degrees C) and pH (4.6-9.2) on the respiration rate during the early phase of composting were investigated in two different composts . A respiration method where small compost samples were incubated at constant temperature was used . The respiration rate was strongly reduced at 46 degrees C and pH below 6, compared to composts with a higher pH or lower temperature . The combination of high temperature and low pH is a possible adverse factor in large-scale composting of food waste. J Appl Microbiol, 1998 Jan, 84(1), 108 - 14 Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus; Escalante A et al.; The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal- ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated . Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source . This was associated with a greater exopolysaccharide production by the ropy strain . The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation . UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain . UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain . The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity. Curr Biol, 2004 Jul 13, 14(13), R514 - 6 Evolution: red algal genome affirms a common origin of all plastids; McFadden GI et al.; Photosynthetic organelles (plastids) come in many forms and were originally thought to have multiple origins . The complete genome of the thermophilic red alga Cyanidioschizon merolae provides further evidence that all plastids derive from a single endosymbiotic event more than 600 million years ago. Structure (Camb), 2004 Jul, 12(7), 1313 - 23 1.2 A crystal structure of the serine carboxyl proteinase pro-kumamolisin; structure of an intact pro-subtilase; Comellas-Bigler M et al.; Kumamolisin, an extracellular proteinase derived from an acido/thermop