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| J Mol Biol, 2002 Jul 12, 320(3), 587 - 96 The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome; Guimaraes BG et al.; Cellobiohydrolase CelS plays an important role in the cellulosome, an active cellulase system produced by the thermophilic anaerobe Clostridium thermocellum . The structures of the catalytic domain of CelS in complex with substrate (cellohexaose) and product (cellobiose) were determined at 2.5 and 2.4 A resolution, respectively . The protein folds into an (alpha/alpha)(6) barrel with a tunnel-shaped substrate-binding region . The conformation of the loops defining the tunnel is intrinsically stable in the absence of substrate, suggesting a model to account for the processive mode of action of family 48 cellobiohydrolases . Structural comparisons with other (alpha/alpha)(6) barrel glycosidases indicate that CelS and endoglucanase CelA, a sequence-unrelated family 8 glycosidase with a groove-shaped substrate-binding region, use the same catalytic machinery to hydrolyze the glycosidic linkage, despite a low sequence similarity and a different endo/exo mode of action . A remarkable feature of the mechanism is the absence, from CelS, of a carboxylic group acting as the base catalyst . The nearly identical arrangement of substrate and functionally important residues in the two active sites strongly suggests an evolutionary relationship between the cellobiohydrolase and endoglucanase families, which can therefore be classified into a new clan of glycoside hydrolases . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jul 12, 320(3), 455 - 74 Sequence and structural conservation in RNA ribose zippers; Tamura M et al.; The "ribose zipper", an important element of RNA tertiary structure, is characterized by consecutive hydrogen-bonding interactions between ribose 2'-hydroxyls from different regions of an RNA chain or between RNA chains . These tertiary contacts have previously been observed to also involve base-backbone and base-base interactions (A-minor type) . We searched for ribose zipper tertiary interactions in the crystal structures of the large ribosomal subunit RNAs of Haloarcula marismortui and Deinococcus radiodurans, and the small ribosomal subunit RNA of Thermus thermophilus and identified a total of 97 ribose zippers . Of these, 20 were found in T . thermophilus 16 S rRNA, 44 in H . marismortui 23 S rRNA (plus 2 bridging 5 S and 23 S rRNAs) and 30 in D . radiodurans 23 S rRNA (plus 1 bridging 5 S and 23 S rRNAs) . These were analyzed in terms of sequence conservation, structural conservation and stability, location in secondary structure, and phylogenetic conservation . Eleven types of ribose zippers were defined based on ribose-base interactions . Of these 11, seven were observed in the ribosomal RNAs . The most common of these is the canonical ribose zipper, originally observed in the P4-P6 group I intron fragment . All ribose zippers were formed by antiparallel chain interactions and only a single example extended beyond two residues, forming an overlapping ribose zipper of three consecutive residues near the small subunit A-site . Almost all ribose zippers link stem (Watson-Crick duplex) or stem-like (base-paired), with loop (external, internal, or junction) chain segments . About two-thirds of the observed ribose zippers interact with ribosomal proteins . Most of these ribosomal proteins bridge the ribose zipper chain segments with basic amino acid residues hydrogen bonding to the RNA backbone . Proteins involved in crucial ribosome function and in early stages of ribosomal assembly also stabilize ribose zipper interactions . All ribose zippers show strong sequence conservation both within these three ribosomal RNA structures and in a large database of aligned prokaryotic sequences . The physical basis of the sequence conservation is stacked base triples formed between consecutive base-pairs on the stem or stem-like segment with bases (often adenines) from the loop-side segment . These triples have previously been characterized as Type I and Type II A-minor motifs and are stabilized by base-base and base-ribose hydrogen bonds . The sequence and structure conservation of ribose zippers can be directly used in tertiary structure prediction and may have applications in molecular modeling and design . (c) 2002 Elsevier Science Ltd. Cent Eur J Public Health, 2002 Jun, 10(1-2), 6 - 10 Comparative investigation of airborne culturable microorganisms in sewage treatment plants; Haas DU et al.; The present study investigated emissions and emmissions of airborne microorganisms (mesophilic bacteria, Escherichia coli, molds, Aspergillus fumigatus, thermophilic actinomycetes/bacilli) in sewage treatment plants . For the aerobiological investigations three sewage treatment facilities with an activated-sludge process, capacities between 2000 and 28,000 PE and different cleaning steps were selected . The measurements of microorganism emission were conducted in the area of the intake (screen), in the area of biological treatment (activated sludge tank) and at a distance of 10 m from the activated sludge tanks . In order to determine the emmission, additional measurements were conducted leeward of the plant at a distance of 200 m . Samples were taken using four parallel six-stage Andersen 1 AFCM volumetric samplers . In the area of the intake counts for bacteria were 7.4 x 10(2) CFU/m3 (median), for thermophilic actinomycetes 1.8 x 10(1) CFU/m3, for thermophilic bacilli 7.1 x 10(1) CFU/m3, for molds 2.4 x 10(3) CFU/m3 and for Aspergillus fumigatus 1.8 x 10(1) CFU/m3 . Only isolated airborne coliform recoveries, i.e . E . coli, were detected . In the area of the activated sludge tank, in the adjoining area (10 m) and in the vicinity of the plants (200 m), the counts for all microorganism groups investigated corresponded to natural conditions . The results show that the counts of culturable aerogenic microorganisms in and in the immediate surrounding of the sewage plants investigated are low . Although the possibility of an infection through inhalation cannot be ruled out, the direct contact with sewage is much more critical. FEBS Lett, 2002 Jul 3, 522(1-3), 35 - 40 Characterization of bacterial homocitrate synthase involved in lysine biosynthesis; Wulandari AP et al.; In Thermus thermophilus homocitrate synthase (HCS) catalyzes the initial reaction of lysine biosynthesis through alpha-aminoadipic acid, synthesis of homocitrate from 2-oxoglutarate and acetyl-CoA . HCS is strongly inhibited by lysine, indicating that the biosynthesis is regulated by the endproduct at the initial reaction in the pathway . HCS also catalyzes the reaction using oxaloacetate in place of 2-oxoglutarate as a substrate, similar to citrate synthase in the tricarboxylic acid cycle . Several other properties of Thermus HCS and an evolutionary relationship of the biosynthetic pathway in the bacterium to other metabolic pathways are also described. J Am Chem Soc, 2002 Jul 10, 124(27), 8152 - 62 Electron-mediating Cu(A) centers in proteins: a comparative high field (1)H ENDOR study; Epel B et al.; High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating Cu(A) center . These include nitrous oxide reductase (N(2)OR), the recombinant water-soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase (COX) ba(3) (M160T9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered "purple" azurin (purpAz) . The three-dimensional (3-D) structures of these proteins, apart from the mutant, are known . The EPR spectra of all samples showed the presence of a mononuclear Cu(II) impurity with EPR characteristics of a type II copper . At W-band, the g( perpendicular) features of this center and of Cu(A) are well resolved, thus allowing us to obtain a clean Cu(A) ENDOR spectrum . The latter consists of two types of ENDOR signals . The first includes the signals of the four strongly coupled cysteine beta-protons, with isotropic hyperfine couplings, A(iso), in the 7-15 MHz range . The second group consists of weakly coupled protons with a primarily anisotropic character with A(zz) < 3 MHz . Orientation selective ENDOR spectra were collected for N(2)OR, M160QT0, and purpAz, and simulations of the cysteine beta-protons signals provided their isotropic and anisotropic hyperfine interactions . A linear correlation with a negative slope was found between the maximum A(iso) value of the beta-protons and the copper hyperfine interaction . Comparison of the best-fit anisotropic hyperfine parameters with those calculated from dipolar interactions extracted from the available 3-D structures sets limit to the sulfur spin densities . Similarly, the small coupling spectral region was simulated on the basis of the 3-D structures and compared with the experimental spectra . It was found that the width of the powder patterns of the weakly coupled protons recorded at g(perpendicular) is mainly determined by the histidine H(epsilon)(1) protons . Furthermore, the splitting in the outer wings of these powder patterns indicates differences in the positions of the imidazole rings relative to the Cu(2)S(2) core . Comparison of the spectral features of the weakly coupled protons of M160QT0 with those of the other investigated proteins shows that they are very similar to those of purpAz, where the Cu(A) center is the most symmetric, but the copper spin density and the H(epsilon)(1)-Cu distances are somewhat smaller . All proteins show the presence of a proton with a significantly negative A(iso) value which is assigned to an amide proton of one of the cysteines . The simulations of both strongly and weakly coupled protons, along with the known copper hyperfine couplings, were used to estimate and compare the spin density distribution in the various Cu(A) centers . The largest sulfur spin density was found in M160T9, and the lowest was found in purpAz . In addition, using the relation between the A(iso) values of the four cysteine beta-protons and the H-C-S-S dihedral angles, the relative contribution of the hyperconjugation mechanism to A(iso) was determined . The largest contribution was found for M160T9, and the lowest was found for purpAz . Possible correlations between the spin density distribution, structural features, and electron-transfer functionality are finally suggested. J Mol Biol, 2002 Jul 19, 320(4), 883 - 97 The structure of Rhodothermus marinus Cel12A, a highly thermostable family 12 endoglucanase, at 1.8 A resolution; Crennell SJ et al.; Cellulose is one of the most abundant polysaccharides in nature and microorganisms have developed a comprehensive system for enzymatic breakdown of this ubiquitous carbon source, a subject of much interest in the biotechnology industry . Rhodothermus marinus produces a hyperthermostable cellulase, with a temperature optimum of more than 90 degrees C, the structure of which is presented here to 1.8 A resolution . The enzyme has been classified into glycoside hydrolase family 12; this is the first structure of a thermophilic member of this family to have been solved . The beta-jelly roll fold observed has identical topology to those of the two mesophilic members of the family whose structures have been elucidated previously . A Hepes buffer molecule bound in the active site may have triggered a conformational change to an active configuration as the two catalytic residues Glu124 and Glu207, together with dependent residues, are observed in a conformation similar to that seen in the structure of Streptomyces lividans CelB2 complexed with an inhibitor . The structural similarity between this cellulase and the mesophilic enzymes serves to highlight features that may be responsible for its thermostability, chiefly an increase in ion pair number and the considerable stabilisation of a mobile region seen in S . lividans CelB2 . Additional aromatic residues in the active site region may also contribute to the difference in thermophilicity . (c) 2002 Elsevier Science Ltd. J Eukaryot Microbiol, 2001 Mar-Apr, 48(2), 147 - 60 An evaluation of Hsp90 as a mediator of cortical patterning in Tetrahymena; Frankel J et al.; This study asks two questions: 1) whether Hsp90 is involved in the regulation of cortical patterning in Tetrahymena, and 2) if it is, whether specific defects in this regulation can be attributed to functional insufficiency of the Hsp90 molecule . To address question 1, we compared the effects of a specific inhibitor of Hsp90, geldanamycin, on population growth and on development of the oral apparatus in two Tetrahymena species, T . pyriformis and T . thermophila . We observed that geldanamycin inhibits population growth in both species at very low concentrations, and that it has far more severe effects on oral patterning in T . pyriformis than in T . thermophila . These effects are parallel to those of high temperature in the same two species, and provide a tentative affirmative answer to the first question . To address question 2, we ascertained the base sequence of the genes that encode the Hsp90 molecules which are induced at high temperatures in both Tetrahymena species, as well as corresponding sequences in Paramecium tetraurelia . Extensive comparative analyses of the deduced amino acid sequences of the Hsp90 molecules of the two Tetrahymena species indicate that on the basis of what we currently know about Hsp90 both proteins are equally likely to be functional . Phylogenetic analyses of Hsp90 amino acid sequences indicate that the two Tetrahymena Hsp90 molecules have undergone a similar number of amino acid substitutions from their most recent common ancestor, with none of these corresponding to any known functionally critical region of the molecule . Thus there is no evidence that the Hsp90 molecule of T . pyriformis is functionally impaired; the flaw in the control of cortical patterning is more likely to be caused by defects in mechanism(s) that mediate the response to Hsp90, as would be expected from the "Hsp90 capacitor" model of Rutherford and Lindquist. J Eukaryot Microbiol, 2001 Mar-Apr, 48(2), 135 - 46 The effects of supraoptimal temperatures on population growth and cortical patterning in Tetrahymena pyriformis and Tetrahymena thermophila: a comparison; Frankel J et al.; In this investigation, we compare the multiplication rates and morphogenetic responses of the two most studied Tetrahymena species, T . pyriformis and T . thermophila, at supraoptimal temperatures . Although the upper temperature limits differ greatly in the two species, the pattern of growth responses to high temperature is for the most part similar, with some differences in detail . The transient recovery of cell division at the highest temperature that allows cell division, characteristic of T . pyriformis, is observed in a less distinct form in T . thermophila . Moreover, there is a remarkable difference in developmental response, with drastic abnormalities in patterning of oral structures during the transient recovery of cell division in T . pyriformis, and far more limited abnormalities under similar conditions in T . thermophila . The abnormalities result from spatial disorder in the alignment and orientation of basal body pairs within the early oral primordium, followed by failures in the realignment that normally occurs as oral structures (membranelles and undulating membrane) mature . Both the initial spatial disorder and the failures in realignment are far more severe in T . pyriformis than in T . thermophila. J Exp Biol, 2002 Jul, 205(Pt 14), 2089 - 97 In the polymorphic ciliate Tetrahymena vorax, the non-selective phagocytosis seen in microstomes changes to a highly selective process in macrostomes; Gronlien HK et al.; Ciliates use phagocytosis to acquire edible particles . The polymorphic ciliate Tetrahymena vorax appears in two forms ('microstomes' and 'macrostomes') . Transformation of microstomes into macrostomes takes place in the presence of the ciliate Tetrahymena thermophila and enables the macrostome to phagocytose the latter species . The non-specific, constitutive phagocytosis in microstomes thereby changes into a specific inducible process in macrostomes . The purpose of this study was to determine whether the phagocytotic process in macrostomes is specifically aimed at catching T . thermophila . The two forms of phagocytosis represent an interesting model system for studying the mechanism whereby phagosomes are formed . The macrostomal form capture deciliated and ciliated Tetrahymena thermophila, latex beads with diameters of 20.3 and 30.0 microm and small microstomal cells . However, the macrostomes select T . thermophila as a prey when they have the opportunity to choose between deciliated T . thermophila and latex beads and between T . thermophila and microstomes . The non-selective formation of phagosomes seen in microstomes changes to a highly selective process during the transformation to macrostomes . Unlike microstomes, macrostomes do not form a closed vacuole after capturing a latex bead, indicating that mechanical stimulation by the prey does not in itself trigger phagocytosis in the macrostomal form of T . vorax . Although macrostomes captured T . thermophila in preference to microstomes, phagocytosis of microstomes started immediately following capture, indicating that the substance/molecule that triggers the formation of the phagosome is not specific for T . thermophila cells . After capturing a T . thermophila cell, the macrostomal cell, which normally swims in a forward direction, reverses direction and swims backwards for a short time before starting to rotate . Macrostomal cells did not change their swimming pattern after capturing a latex bead . We believe, therefore, that backward swimming is more likely to be related to signals resulting from phagocytosis than from mechanical stimulation of the pouch . Cytochalasin B (10 microg ml(-1)) inhibits phagocytosis in both microstomes and macrostomes, indicating that actin filaments play an active role in phagocytosis in both cell types . The antitubulin drug nocodazole (0.3-30 micromol l(-1)) inhibits the formation of more than one phagosome in the macrostome, indicating that membrane transport to the oral apparatus in macrostomes is guided by microtubules . Nocodazole has no effect on the process of phagocytosis in microstomes. Ann Agric Environ Med, 2002, 9(1), 41 - 8 Air contaminants in different European farming environments; Radon K et al.; Farmers are known to be at high risk from the development of occupational airway disease . The first stage of the European farmers' study has shown that pig farmers in Denmark and Germany, poultry farmers in Switzerland and greenhouse workers in Spain were at highest risk for work-related respiratory symptoms . Therefore, the aim of this study was to determine exposure levels at relevant farm workplaces . Dust and endotoxin levels as well as microbiological concentrations were determined in 213 crop and animal farming environments by personal sampling . The highest total dust concentrations were found in poultry houses in Switzerland with median concentrations of 7.01 mg/m(3) . The median airborne endotoxin concentrations in total dust ranged between 0.36 ng/m(3) in Spanish greenhouses and 257.58 ng/m(3) in poultry houses in Switzerland . Likewise, the highest median concentrations of total (2.0 x (7) cells/m(3)) and active fungi (4.4 x (5) cfu/m(3)) have been found in Swiss poultry houses . The predominant fungus taxa discovered in poultry houses were Eurotium spp . and thermophilic fungi . Cladosporium and Botrytis were mainly detected in greenhouses . The exposure level found in this study might put the farmers at risk from respiratory diseases. Mol Cell, 2002 Jun, 9(6), 1263 - 72 Elongation factor G participates in ribosome disassembly by interacting with ribosome recycling factor at their tRNA-mimicry domains; Ito K et al.; Elongation factor G (EF-G) is a G protein with motor function that drives two target molecules, a tRNA in the translating ribosome and the ribosome recycling factor (RRF) in the post-termination complex . How G protein motor action is transmitted to RRF is unknown . Thermus thermophilus RRF is nonfunctional in Escherichia coli . It became functional upon introducing a plasmid expressing E . coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E . coli EF-G tRNA-mimic domain by the Thermus domain . Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region . These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles . These mutational studies suggest that EF-G motor action is transmitted to RRF by specific surface contacts between the domains that mimic the anticodon arm. Syst Appl Microbiol, 2002 Apr, 25(1), 52 - 9 Study of the intra- and interlaboratory reproducibility of partial single base C-sequencing of the 16S rRNA gene and its applicability for the identification of members of the genus Streptococcus; Storms V et al.; The use of Single Base C-Sequencing of the first 500 bases of the 16S rRNA-gene (SBCS) combined with capillary electrophoresis was evaluated for the identification of reference strains of 30 different species within the genus Streptococcus . For SBCS, only dd-CTP's are used in the sequencing reactions instead of the four dideoxy bases and the primer is fluorescently labeled . The reproducibility, interlaboratory exchangeability and discriminative power of this method were studied by comparing the patterns obtained in three laboratories under highly standardized conditions . The interlaboratory reproducibility proved to be high, enabling the construction of a common database for the identification of strains belonging to the streptococcal species studied . Most of the examined species generated distinguishable profiles . SBCS did not differentiate between the closely related species S . constellatus and S . intermedius . Also S . thermophilus and S . vestibularis as well as S . mitis and S . pneumoniae showed highly resembling profiles . The previously reported heterogeneity within the species S . equinus was reflected by SBCS . For all other species, strains belonging to the same species generated indistinguishable patterns . In conclusion, Single Base C-sequencing of the first 500 bases of the 16S rRNA-gene could be a useful and widely applicable method for the identification of bacteria at the species level, with the added advantage of being more rapid and easier to automatize than full sequence determination. J Dairy Sci, 2002 May, 85(5), 1031 - 8 Influence of a Spirulina platensis biomass on the microflora of fermented ABT milks during storage (R1); Varga L et al.; The objective of this research was to investigate the effect of a cyanobacterial (Spirulina platensis) biomass on the microflora of a probiotic fermented dairy product during storage at two temperatures . Spirulina-enriched and control (plain) fermented acidophilus-bifidus-thermophilus (ABT) milks were produced using a fast fermentation starter culture (ABT-4) as the source of Lactobacillus acidophilus (A), bifidobacteria (B), and Streptococcus thermophilus (T) . Incubation took 6 h at 40 degrees C . As for the cyanobacterial product, the S . platensis biomass was added to the process milk during stirring at pH 4.5 to 4.6 . Thereafter, the ABT-type fermented milks were cooled to 25 degrees C in ice water, filled into sterile, tightly capped centrifuge tubes, further cooled at 4 degrees C for 24 h, and then stored either at 15 degrees C for 18 d or at 4 degrees C for 42 d . Microbiological analyses and acidity measurements were performed at regular intervals . Our results showed that the counts of the starter organisms were satisfactory during the entire storage period at both temperatures applied in this research . The S . platensis biomass had a beneficial effect on the survival of ABT starter bacteria regardless of storage temperature . Postacidification was observed at 15 degrees C, whereas pH remained stable during refrigerated storage at 4 degrees C . The abundance of bioactive substances in S . platensis is of great importance from a nutritional point of view because thus the cyanobacterial biomass provides a new opportunity for the manufacture of functional dairy foods. Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8494 - 9 Omnipotent decoding potential resides in eukaryotic translation termination factor eRF1 of variant-code organisms and is modulated by the interactions of amino acid sequences within domain 1; Ito K et al.; In eukaryotes, a single translational release factor, eRF1, deciphers three stop codons, although its decoding mechanism remains puzzling . In the ciliate Tetrahymena thermophila, UAA and UAG codons are reassigned to Gln codons . A yeast eRF1-domain swap containing Tetrahymena domain 1 responded only to UGA in vitro and failed to complement a defect in yeast eRF1 in vivo at 37 degrees C . This finding demonstrates that decoding specificity of eRF1 from variant code organisms resides at domain 1 . However, the wild-type eRF1 hybrid fully restored the growth of eRF1-deficient yeast at 30 degrees C . Tetrahymena eRF1 contains a variant sequence, KATNIKD, at the tip of domain 1 . The TASNIKD variant of hybrid eRF1 rendered the eRF1-nullified yeast viable, although in an in vitro assay, the same hybrid eRF1 responded only to UGA . Nevertheless, the yeast eRF1 bearing the KATNIKD motif instead of the TASNIKS heptapeptide present in higher eukaryotes remains omnipotent in vivo . Collectively, these data suggest that variant genetic code organisms like Tetrahymena have an intrinsic potential to decode three stop codons in vivo, and that interaction within domain 1 between the KAT tripeptide and other sequences modulates the decoding specificity of Tetrahymena eRF1. Eur J Biochem, 2002 Jul, 269(13), 3113 - 21 Environmentally coupled hydrogen tunneling . Linking catalysis to dynamics; Knapp MJ et al.; Many biological C-H activation reactions exhibit nonclassical kinetic isotope effects (KIEs) . These nonclassical KIEs are too large (kH/kD > 7) and/or exhibit unusual temperature dependence such that the Arrhenius prefactor KIEs (AH/AD) fall outside of the semiclassical range near unity . The focus of this minireview is to discuss such KIEs within the context of the environmentally coupled hydrogen tunneling model . Full tunneling models of hydrogen transfer assume that protein or solvent fluctuations generate a reactive configuration along the classical, heavy-atom coordinate, from which the hydrogen transfers via nuclear tunneling . Environmentally coupled tunneling also invokes an environmental vibration (gating) that modulates the tunneling barrier, leading to a temperature-dependent KIE . These properties directly link enzyme fluctuations to the reaction coordinate for hydrogen transfer, making a quantum view of hydrogen transfer necessarily a dynamic view of catalysis . The environmentally coupled hydrogen tunneling model leads to a range of magnitudes of KIEs, which reflect the tunneling barrier, and a range of AH/AD values, which reflect the extent to which gating modulates hydrogen transfer . Gating is the primary determinant of the temperature dependence of the KIE within this model, providing insight into the importance of this motion in modulating the reaction coordinate . The potential use of variable temperature KIEs as a direct probe of coupling between environmental dynamics and the reaction coordinate is described . The evolution from application of a tunneling correction to a full tunneling model in enzymatic H transfer reactions is discussed in the context of a thermophilic alcohol dehydrogenase and soybean lipoxygenase-1. J Mol Biol, 2002 May 17, 318(5), 1341 - 9 Origins of the high stability of an in vitro-selected cold-shock protein; Martin A et al.; In previous work, we had identified stabilized forms of the cold-shock protein Bs-CspB from Bacillus subtilis in a combinatorial library by an in vitro selection procedure . In this library, the sequence positions 2, 3, 46, 64, 66, and 67 had been randomized, because Bs-CspB differs from the naturally thermostable homolog Bc-Csp from Bacillus caldolyticus, among others, at these six positions . For the most stable selected variant, the midpoint of thermal unfolding (tM) increased by 28.2 deg . C and the Gibbs free energy of unfolding (deltaG(D)) by 19 kJ/mol . Here, we analyzed by site-directed mutagenesis how the selected residues contribute individually to this strong stabilization . Val3 and Val66, which replace Glu3 and Glu66 of wild-type Bs-CspB, each contribute about 7 kJ/mol to stability, the Thr64Arg substitution contributes 4.5 kJ/mol, and 3.2 kJ/mol originate from the Ala46Leu replacement . Gly67 at the carboxy terminus is unimportant for stability, the Arg selected at position 2 is overall slightly destabilizing but improves the coulombic interactions . The best variant differs from Bc-Csp at all six positions; nevertheless, natural and in vitro selection followed similar principles . In both cases, negatively charged residues at the adjacent positions 3 and 66 are avoided, and a positively charged residue is introduced into this area of the protein surface . Its exact location is unimportant . It can be at position 3, as in the thermophilic Bc-Csp, or at positions 2 or 64, as in the most stable selected variant . These positively charged residues contribute to stability not by engaging in pairwise coulombic interactions with a specific carboxyl group, but by generally improving the charge distribution in this particular region of the protein surface . These coulombic effects contribute significantly to the thermostability of the cold-shock proteins . They are only weakly interdependent and best explained by the presence of a flexible ion network at the protein surface . Our results emphasize that surface positions are very good candidates for optimizing protein stability. Protein Eng, 2002 Jun, 15(6), 471 - 6 Cold-adaptation mechanism of mutant enzymes of 3-isopropylmalate dehydrogenase from Thermus thermophilus; Suzuki T et al.; Random mutagenesis of Thermus thermophilus 3-isopropylmalate dehydrogenase revealed that a substitution of Val126Met in a hinge region caused a marked increase in specific activity, particularly at low temperatures, although the site is far from the binding residues for 3-isopropylmalate and NAD . To understand the molecular mechanism, residue 126 was substituted with one of eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile or Phe . Circular dichroism analyses revealed a decreased thermal stability of the mutants (Delta T ((1/2))= 0-13 degrees C), indicating structural perturbations caused by steric conflict with surrounding residues having larger side chains . Kinetic parameters, k(cat) and K(m) values for isopropylmalate and NAD, were also affected by the mutation, but the resulting k(cat)/K(m) values were similar to that of the wild-type enzyme, suggesting that the change in the catalytic property is caused by the change in free-energy level of the Michaelis complex state relative to that of the initial state . The kinetic parameters and activation enthalpy change (Delta H (double dagger)) showed good correlation with the van der Waals volume of residue 126 . These results suggested that the artificial cold adaptation (enhancement of k(cat) value at low temperatures) resulted from the destabilization of the ternary complex caused by the increase in the volume of the residue at position 126. Protein Eng, 2002 Jun, 15(6), 455 - 62 Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart; Uchiyama S et al.; Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6 . The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile . The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains . Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions . The obtained differences in the Gibbs free energy changes of unfolding {Delta(DeltaG)} showed that the three regions contributed to the overall stability in an additive manner . HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552 . Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization. Biophys J, 2002 Jul, 83(1), 433 - 57 Light harvesting in photosystem I: modeling based on the 2.5-A structure of photosystem I from Synechococcus elongatus; Byrdin M et al.; The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-A resolution . Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules . It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state . Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer . On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function . A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S . elongatus . The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center . For the Forster radius and the intrinsic primary charge separation rate, the following values have been obtained: R(0) = 7.8 nm and k(CS) = 0.9 ps(-1) . Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1232 - 3 Epub 2002 Jun 20. Crystallization and preliminary X-ray crystallographic studies of monoacylglycerol lipase of the moderately thermophilic Bacillus sp . H-257; Yoneda K et al.; Thermostable monoacylglycerol lipase (MGLP; EC 3.1.1.23) from the moderately thermophilic Bacillus sp . H-257 has a unique substrate specificity . It hydrolyzes monoacylglycerols but does not hydrolyze di- or triacylglycerols . Crystals of the enzyme were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant and benzamidine as an additive . The orthorhombic crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 43.53, b = 100.82, c = 108.17 A . The crystals diffract to at least 2.3 A resolution and a native data set has been collected to 2.6 A resolution on a CCD detector using synchrotron radiation. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1129 - 37 Epub 2002 Jun 20. An enzyme with a deep trefoil knot for the active-site architecture; Nureki O et al.; Knots in polypeptide chains have been found in very few proteins . Only two proteins are considered to have a shallow 'trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface . Recently, another protein was found by a mathematical algorithm to have a deep 'figure-of-eight' knot which had not been visually identified . In the present study, the crystal structure of a hypothetical RNA 2'-O-ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 A resolution and a deep trefoil knot was found for the first time . The present knot is formed by the threading of a 44-residue polypeptide chain through a 41-residue loop and is better defined than the previously reported knots . Two of the three catalytic residues conserved in the 2'-O-ribose methyltransferase family are located in the knotting loop and in the knotted carboxy-terminal chain, which is the first observation that the enzyme active site is constructed right on the knot . On the other hand, the amino-terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA. FEMS Microbiol Lett, 2002 Jun 18, 212(1), 121 - 6 Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: bioconversion of D-galactose to D-tagatose using the enzyme; Kim BC et al.; Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli . The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da . The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima . The recombinant enzyme expressed in E . coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration . The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability . The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+) . A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C. Microb Cell Fact . 2002 Apr 18;1(1):1. Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis; Martirani L et al.; BACKGROUND: Applications of bacteriocins as food preservatives have been so far limited, principally because of their low antimicrobial activity in foods . Nisin is the only bacteriocin of significant use, but applications are restricted principally because of its very low activity at neutral or alkaline pH . Thus the isolation of new bacteriocins active in foods is desirable . RESULTS: We isolated a Bacillus licheniformis thermophilic strain producing a bacteriocin with some novel features, named here bacillocin 490 . This bacteriocin was inactivated by pronase E and proteinase K and was active against closely related Bacillus spp . both in aerobic and in anaerobic conditions . Bactericidal activity was kept during storage at 4 degrees C and was remarkably stable in a wide pH range . The bacteriocin was partially purified by elution after adhesion to cells of the food-isolated strain Bacillus smithii and had a rather low mass (2 KDa) . Antimicrobial activity against B . smithii was observed also when this organism was grown in water buffalo milk . CONCLUSIONS: Bacillocin 490 is a novel candidate as a food anti-microbial agent since it displays its activity in milk, is stable to heat treatment and during storage, is active in a wide pH range and has bactericidal activity also at high temperature . These features may allow the use of bacillocin 490 during processes performed at high temperature and as a complementary antimicrobial agent of nisin against some Bacillus spp . in non-acidic foods . The small size suggests its use on solid foods. J Ind Microbiol Biotechnol, 2002 Mar, 28(3), 134 - 6 The resistance to heat of thermo-resistant streptococci attached to stainless steel in the presence of milk; Flint S et al.; Skim milk residues had a significant impact on the sensitivity to heat of a dairy isolate of the thermo-resistant, Streptococcus thermophilus . Cells of S . thermophilus (H) suspended in water or in milk had D values at 60 degrees C of 2.0 and 14 min, respectively . Cells of S . thermophilus (H) attached to stainless steel in the presence of water or milk had D values at 60 degrees C of 2.2 and 8.1 min, respectively . The attached cells in both experiments were heat-treated in the presence of water . The increase in heat resistance could not be fully attributed to individual components (caseinate or whey) in the milk . The potential for thermo-resistant streptococci to survive heat treatment in a dairy manufacturing plant is therefore greater than may be expected for the organism in less complex environments. Appl Microbiol Biotechnol, 2002 Jun, 59(1), 105 - 11 Epub 2002 Apr 04. Assessment of effluent turbidity in mesophilic and thermophilic activated sludge reactors - origin of effluent colloidal material; Vogelaar JC et al.; Two lab-scale plug flow activated sludge reactors were run in parallel for 4 months at 30 and 55 degrees C . Research focussed on: (1) COD (chemical oxygen demand) removal, (2) effluent turbidity at both temperatures, (3) the origin of effluent colloidal material and (4) the possible role of protozoa on turbidity levels . Total COD removal percentages over the whole experimental period were 66+/-7% at 30 degrees C and 53+/-11% at 55 degrees C . Differences in total COD removal between both systems were due to less removal of soluble and colloidal COD at 55 degrees C compared to the reference system . Thermophilic effluent turbidity was caused by a combination of influent colloidal particles that were not effectively retained in the sludge flocs, and erosion of the thermophilic activated sludge itself, as shown by denaturing gradient gel electrophoresis (DGGE) profiles . DGGE analysis of PCR-amplified 16S rDNA fragments from mesophilic and thermophilic sludge differed, indicating that different microbial communities were present in the two reactor systems . The effects of protozoal grazing on the effluent turbidity of both reactors was negligible and thus could not account for the large turbidity differences observed. Curr Genet, 2002 May, 41(2), 89 - 98 Epub 2002 May 07. Cloning and relational analysis of 15 novel fungal endoglucanases from family 12 glycosyl hydrolase; Goedegebuur F et al.; Cellulases belong to the large family of glycosyl hydrolases (GHs) and are produced by a variety of bacteria and fungi . These extracellular enzymes act as endoglucanases (EGs), cellobiohydrolases or beta-glucosidases . In this paper, we describe molecular screening for EGs from the GH family 12 . Using three homologous sequence boxes deduced from five previously known members of the family, we analysed 22 cellulase-producing fungal strains obtained from a diverse area of the fungal kingdom . Polymerase chain reactions using degenerate primers designed to the homologous protein boxes were used to identify the family 12 homologues . Several fungi showed the presence of multiple versions of the gene, while amino acid sequence analysis showed diversity in 15 novel members of the family, ranging from 26% to 96% similarity . Our sequence analysis shows that the phylogenetic tree of family 12 EGs can be divided into four subfamilies: 12-1 (fungal group I), 12-2 (fungal group II), 12-3 ( Streptomyces group in which Rhodothermus marinus fits) and 12-4 ( Thermophiles group) . Erwinia carotovora may form a new subgroup. Extremophiles, 2002 Jun, 6(3), 233 - 43 Epub 2002 Feb 20. A novel subtilisin-like serine protease from Thermoanaerobacter yonseiensis KB-1: its cloning, expression, and biochemical properties; Jang HJ et al.; A gene, tayI, encoding a novel subtilisin-like protease, designated thermicin, from the extremely thermophilic bacterium Thermoanaerobacter yonseiensis KB-1 (DSM 13777) was cloned by using a sequence tag containing the consensus sequence of proteases . The gene consisted of 1,239 nucleotides, and the deduced amino acid sequence indicated that it is a preproenzyme with a 311-residue mature protein composed of canonical catalytic residues (Asp29, His64, and Ser252) . Thermicin was overproduced in E . coli as a fusion protein with a histidine tag and purified by nickel nitrilotriacetic acid affinity chromatography . Thermicin from E . coli showed maximum proteolytic activity at 92.5 degrees C and pH 9.0, and its half-life was 30 h at 80 degrees C . In order to determine cleavage specificity, thermicin was incubated with insulin beta chain, and the resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry . The carboxyl group side of the Val12, Leu15,17, Gly23, and Pro28 residues was cleaved . Thermicin is well known to hydrolyze Gly- and Pro-rich collagens . The K (m) and k (cat)/ K (m) values of thermicin for the hydrolysis of the synthetic substrate L-Gly-Pro- p-nitroaniline were 54.16 microM and 142.05 (10(5) s(-1) M(-1)), respectively, at 92.5 degrees C and pH 9.0 . Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme belongs to a new subgroup with respect to its molecular evolution, when compared with previously characterized subtilisins . This result indicates that thermicin is a novel enzyme different from other thermostable proteases. Extremophiles, 2002 Jun, 6(3), 225 - 32 Epub 2002 Feb 27. The periplasmic space in Thermus thermophilus: evidence from a regulation-defective S-layer mutant overexpressing an alkaline phosphatase; Castan P et al.; The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer) . This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase . Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan . A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components . To demonstrate this, we cloned a gene ( phoA) from T . thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector . Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction . All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria . The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed. Extremophiles, 2002 Jun, 6(3), 201 - 7 Epub 2002 Jan 22. Acidophiles of saline water at thermal vents of Vulcano, Italy; Simmons S et al.; DNA was extracted from samples taken from close to acidic hydrothermal vents on shore of the Aeolian Island of Vulcano (Italy) . RNA gene sequences were amplified by PCR, cloned, and sequenced . A sequence with an origin in samples at 35 degrees and 45 degrees C corresponded to that of a novel Acidithiobacillus species that was isolated from water close to the vents . Novel, iron-oxidizing mesophilic acidophiles were isolated through enrichment cultures with ferrous iron but were not represented in the clone banks of environmental rDNA . These acidophiles were related to Thiobacillus prosperus, which was isolated previously from Vulcano . The archaeal sequences that comprised a clone bank representing a high-temperature sample (75 degrees C) corresponded to those of Acidianus brierleyi and of thermophiles previously isolated from Vulcano, Thermoplasma volcanium and Acidianus infernus. Extremophiles, 2002 Jun, 6(3), 185 - 94 Epub 2002 Feb 14. Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans; Kluskens LD et al.; The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction . Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases . It was cloned into a pET9d vector, without its signal sequence, and expressed in Escherichia coli . The heterologously produced fervidolysin was purified by heat incubation followed by ion exchange chromatography and emerged in the soluble fraction as three distinct protein bands, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Amino-terminal-sequence analysis of these bands and their comparison with that determined from biochemically purified keratinase and its predicted protein sequence, identified them as a 73-kDa fervidolysin precursor, a 58-kDa mature fervidolysin, and a 14-kDa fervidolysin propeptide . Using site-directed mutagenesis, the active-site histidine residue at position 79 was replaced by an alanine residue . The resulting fervidolysin showed a single protein band corresponding in size to the 73-kDa fervidolysin precursor, indicating that its proteolytic cleavage resulted from an autoproteolytic process . Knowledge-based modeling experiments showed a distinctive binding region for subtilases, in which binding of the propeptide could take place prior to autoproteolysis . Assays using keratin and other proteinaceous substrates did not display fervidolysin activity, perhaps because of the tight binding of the propeptide in the substrate-binding site, where it could then function as an inhibitor. Extremophiles, 2002 Jun, 6(3), 177 - 84 Epub 2002 Mar 08. Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts; Bergquist P et al.; Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes . As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production . We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry . The K . lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture . The T . reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin . We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression . A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification . Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host . Comparison of the expression of three thermophilic heterologous microbial xylanases in T . reesei demonstrates the need for addressing each case individually. Curr Microbiol, 2002 Aug, 45(2), 144 - 50 Thermophilic protease-producing Geobacillus from Buranga hot springs in Western Uganda; Hawumba JF et al.; Two proteolytic thermophilic aerobic bacterial strains, PA-9 and PA-5, were isolated from Buranga hot springs in western Uganda . The cells were rods, approximately 10-12 microm in length and 3 microm in width . Isolate PA-9 grew at between 38 degrees C and 68 degrees C (optimum, 62 degrees C), and PA-5 grew at between 37 degrees C and 72 degrees C (optimum, 60 degrees C) . Both isolates grew optimally at pH 7.5-8.5 . Their 16S rRNA gene sequences indicated that they belong to the newly described genus Geobacillus . Zymogram analysis of the crude enzyme extracts revealed the presence of two extracellular proteases for isolate PA-5, and at least eight for isolate PA-9 . The optimum temperature and pH for casein-degrading activity were 70 degrees C, pH 6.5 for isolate PA-9, but caseinolytic activity could also be observed at 2 degrees C . In the case of isolate PA-5, optimal activity was observed over a temperature and pH range of 50-70 degrees C and pH 5-10, respectively. Biochemistry, 2002 Jun 25, 41(25), 8152 - 61 Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study; Chakravarty S et al.; Understanding the molecular basis for the enhanced stability of proteins from thermophiles has been hindered by a lack of structural data for homologous pairs of proteins from thermophiles and mesophiles . To overcome this difficulty, complete genome sequences from 9 thermophilic and 21 mesophilic bacterial genomes were aligned with protein sequences with known structures from the protein data bank . Sequences with high homology to proteins with known structures were chosen for further analysis . High quality models of these chosen sequences were obtained using homology modeling . The current study is based on a data set of models of 900 mesophilic and 300 thermophilic protein single chains and also includes 178 templates of known structure . Structural comparisons of models of homologous proteins allowed several factors responsible for enhanced thermostability to be identified . Several statistically significant, specific amino acid substitutions that occur going from mesophiles to thermophiles are identified . Most of these are at solvent-exposed sites . Salt bridges occur significantly more often in thermophiles . The additional salt bridges in thermophiles are almost exclusively in solvent-exposed regions, and 35% are in the same element of secondary structure . Helices in thermophiles are stabilized by intrahelical salt bridges and by an increase in negative charge at the N-terminus . There is an approximate decrease of 1% in the overall loop content and a corresponding increase in helical content in thermophiles . Previously overlooked cation-pi interactions, estimated to be twice as strong as ion-pairs, are significantly enriched in thermophiles . At buried sites, statistically significant hydrophobic amino acid substitutions are typically consistent with decreased side chain conformational entropy. Int J Hyg Environ Health, 2002 May, 205(4), 321 - 4 First isolation of urease-positive thermophilic Campylobacter (UPTC) from crows (Corvus levaillantii) in Japan; Matsuda M et al.; Two strains of urease-positive thermophilic Campylobacter (UPTC), designated YC98-1 and YC98-2, were identified by biochemical characterization after isolation from the intestinal contents of crows around Yokohama City, Japan, in 1998 . The biochemical characteristics of these strains were identical to those of strains described previously . Pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI, SalI, and SmaI of the genomic DNA from the two strains indicated that respective PFGE profiles were distinctly different and distinguishable from each other . This is the first report of the isolation of UPTC from crows (Corvus levaillantii). Prikl Biokhim Mikrobiol, 2002 May-Jun, 38(3), 261 - 7 {Secondary antimicrobial metabolites produced by thermophilic Bacillus spp . strains VK2 and VK21}; Esikova TZ et al.; A collection of thermophilic strains of the genus Bacillus was made . The strains were screened for antimicrobial activity . Strains VK2 and VK21 isolated from thermal springs of the Kamchatka Peninsula, and antagonistic to several gram-positive bacterial species were chosen for further investigation of antibiotics produced by them . Restriction analysis of DNA coding for 16S rRNA showed that both strains can be assigned to Bacillus licheniformis . It was shown that the lytic activity of strains VK2 and VK21 was not related to the synthesis of hydrolytic enzymes . The maximum level of antimicrobial activity in the growth medium was found to correspond to the beginning of the stationary growth phase . Addition of manganese sulfate induced sporulation and altered significantly the time course of antibiotic production in both strains . Active metabolites were extracted with n-butanol . They survived boiling for 30 min and were resistant to trypsin and chymotrypsin but were partly hydrolyzed by pronase . They were stable at a pH range of 2.0-9.0. J Appl Microbiol, 2002, 93(1), 52 - 9 Cloning and sequencing of the gene encoding X-prolyl-dipeptidyl aminopeptidase (PepX) from Streptococcus thermophilus strain ACA-DC 4; Anastasiou R et al.; AIMS: To clone and sequence the pepX gene from Streptococcus thermophilus . METHODS AND RESULTS: Three pairs of primers were used in polymerase chain reactions using as template the total DNA from Strep . thermophilus ACA-DC 4 in order to amplify, clone and sequence the pepX gene . Sequence analysis revealed an open reading frame of 2268 nucleotides encoding a protein of 755 amino acids . The calculated molecular mass of 85 632 Da agreed well with the apparent molecular mass of 80 000 Da previously determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration for the monomeric form of the purified enzyme . CONCLUSIONS: The pepX gene from Strep . thermophilus ACA-DC 4 was cloned and sequenced . The PepX protein showed significant sequence similarity with PepX enzymes from other lactic acid bacteria and contained a motif which was almost identical with the active site motif of the serine-dependent PepX family . SIGNIFICANCE AND IMPACT OF THE STUDY: There are economic and technological incentives for accelerating and controlling the process of cheese ripening . To achieve this, starters may be modified by introducing appropriate genes from other food-grade bacteria . New or additional peptidase activities may alter or improve the proteolytic properties of lactic acid bacteria. J Biol Chem, 2002 Aug 23, 277(34), 30649 - 55 Epub 2002 Jun 13. A novel candidate for the true fructose-1,6-bisphosphatase in archaea; Rashid N et al.; Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes of the gluconeogenic pathway . Although enzyme activity had been detected in Archaea, the corresponding gene had not been identified until a presumable inositol monophosphatase gene from Methanococcus jannaschii was found to encode a protein with both inositol monophosphatase and FBPase activities . Here we display that a gene from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, which does not correspond to the inositol monophosphatase gene from M . jannaschii, displays high FBPase activity . The FBPase from strain KOD1 was partially purified, its N-terminal amino acid sequence was determined, and the gene (Tk-fbp) was cloned . Tk-fbp encoded a protein of 375 amino acid residues with a molecular mass of 41,658 Da . The recombinant Tk-Fbp was purified and characterized . Tk-Fbp catalyzed the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate following Michaelis-Menten kinetics with a K(m) value of 100 microm toward fructose 1,6-bisphosphate, and a k(cat) value of 17 s(-1) subunit(-1) at 95 degrees C . Unlike the inositol monophosphatase from M . jannaschii, Tk-Fbp displayed strict substrate specificity for fructose 1,6-bisphosphate . Activity was enhanced by Mg(2+) and dithioerythritol, and was slightly inhibited by fructose 2,6-bisphosphate . AMP did not inhibit the enzyme activity . We examined whether expression of Tk-fbp was regulated at the transcription level . High levels of Tk-fbp transcripts were detected in cells grown on pyruvate or amino acids, whereas no transcription was detected when starch was present in the medium . Orthologue genes corresponding to Tk-fbp with high similarity are present in all the complete genome sequences of thermophilic Archaea, including M . jannaschii, Pyrococcus furiosus, Sulfolobus solfataricus, and Archaeoglobus fulgidus, but are yet to be assigned any function . Taking into account the high FBPase activity of the protein, the strict substrate specificity, and its sugar-repressed gene expression, we propose that Tk-Fbp may represent the bona fide FBPase in Archaea. J Med Entomol, 2002 May, 39(3), 485 - 92 Geographical information systems and bootstrap aggregation (bagging) of tree-based classifiers for Lyme disease risk prediction in Trentino, Italian Alps; Rizzoli A et al.; The risk of exposure to Lyme disease in the province of Trento, Italian Alps, was predicted through the analysis of the distribution of Ixodes ricinus (L.) nymphs infected with Borrelia burgdorferi s.l . with a model based on bootstrap aggregation (bagging) of tree-based classifiers within a geographical information system (GIS) . Data on L ricinus density assessed by dragging the vegetation in 438 sites during 1996 were cross-correlated with the digital cartography of a GIS, which included the variables altitude, exposure and slope, substratum, vegetation type and roe deer density . Ticks were more abundant at altitudes below 1,300 m a.s.l., in the presence of limestone and vegetation cover with thermophile deciduous forests and high densities of roe deer . A bootstrap aggregation procedure (bagging) was used to produce a model for the prediction of tick occurrence, the accuracy of which was tested on actual tick counts assessed by a further dragging campaign carried out during 1997 to determine infection prevalence and resulted in average 77% . Other tests of the model were made on additional and independent data sets . The prevalence of infection with Borrelia burgdorferi s.l, determined by polymerase chain reaction on 2,208 nymphs collected by random dragging in 245 transects selected within eight areas where the model predicted the occurrence of I . ricinus during 1997, was 17.5% and was positively correlated to tick abundance and roe deer density . These findings were used to relate the output of the bagged model (probability of tick occurrence) to the density of infected nymphs through a stepwise model selection procedure and thus to produce a GIS digital map of the probability distribution of infected nymphs in the Province of Trento at high resolution scale (50 by 50-m cell resolution) . The application of the bagging procedure increased the accuracy of the prediction made by a single classification tree, a well-known classification method for the analysis of epidemiological data. Nucleic Acids Res, 2002 Jun 15, 30(12), 2656 - 62 DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus; Napoli A et al.; Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea SULFOLOBUS: Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending . On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones . We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology . We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex . Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology . Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones . Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase . The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments. J Am Chem Soc, 2002 Jun 19, 124(24), 7235 - 9 Determination of (1)H homonuclear scalar couplings in unlabeled carbohydrates; Zwahlen C et al.; The scarcity of structural information on carbohydrates results from combined difficulties to crystallize and the limitations in NMR analysis . Current methods for determining basic NMR parameters such as (1)H homonuclear scalar couplings are very limited, especially for large molecules such as polysaccharides, oligonucleotides, and the carbohydrate part of glycoproteins . In this paper, a NMR experiment for the determination of endocyclic (1)H homonuclear scalar couplings ((3)J(HH)) in unlabeled carbohydrates is presented . In addition to scalar couplings, cross-correlated dipole-dipole relaxation rates were measured for large polysaccharides . The measurement of all endocyclic homonuclear coupling constants within monosaccharide units is presented for lactose, a model disaccharide, and for a natural-abundance 2 MDa bacterial polysaccharide excreted by Streptococcus thermophilus Sfi39. J Mol Biol, 2002 Jun 7, 319(3), 673 - 83 Directed evolution of restriction endonuclease BstYI to achieve increased substrate specificity; Samuelson JC et al.; Restriction endonucleases have proven to be especially resistant to engineering altered substrate specificity, in part, due to the requirement of a cognate DNA methyltransferase for cellular DNA protection . The thermophilic restriction endonuclease BstYI recognizes and cleaves all hexanucleotide sequences described by 5'-R GATCY-3' (where R=A or G and Y=C or T) . The recognition of a degenerate sequence is a relatively common feature of the more than 3000 characterized restriction endonucleases . However, very little is known concerning substrate recognition by such an enzyme . Our objective was to investigate the substrate specificity of BstYI by attempting to increase the specificity to recognition of only AGATCT . By a novel genetic selection/screening process, two BstYI variants were isolated with a preference for AGATCT cleavage . A fundamental element of the selection process is modification of the Escherichia coli host genomic DNA by the BglII N4-cytosine methyltransferase to protect AGATCT sites . The amino acid substitutions resulting in a partial change of specificity were identified and combined into one superior variant designated NN1 . BstYI variant NN1 displays a 12-fold preference for cleavage of AGATCT over AGATCC or GGATCT . Moreover, cleavage of the GGATCC sequence is no longer detected . This study provides further evidence that laboratory evolution strategies offer a powerful alternative to structure-guided protein design . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 May 3, 318(3), 707 - 21 Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases; Dalhus B et al.; The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared . In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases . The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases . The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C . Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified . It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit . Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored . This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer . A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions . (c) 2002 Elsevier Science Ltd. Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 945 - 51 Comparison between Streptococcus macedonicus and Streptococcus waius strains and reclassification of Streptococcus waius (Flint et at . 1999) as Streptococcus macedonicus (Tsakalidou et al . 1998); Manachini PL et al.; Two species of dairy streptococci, Streptococcus waius and Streptococcus macedonicus, were originally characterized by 16S-23S intergenic spacer sequence analysis, random amplified polymorphic DNA fingerprinting, PFGE analysis and DNA-DNA reassociation experiments . All genetic data suggested that S . waius strains belong to the previously described species S . macedonicus . Likewise, the phenotypic characterization showed that strains of S . macedonicus and S . waius were highly related and easily differentiated from the closest phylogenetic neighbour, Streptococcus bovis, principally by their failure to produce a blackening reaction in medium containing aesculin . The utilization of maltose and cellobiose by S . macedonicus/S . waius strains allowed their differentiation from the most studied dairy species, Streptococcus thermophilus . On the basis of genetic and phenotypic data S . macedonicus and S . waius species should be considered synonyms and S . macedonicus has the priority. Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 895 - 900 Caldimonas manganoxidans gen . nov., sp . nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile; Takeda M et al.; A poly(3-hydroxybutyrate) (PHB)-degrading, gram-negative, aerobic bacterium, strain HS(T), was isolated from a hot spring and chemotaxonomically and phylogenetically characterized . The oxidase-positive, weakly catalase-positive, non-pigmented cells (0.6 x 2.6 microm) exhibited a single polar flagellum and accumulated PHB granules . Strain HS(T) was capable of manganese oxidation . Highest growth rate was attained at 50 degrees C . The optimum pH for growth was 7-8 . The major respiratory quinone was ubiquinone-8 and major cellular fatty acids were C16:0, C16:1 and C18:1 . The G+C content of the DNA was 66.2 mol% . Comparative 16S rDNA analysis indicated that strain HS(T) is related to the Rubrivivax subgroup and the family Comamonadaceae . The nearest phylogenetic relatives were Ideonella dechloratans (92.1% similarity), Leptothrix discophora (93.6%), Roseateles depolymerans (92.4%) and Rubrivivax gelatinosus (92.2%) . On the basis of its phylogenetic and phenotypic properties, it is proposed that this isolate be designated Caldimonas manganoxidans gen . nov., sp . nov.; the type strain is HS(T) (= JCM 10698T = IFO 16448T = ATCC BAA-369T). Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 889 - 94 Amycolatopsis eurytherma sp . nov., a thermophilic actinomycete isolated from soil; Kim B et al.; The taxonomic positions of two thermophilic actinomycetes isolated from soil were established in a polyphasic taxonomic study . The organisms were shown to have phenotypic properties typical of members of the genus Amycolatopsis and formed a distinct phyletic line in the Amycolatopsis methanolica 16S rDNA subclade . They also had many phenotypic properties in common and formed a genomic species that was closely related to, albeit distinct from, the type strain of A . methanolica . A range of phenotypic properties distinguished the isolates from representatives of all validly described species of Amycolatopsis . Genotypic and phenotypic data show that the two strains should be classified in the genus Amycolatopsis as a novel species, Amycolatopsis eurytherma sp . nov.; the type strain is strain NT202T (= DSM 44348T = NCIMB 13795T). Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 795 - 800 Thermaerobacter subterraneus sp . nov., a novel aerobic bacterium from the Great Artesian Basin of Australia, and emendation of the genus Thermaerobacter; Spanevello MD et al.; A strictly aerobic, thermophilic, gram-positive, spore-producing, rod-shaped bacterium (2.0-10.0 x 0.3 microm), designated isolate C21T, was isolated from a sample collected from an open drain run-off channel of a bore in the Great Artesian Basin of Australia (New Lorne Bore, registered number 17263) . Isolate C21T grew optimally at 70 degrees C (temperature range for growth was 55-80 degrees C) and pH 8.5 (pH range for growth was 6.0-10.5), with a generation time of 90 min . The isolate was strictly heterotrophic and grew on yeast extract and/or tryptone as carbon and energy sources . An increase in growth was not observed with carbohydrates (sucrose, cellobiose, glucose, dextrin, amylopectin, chitin, carboxymethylcellulose, xylan, inositol, arabinose, mannose, fructose, gelatin, starch, amylose, galactose, dextrose, xylose, maltose, L-sorbose or raffinose), organic acids (lactic acid, pyruvic acid or benzoic acid) or Casamino acids as sole carbon sources or in the presence of yeast extract and/or tryptone . The G+C content of the chromosomal DNA, as measured by the thermal denaturation method, was 71 mol% . Phylogenetic analysis of the 16S rRNA gene of isolate C21T placed it as a member of the phylum Firmicutes, with Thermaerobacter marianensis as the closest relative (similarity value of 98%) . However, isolate C21T and T . marianensis differed in a number of key physiological and phenotypic properties and also had a DNA-DNA hybridization value of less than 5% . Based on this evidence, it is proposed that strain C21T be designated Thermaerobacter subterraneus sp . nov . (type strain C21T = ATCC BAA-137T = DSM 13965T). Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 765 - 72 Thermodesulfobacterium hydrogeniphilum sp . nov., a thermophilic, chemolithoautotrophic, sulfate-reducing bacterium isolated from a deep-sea hydrothermal vent at Guaymas Basin, and emendation of the genus Thermodesulfobacterium; Jeanthon C et al.; A thermophilic, non-spore-forming, marine, sulfate-reducing bacterium, strain SL6T, was isolated from deep-sea hydrothermal sulfides collected at Guaymas Basin . The gram-negative-staining cells occurred singly or in pairs as small, highly motile rods . The temperature range for growth was 50-80 degrees C with an optimum at 75 degrees C . The pH range for growth at 70 degrees C was 6.3-6.8, with an optimum at 6.5 . The NaCl concentration range for growth was 5-55 g l(-1), with an optimum at 30 g l(-1) . H2 and CO2 were the only substrates for growth and sulfate reduction . However, growth was stimulated by several organic compounds . Sulfur, thiosulfate, sulfite, cystine, nitrate and fumarate were not used as electron acceptors . Pyruvate, lactate and malate did not support fermentative growth . Desulfoviridin was not detected . The G+C content of the genomic DNA was 28 mol% . On the basis of 16S rRNA sequence analysis, strain SL6T is related to members of the genus Thermodesulfobacterium . However, the novel organism possesses phenotypic and phylogenetic traits that differ from those of its closest relatives . Therefore, it is proposed that this isolate, which constitutes the first marine representative of this genus, should be described as the type strain of a novel species, Thermodesulfobacterium hydrogeniphilum sp . nov . The type strain is SL6T (= DSM 14290T = JCM 11239T) . Because of the phenotypic characteristics of the novel species, it is also proposed that the description of the genus Thermodesulfobacterium requires emendation. J Parasitol, 2002 Feb, 88(1), 41 - 6 Comparing tolerance of Ichthyophthirius multifiliis and Tetrahymena thermophila for new cryopreservation methods; Everett KD et al.; Ichthyophthirius multifiliis is an obligate protozoan parasite of freshwater fishes that has a complex developmental cycle . It has not been successfully cryopreserved, so management studies are restricted to parasites obtained during outbreaks or perpetuated by passage in live fishes . To overcome this serious limitation, free-swimming I . multifiliis parasites were tested in a cryopreservation protocol routinely used for a related ciliate, Tetrahymena . In this protocol, I . multifiliis theronts retained infectivity for 3 days, although the protocol itself was ultimately lethal . Exposure of I . multifiliis and Tetrahymena thermophila to a battery of media and cryopreservative reagents showed that I . multifiliis was less hardy than T . thermophila and likely had significant biological and cytoskeletal differences . No combination of reagents, media, freezing rates, or dilution media permitted cryopreservation of I . multifiliis parasites that could then undergo development or infect fish . However, a vitrification protocol was formulated using Ficoll, 1,2-propanediol, and N,N-dimethylacetamide from which intact cryopreserved theronts with some motility were recovered . Understanding the effects of these reagents may lead to both a cryopreservation method for I . multifiliis and to improved understanding of the biology of ciliates. EMBO Rep, 2002 Jun, 3(6), 537 - 42 Epub 2002 Jun 01. NurA, a novel 5'-3' nuclease gene linked to rad50 and mre11 homologs of thermophilic Archaea; Constantinesco F et al.; We isolated and characterized a new nuclease (NurA) exhibiting both single-stranded endonuclease activity and 5'-3' exonuclease activity on single-stranded and double-stranded DNA from the hyperthermophilic archaeon Sulfolobus acidocaldarius . Nuclease homologs are detected in all thermophilic archaea and, in most species, the nurA gene is organized in an operon-like structure with rad50 and mre11 archaeal homologs . This nuclease might thus act in concert with Rad50 and Mre11 proteins in archaeal recombination/repair . To our knowledge, this is the first report of a 5'-3' nuclease potentially associated with Rad50 and Mre11-like proteins that may lead to the processing of double-stranded breaks in 3' single-stranded tails. FEMS Microbiol Lett, 2002 May 21, 211(1), 97 - 103 Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters; Moreno Y et al.; In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples . 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability . Eleven different types were defined at 100% linkage level following numerical analysis of band patterns . Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains . Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns . The resulting dendrogram showed a high diversity among the strains studied . All the isolates were grouped within eight main types with a 69% homology degree among them . Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains . When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters. J Mol Biol, 2002 May 31, 319(2), 541 - 54 The effects of ionic strength on protein stability: the cold shock protein family; Dominy BN et al.; Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations . Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods . The model is validated by comparison with experimental data collected for these proteins . In addition, a number of single point mutants and three designed sequences are examined . The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family . The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized . A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability . This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt . Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins . Med Pr, 2002, 53(1), 29 - 39 {Biologic factors hazardous to health: classification and criteria of exposure assessment}; Dutkiewicz J et al.; Occupational biohazards include not only factors that have long been known (viruses, bacteria, fungi, parasites), but also agents exerting allergic and toxic effects, which are directly responsible for the development of various diseases in many occupational groups . Numerous agents of this group (allergens, microbial toxins, pollen allergens and allergens of animal origin) are components of bioacrosols--potential hazards inducing occupational respiratory diseases among farmers and people involved in other occupations . Contrary to the majority of chemical and physical factors, neither commonly approved criteria for assessing exposure to biological factors, nor threshold values and methodological recommendations are as yet available . The lack of these criteria renders it difficult to implement in Poland and in other countries Directive 2000/54/EC on the protection of workers against the risk of occupational exposure to biohazards, issued by the European Community . The Institute of Rural Medicine in Lublin has drafted the proposals for threshold limit values of occupational exposure to bioaerosols associated with plant and animal dusts, including: mesophilic bacteria, Gram-negative bacteria, thermophilic actinomycetes, fungi and bacterial endotoxins . These proposals could be considered as a starting point for developing appropriate facultative standards that would facilitate the practical implementation of the aforesaid Directive . Meantime it is essential to be strict in following the binding concentration limits of the plant and animal dusts in the air. J Biol Chem, 2002 Sep 13, 277(37), 33559 - 63 Epub 2002 Jun 04. The Escherichia coli cytochrome c maturation (Ccm) system does not detectably attach heme to single cysteine variants of an apocytochrome c; Allen JW et al.; Cytochromes c are typically characterized by the covalent attachment of heme to polypeptide through two thioether bonds with the cysteine residues of a Cys-Xaa-Xaa-Cys-His peptide motif . In many Gram-negative bacteria, the heme is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins . Exceptionally, Hydrogenobacter thermophilus cytochrome c(552), which has a normal CXXCH heme-binding motif, and variants with AXXCH, CXXAH, and AXXAH motifs, can be expressed as stable holocytochromes in the cytoplasm of Escherichia coli . By targeting these proteins to the periplasm using a signal peptide, with or without co-expression of the Ccm proteins, we have assessed the ability of the Ccm system to attach heme to proteins with no, one, or two cysteine residues in the heme-binding motif . Only the wild-type protein, with two cysteines, was effectively processed and thus accumulated in the periplasm as a holocytochrome . This is strong evidence for disulfide bond formation involving the two cysteine residues of apocytochrome c as an intermediate in Ccm-type Gram-negative bacterial cytochrome c biogenesis and/or that only a pair of cysteines can be recognized by the heme attachment apparatus. Trends Genet, 2002 May, 18(5), 236 - 7 A hot story from comparative genomics: reverse gyrase is the only hyperthermophile-specific protein; Forterre P; I have looked for proteins that are present in all hyperthermophile genomes, but absent from all mesophile or thermophile genomes by using the phylogenetic pattern search program of the COG database . Surprisingly, this search retrieved only one such hyperthermophile-specific protein: reverse gyrase . This result emphasizes the importance of reverse gyrase in the adaptation of life to very high temperatures, and strengthens the idea that evolution of this enzyme was crucial in the origin of hyperthermophiles. J Dairy Res, 2002 Feb, 69(1), 125 - 37 Evolution of carbohydrate fraction in carbonated fermented milks as affected by beta-galactosidase activity of starter strains; Guetmonde M et al.; The influence of carbonation on the evolution of lactose, galactose and glucose in fermented milks with added probiotic bacteria (Lactobacillus casei, Lactobacillus acidophilus and/or Bifidobacterium bifidum) was evaluated and related to beta-galactosidase activity of starter strains . During incubation and first days of refrigeration, lactose hydrolysis resulting in the liberation of galactose and glucose occurred in CT (Streptococcus thermophilus/Lb . casei), AT (Str . thermophilus/Lb . acidophilus) and ABT fermented milks (Str . thermophilus/Lb . acidophilus/Bifid . bifidum) . Levels of galactose were higher than those of glucose and could be related to the preferential consumption of glucose by actively growing bacteria . Through the incubation, lactose and monosaccharide levels were not affected by milk carbonation . However, during refrigerated storage the presence of this gas was associated with slightly lower content of lactose and higher levels of galactose and glucose in AT and ABT products but not in CT fermented milks . Through the refrigeration galactose was moderately utilised by Lb . acidophilus in AT products whereas the presence of Bifid . bifidum seems to prevent the consumption of this sugar in ABT fermented milks . Glucose remained constant, with minor variations in CT products but a continuous increase of this sugar occurred in carbonated AT and ABT fermented milks during storage . Beta-galactosidase activity displayed by Str . thermophilus strains was similar at pH 6.5 (initial pH of non-carbonated samples) and pH 6.3 (initial pH of carbonated samples) whereas Lb . acidophilus LaA3 showed greater beta-galactosidase activity at pH 6.3 than at higher pH values . Thus, the enhanced metabolic activity of Lb . acidophilus caused by the low initial pH of carbonated milk also promoted higher cellular beta-galactosidase activity that could have released greater amounts of galactose and glucose from lactose in AT and ABT fermented milks through the refrigerated period . In CT fermented milks, similar beta-galactosidase activity levels of Str . thermophilus at pH 6.5 and 6.3 together with the absence of beta-galactosidase activity in Lb . casei could explain the lack of differences on glucose and galactose content between carbonated and non-carbonated samples. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(4), 563 - 71 Quantitative structure-activity relationships for the toxicity of nitrobenzenes to Tetrahymena thermophila; Xu JB et al.; In this study IGC50 (50% inhibitory growth concentration) values of 26 nitrobenzenes were determined for population growth endpoint of Tetrahymena thermophila . The toxicity order of the observed compounds has been found as follows: dinitro compounds > mono-nitro compounds; dichloronitrobenzenes > monochloronitrobenzenes; and meta-substituted nitrobenzenes > ortho-/para-substituted nitrobenzenes (NT, NPh, NAnis) except for the dinitrobenzenes and nitroanilines (DNB, NAn) . Quantitative structure activity relationships (QSARs) were developed using log of the inverse of the IGC50 (logIGC50(-1)) in mole liter as the dependent variable and six molecular descriptors--logP, 1X(V), I, K alpha, sigma sigma- and E(LUMO) as the independent variables . Through multiplicate regression analysis, one best equation was obtained: log IGC50(-1) = 2.93 + 0.830sigma sigma- + 0.350I, n = 26, r = 0.923, r2 = 0.852, s = 0.265, f = 66.4 The equation was used to estimate IGC50 for seven analogues. Trends Genet, 2002 Jun, 18(6), 278 - 81 Evidence for cysteine clustering in thermophilic proteomes; Rosato V et al.; Through linguistic analysis, we show that the presence of an amino acid at a given position within a proteome positively influences the presence of identical amino acids at nearby positions . We call this phenomenon 'amino acid clustering' . Clustering extends well beyond the closest neighbouring sites and is particularly pronounced for cysteine and tryptophan . Cysteine clusters preferentially form CXXC structures, and they are often involved in metal coordination or disulfide bond formation . Cysteine clustering shows a clear correlation with the growth temperature of the organism . This seems to be a general property of living organisms. Water Res, 2002 Apr, 36(7), 1869 - 79 Mesophilic and thermophilic activated sludge post-treatment of paper mill process water; Vogelaar JC et al.; Increasing system closure in paper mills and higher process water temperatures make the applicability of thermophilic treatment systems increasingly important . The use of activated sludge as a suitable thermophilic post-treatment system for anaerobically pre-treated paper process water from a paper mill using recycled wastepaper was studied . Two lab-scale plug flow activated sludge reactors were run in parallel for 6 months; a thermophilic reactor at 55 degrees C and a reference reactor at 30 degrees C . Both reactors were operated simultaneously at 20, 15 and 10 days SRT . The effects of temperature and SRT on sludge settleability and chemical oxygen demand (COD) removal efficiencies of different fractions were studied . Total COD removal percentages over the whole experimental period were 58+/-5% at 30 degrees C and 48 +/- 10% at 55 degrees C . The effect of the SRT on the total COD removal was negligible . Differences in total COD removal between both systems were due to a lesser removal of soluble and colloidal COD at 55 degrees C compared to the reference system . At 30 degrees C, colloidal COD removal percentages were 65+/-25%, 75+/-17% and 86+/-22% at 20, 15 and 10 days SRT, respectively . At 55 degrees C, these percentages were 48+/-34%, 40+/-28% and 70+/-25%, respectively . The effluent concentrations of colloidal COD in both systems were related to the influent concentration of colloidal material . The thermophilic sludge was not able to retain influent colloidal material as well as the mesophilic sludge causing a higher thermophilic effluent turbidity . Sludge settling properties were excellent in both reactor systems . These were neither temperature nor SRT dependent but were rather caused by extensive calcium precipitation in the aeration tanks creating a very dense sludge . For application in the board industry, a thermophilic in line treatment system seems feasible . The higher effluent turbidity is most likely offset by the energy gains of treatment under thermophilic conditions. Water Res, 2002 Apr, 36(7), 1825 - 33 Optimisation of sulphate reduction in a methanol-fed thermophilic bioreactor; Weijma J et al.; Several methods were tested to optimise sulphate reduction and minimise methane formation in thermophilic (65 degrees) expanded granular sludge bed reactors fed with a medium containing sulphate and methanol . Lowering the pH from 7.5 to 6.75 resulted in a rapid decrease of methane formation and a concomitant increase in sulphate reduction . The inhibition of methane formation was irreversible on the short-term . Lowering the COD/SO4(2-) ratio (COD: chemical oxygen demand) from 6 to 0.34 (g/g) rapidly favoured sulphate reduction over methanogenesis . Continuous addition of 2 g L(-1) 2-bromoethanesulphonate was ineffective as complete inhibition of methanogenesis was obtained only for two days . Inhibition of methanogens by sulphide at pH 7.5 was only effective when the total sulphide concentration was above 1200 mg S L(-1) . For practical applications, a relatively short exposure to a slightly acidic pH in combination with operating the reactor at a volumetric methanol-COD loading rate close to the maximum volumetric sulphide-COD formation rate. J Eukaryot Microbiol, 2002 Mar-Apr, 49(2), 99 - 107 Analysis of expressed sequence tags (ESTs) in the ciliated protozoan Tetrahymena thermophila; Fillingham JS et al.; To assess the utility of expressed sequence tag (EST) sequencing as a method of gene discovery in the ciliated protozoan Tetrahymena thermophila, we have sequenced either the 5' or 3' ends of 157 clones chosen at random from two cDNA libraries constructed from the mRNA of vegetatively growing cultures . Of 116 total non-redundant clones, 8.6% represented genes previously cloned in Tetrahymena . Fifty-two percent had significant identity to genes from other organisms represented in GenBank, of which 92% matched human proteins . Intriguing matches include an opioid-regulated protein, a glutamate-binding protein for an NMDA-receptor, and a stem-cell maintenance protein . Eleven-percent of the non-Tetrahymena specific matches were to genes present in humans and other mammals but not found in other model unicellular eukaryotes, including the completely sequenced Saccharomyces cerevisiae . Our data reinforce the fact that Tetrahymena is an excellent unicellular model system for studying many aspects of animal biology and is poised to become an important model system for genome-scale gene discovery and functional analysis. Aust Fam Physician, 2002 Apr, 31(4), 399 - 400 The history of the discovery of primary amoebic meningoencephalitis; Cooter R; BACKGROUND: Primary amoebic meningoencephalitis was first recognised by a South Australian pathologist . The histopathological appearances indicated that the organism, Naegleria fowleri, entered the central nervous system from the nasal cavity via the cribriform plate . But the mode of transmission remained unknown . AIMS: To describe how the pathogenesis of this condition was discovered, and correct misinformation about the events and persons involved in this process . HYPOTHESIS: We hypothesised that pipeline water supplying northern centres in South Australia was responsible for transmitting thermophilic amoebae during the summer months . EVIDENCE: The evidence supporting our hypothesis was: domestic water pipelines were exposed to sunlight and became heated to 35-45 degrees C in summer which promoted the formation of vegetative forms of the amoebae; some patients described using tap water to flush their nasal cavities; and Naegleri fowleri were eventually recovered from domestic tap water supplies . CONCLUSION: A successful collaboration between general practitioners and laboratory scientists elucidated the pathogenesis of primary amoebic encephalomyelitis, a serious public health hazard in South Australia from 1947 until the early 1970s. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(5), 577 - 581 Effect of Thermozeaxanthins on Liposome Membranes in Acidic and Basic Conditions; Yuan HQ et al.; Thermozeaxanthins (TZS), a group of carotenoid-glucoside esters extracted from the lipids fraction of thermophilic bacterium, are amphiphilic compounds . The effects of TZS on membrane permeability were examined on large unilamellar liposomes (LUVs) in different pH buffers . The LUVs were composed of 0.01 molar of TZS and phosphatidylcholine (PC) of various lengths and saturation degrees of hydrocarbon chains . The results showed that the LUVs containing TZS were more stable than that liposomes without TZS in pH 5.0, and no significant effects in pH 8.2 and 9.0 buffer solutions . The liposomes composed of TZS and egg PC or E.coli PE were better than those DPPC or DOPC LUVs containing TZS at same experimental conditions . There was no observed difference in stability between the liposomes with or without TZS in pH 6.5 solution . Conclusions (1) Matching of rigid TZS in the lipid bilayer was important in stabilization effects on liposomes, (2)The TZS showed some stabilization effects on liposome membranes in acidic conditions and no significant effects in basic environment. Plant Cell Physiol, 2002 May, 43(5), 484 - 93 The complete purification and characterization of three forms of ferredoxin-NADP(+) oxidoreductase from a thermophilic cyanobacterium Synechococcus elongatus; Nakajima M et al.; The petH gene, encoding ferredoxin-NADP(+) oxidoreductase (FNR), was isolated from a thermophilic cyanobacterium, Synechococcus elongatus (the same strain as Thermosynechococcus elongatus) . The petH gene of S . elongatus was a single copy gene, and the N-terminal region of PetH showed a sequence similarity to the CpcD-phycobilisome linker polypeptide . The amino acid sequence of the catalytic domains of PetH was markedly similar to those from mesophilic cyanobacterial PetH and higher plant FNR . The enzymatically active FNR protein was purified to homogeneity from S . elongatus as three forms corresponding to the 45-kDa form retaining the CpcD-like domain, the 34-kDa form lacking the CpcD-like domain, and the 78-kDa complex with phycocyanin . The FNR in the 78-kDa complex was tolerant to proteolytic cleavage . However, the dissociation of phycocyanin from the 78-kDa complex induced to specific proteolysis between the CpcD-like domain and the FAD-binding domain to give rise to the 34-kDa form of FNR . The enzymatic activity of the 45-kDa form was thermotolerant, but the 45-kDa form readily aggregated under the storage at -30 degrees C . These results suggest that the association with phycocyanin via CpcD-like domain gives remarkable stability to S . elongatus FNR. Appl Environ Microbiol, 2002 Jun, 68(6), 3176 - 9 Chi18A, the endochitinase in the cellulosome of the thermophilic, cellulolytic bacterium Clostridium thermocellum; Zverlov VV et al.; The chitinase gene chiA was identified on the Clostridium thermocellum genome downstream of the endoglucanase gene celA . It contains a catalytic module of glycosyl hydrolase family 18 and a cellulosomal dockerin module . Chi18A hydrolyzes aryl-acetyl-chito-oligosaccharides preferentially . In denaturing electrophoresis of purified cellulosomes, a single chitinase activity band was identified in zymograms and Western blots, indicating that Chi18A is the only chitinase in the cellulosome. Appl Environ Microbiol, 2002 Jun, 68(6), 3162 - 5 Casein utilization by Streptococcus thermophilus results in a diauxic growth in milk; Letort C et al.; In milk, Streptococcus thermophilus displays two distinct exponential growth phases, separated by a nonexponential one, during which proteinase synthesis was initiated . During the second exponential phase, utilization of caseins as the source of amino acids resulted in a decrease in growth rate, presumably caused by a limiting peptide transport activity. Appl Environ Microbiol, 2002 Jun, 68(6), 3046 - 54 Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine; Takai K et al.; Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine . Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated . 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer . The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors . Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine. Curr Opin Chem Biol, 2002 Apr, 6(2), 151 - 60 Starch-hydrolyzing enzymes from thermophilic archaea and bacteria; Bertoldo C et al.; Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions . For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes . This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer . In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, and cyclodextrin glycosyltransferase . Comparison of the primary sequence of the pyrococcal pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes. J Biochem (Tokyo), 2002 Jun, 131(6), 849 - 53 Polypeptide synthesis directed by DNA as a messenger in cell-free polypeptide synthesis by extreme thermophiles, Thermus thermophilus HB27 and Sulfolobus tokodaii strain 7; Uzawa T et al.; Polypeptide synthesis at high temperature directed by single strand DNA as a messenger was investigated using cell-free extracts of an extremely thermophilic bacterium, Thermus thermophilus strain HB27, and a hyperthermophilic, acidophilic archaeon, Sulfolobus tokodaii strain 7 . Aminoglycoside antibiotics enhanced the reaction; neomycin stimulated it most effectively when the extract of the thermophilic bacterium was used, and paromomycin was the best among the antibiotics tested for the extract of the hyperthermophilic archaeon . A common correlation was found between the stimulation of DNA-directed polypeptide synthesis and the misreading rate in RNA-directed polypeptide synthesis . Spermine stimulated the reaction directed by DNA like in the case of poly(Phe) synthesis directed by poly (rU) . The cell-free systems can be used for direct production of proteins from genes in high throughput studies on the structural genomics of thermophilus. Int J Food Microbiol, 2002 Jun 5, 76(1-2), 93 - 105 Effect of Enterococcus faecium on microbiological, physicochemical and sensory characteristics of Greek Feta cheese; Sarantinopoulos P et al.; Greek Feta cheese was prepared using as adjunct starter cultures Enterococcus faecium FAIR-E 198, E . faecium FAIR-E 243, and their combination . Numbers of enterococci in the control and in the batches containing E . faecium strains as adjunct starters rapidly increased until day 15 of ripening, and then remained constant . Both E . faecium strains positively affected the counts of non-starter lactic acid bacteria (NSLAB), micrococci and coliforms, while thermophilic cocci were not influenced . Moreover . E . faecium FAIR-E 243 enhanced the growth of mesophilic cocci and thermophilic bacilli . Physicochemical characteristics, such as pH, moisture, ash, salt in moisture and fat in dry matter (FDM) were not influenced by the addition of the E . faecium strains . The most pronounced effect was observed in the case of proteolysis . Both E . faecium strains, either as sole adjunct starter or in combination, increased the proteolytic index and the free amino groups concentration, and enhanced degradation of alpha(s1)- and beta-caseins in comparison to the control . Furthermore, the reverse-phase (RP)-HPLC peptide profiles of the water-soluble nitrogen (WSN) fractions were significantly affected by the addition of enterococci . The main volatile compounds produced were ethanol, acetate, acetone, acetaldehyde, acetoin and diacetyl, with highest amounts determined for ethanol, followed by acetate . Both E . faecium strains positively affected taste, aroma, colour and structure of the full-ripened cheeses, as well as the overall sensory profile . The present work emphasizes the technological significance of E . faecium strains and supports their use as adjunct cultures in the manufacture of Feta cheese. Int J Food Microbiol, 2002 Jun 5, 76(1-2), 27 - 38 Use of PCR-based methods and PFGE for typing and monitoring homofermentative lactobacilli during Comté cheese ripening; Bouton Y et al.; This study investigated the genotypic characteristics of selected and wild homofermentative thermophilic lactobacilli strains during ripening of Comte cheeses, made into two cheese plants . Both amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) and classical biochemical tests were used to identify isolates . Diversity within homofermentative lactobacilli was not found in their species composition since the same two species Lactobacillus helveticus and L . delbrueckii susbp . lactis were isolated from cheeses . In cheeses made with natural whey starter, it appeared that the most likely sources of L . helveticus and L . delbrueckii susbp . lactis were the starter and raw milk, respectively . The examination of RAPD profiles of lactobacilli strains revealed 19 RAPD groups among 50 isolates, which were different from selected starter strains . Using RAPD, REP-PCR, and PFGE to identify selected starter strains during cheese ripening, we showed that L . helveticus decreased quickly while L . delbrueckii susbp . lactis sustained high viability during ripening . The use of selected L . delbrueckii susbp . lactis strains diminished the genetic diversity among strains isolated from cheese, probably in preventing the raw milk microflora from growing. Microb Ecol, 2002 Apr, 43(3), 378 - 87 Epub 2002 Mar 28. Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor; Plugge CM et al.; A dialysis cultivation system was used to enrich slow-growing moderately thermophilic anaerobic bacteria at high cell densities . Bicarbonate buffered mineral salts medium with 5 mM glutamate as the sole carbon and energy source was used and the incubation temperature was 55 degrees C . The reactor inoculum originated from anaerobic methanogenic granular sludge bed reactors . The microbial population was monitored over a period of 2 years using the most probable number (MPN) technique . In the reactor glutamate was readily degraded to ammonium, methane, and carbon dioxide . Cell numbers of glutamate-degrading organisms increased 400-fold over the first year . In medium supplemented with bromoethane sulfonic acid (BES, an inhibitor of methanogenesis), tenfold lower cell numbers were counted, indicating the syntrophic nature of glutamate degradation . After 2 years of reactor operation the predominant organisms were isolated and characterized . Methanobacterium thermoautotrophicum (strain R43) and a Methanosaeta thermophila strain (strain A) were the predominant hydrogenotrophic and acetoclastic methanogens, respectively . The numbers in which the organisms were present in the reactor after 24 months of incubation were 8.6 x 10(9) and 3.8 x 10(7) mL(-1) sludge, respectively . The most predominant glutamate-degrading organism (8.6 x 10(7) mL(-1) sludge), strain Z, was identified as a new species, Caloramator coolhaasii . It converted glutamate to hydrogen, acetate, some propionate, ammonium, and carbon dioxide . Growth of this syntrophic organism on glutamate was strongly enhanced by the presence of methanogens. Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1039 - 41 Epub 2002 May 29. Expression, refolding and crystallization of Aquifex aeolicus elongation factor P; Kristensen O et al.; Elongation factor P is a universally conserved protein stimulating peptidyltransferase activity during protein synthesis . The factor is sensitive to classical inhibitors of the ribosomal peptidyltransferase activity and is possibly involved in alignment of the substrate tRNAs in the catalytic centre of 70S ribosomes . Elongation factor P from the thermophilic Aquifex aeolicus was overexpressed as a soluble protein in Escherichia coli and crystallized . A fast generally applicable refolding protocol was developed to improve crystal quality and circumvent strong binding of oligonucleotides to the protein . Diffraction data collected to 2.7 A resolution present twinning. Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1023 - 9 Epub 2002 May 29. Structure of ribosomal protein L1 from Methanococcus thermolithotrophicus . Functionally important structural invariants on the L1 surface; Nevskaya N et al.; The crystal structure of ribosomal protein L1 from the archaeon Methanococcus thermolithotrophicus has been determined at 2.7 A resolution . The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 67.0, b = 70.1, c = 106.3 A and two molecules per asymmetric unit . The structure was solved by the molecular-replacement method with AMoRe and refined with CNS to an R value of 18.9% and an R(free) of 25.4% in the resolution range 30-2.7 A . Comparison of this structure with those obtained previously for two L1 proteins from other sources (the bacterium Thermus thermophilus and the archaeon M . jannaschii) as well as detailed analysis of intermolecular contacts in the corresponding L1 crystals reveal structural invariants on the molecular surface which are probably important for binding the 23S ribosomal RNA and protein function within the ribosome. Virology, 2002 Apr 25, 296(1), 62 - 76 Transcription mapping as a tool in phage genomics: the case of the temperate Streptococcus thermophilus phage Sfi21; Ventura M et al.; For the lytic growth cycle of the temperate cos-site Streptococcus thermophilus phage Sfi21 a transcription map was developed on the basis of systematic Northern blot hybridizations . All deduced 5' ends were confirmed by primer extension analysis . Three time classes of transcripts were observed . Early transcripts were identified in four different genome regions . One prominent early mRNA of 4.8 kb length covered a group of 12 genes located between the origin of replication and the cos-site . Two short early mRNAs represented a single gene from the direct vicinity of the cos-site and the superinfection immunity gene from the lysogeny module, respectively . A fourth early transcript covered a group of four genes located between the lysin and the integrase gene . Middle transcripts of 2.1 and 5.8 kb length covered cro-like and ant-like repressor genes and the DNA replication module, respectively . Four types of late transcripts were identified . The transcripts covered the likely DNA packaging genes, the head morphogenesis module plus the major tail gene, the remainder of the tail genes, and the putative tail fiber plus lysis genes, respectively . Only the transcript from the head morphogenesis genes yielded defined late mRNA species . The transcription map concurred with most of the in silico predictions for the genome organization of phage Sfi21 except for the separation of the DNA replication module from a possible transcription regulation module . Most 5' ends of the transcripts determined in primer-extension experiments were not preceded by a consensus promoter sequence . The involvement of phage-encoded regulators for middle and late transcription was suggested by chloramphenicol-inhibition experiments. Microb Ecol, 2000 Apr, 39(3), 246 - 262 Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes; Zheng D et al.; A BSTRACTTo evaluate the role of Methanosaeta spp . in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized . The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila . The temperature of dissociation was determined for each probe . Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors . Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated . Although permeability of methanogens was variable, M . concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions . Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp . were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low . Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge . In lab-scale upflow anaerobic sludge blanket reactors, the levels of M . concilii rRNA were as high as 30% of the total rRNA. Nucleic Acids Res . 2002 Jun 1;30(11):e49. A Tetrahymena thermophila ribozyme-based indicator gene to detect transposition of marked retroelements in mammalian cells; Esnault C et al.; We devised an indicator gene for retrotransposition based on an autocatalytic ribozyme element--the Tetrahymena thermophila 23S rRNA group I intron--which can self-splice in vitro and does not require--at variance with nuclear mRNA introns--any specific pathway and cellular component for the completion of the splicing process . Several constructs, with the Tetrahymena intron adequately modified so as to be inserted at various positions within a neomycin-containing cassette under conditions that restore the neomycin-coding sequence after splicing out of the intron, were assayed for splicing efficiency in mammalian cells in culture . We show, both by northern blot analysis and by the recovery of neomycin activity upon retroviral transduction of the cassettes, that splicing efficiency depends on both the local base pairing and the global position of the intron within the neomycin transcript, and that some constructs are functional . We further show that they allow the efficient sorting out of retrotransposition events when assayed, as a control, with a human LINE retrotransposon . These indicator genes should be of great help in elucidating the mechanisms of transposition of a series of retroelements associated with transcripts not prone to nuclear mRNA intron splicing and previously not opened to any retrotransposition assay. Nucleic Acids Res, 2002 Jun 1, 30(11), 2524 - 37 A novel family of mobile genetic elements is limited to the germline genome in Tetrahymena thermophila; Wuitschick JD et al.; In the ciliated protozoan Tetrahymena thermophila, extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germline micronucleus . This study describes the isolation and complete characterization of Tlr elements, a family of approximately 30 micronuclear DNA sequences that are efficiently eliminated from the developing macronucleus . The data indicate that Tlr elements are comprised of an approximately 22 kb internal region flanked by complex and variable termini . The Tlr internal region is highly conserved among family members and contains 15 open reading frames, some of which resemble genes encoded by transposons and viruses . The Tlr termini appear to be long inverted repeats consisting of (i) a variable region containing multiple direct repeats which differ in number and sequence from element to element and (ii) a conserved terminal 47 bp sequence . Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the Tetrahymena germline genome . Possible mechanisms of developmentally programmed Tlr elimination are discussed. Biochim Biophys Acta, 2002 Apr 22, 1554(1-2), 29 - 35 Increased tolerance to thermal inactivation of oxygen evolution in spinach Photosystem II membranes by substitution of the extrinsic 33-kDa protein by its homologue from a thermophilic cyanobacterium; Pueyo JJ et al.; Photosynthetic oxygen evolution is an extremely heat-sensitive process and incubation of spinach Photosystem II (PSII) membranes at 40 degrees C for only several minutes leads to its complete inactivation . Substitution experiments of the spinach 33-kDa manganese stabilizing protein by a homologue protein, isolated either from the thermophilic cyanobacterium Phormidium laminosum, or from Escherichia coli as a recombinant thermophilic cyanobacterial protein, showed a significant increase in tolerance to heat inactivation of the oxygen-evolving activity . The results allow us to suggest that thermal inactivation of oxygen evolution in higher plant PSII membranes is due to dissociation of the 33-kDa protein as a consequence of temperature-induced conformational changes, and stabilization can be provided by substitution by a thermostable homologue whose secondary structure and binding to PSII remain unaltered at moderately high temperatures. Biophys Chem, 2002 May 2, 96(2-3), 229 - 41 Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study; Nemeth A et al.; Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies . We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins . Cellulase C has a TIM barrel fold with an additional helical subdomain . We designed and produced a number of mutants with the aim to increase its thermostability . Five mutants were designed to create new electrostatic interactions . They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme . Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains . In another mutant, the small helical subdomain was deleted . This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function . A double mutant was designed to introduce a new disulfide bridge into the enzyme . This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme . Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior . We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein . Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects. J Agric Food Chem, 2002 Jun 5, 50(12), 3507 - 11 Formation of bound residues of 8-hydroxybentazon by oxidoreductive catalysts in soil; Kim JS et al.; This study was performed to determine which oxidoreductive catalysts were most efficient in catalyzing the binding of 8-hydroxybentazon to soil humic substances . 8-Hydroxybentazon was completely transformed by an oxidoreductive enzyme, laccase of Myceliophthora thermophila, at pH 3.0-7.0 within 30 min . When abiotic catalysts, manganese(IV), iron(III), and aluminum oxides were used in the same pH range, 8-hydroxybentazon was completely transformed only by manganese(IV) oxide (delta-MnO2), but a relatively small amount of 8-hydroxybentazon was transformed by iron(III) oxide and aluminum oxide . The adsorption of 8-hydroxybentazon in the soil showed an H-type and coincided well with the Langmuir isotherm . To better understand the factors involved in the rapid and strong binding of 8-hydroxybentazon with soil humic substances, 8-hydroxybentazon transformation by oxidoreductive catalysts was studied in various soil conditions: air-dried, preincubated, sterilized, and iron(III) oxide and manganese(IV) oxide free . 8-Hydroxybentazon was completely transformed within 24 h in the decreasing order of preincubated, air-dried, and sterilized soils . However, little transformation was observed in the iron(III) oxide and manganese(IV) oxide free soils . These results suggest that the major catalyst responsible for the rapid and strong binding of 8-hydroxybentazon to soil humic substances is a metal oxide, manganese(IV) oxide, not a soil oxidoreductive enzyme. J Agric Food Chem, 2002 Jun 5, 50(12), 3479 - 85 Proteolysis in Hispánico cheese manufactured using a mesophilic starter, a thermophilic starter, and bacteriocin-producing Lactococcus lactis subsp . lactis INIA 415 adjunct culture; Garde S et al.; Lactococcus lactis subsp . lactis INIA 415, a strain harboring the structural genes of bacteriocins nisin Z and lacticin 481, was used as adjunct culture in the manufacture of Hispanico cheese with a mesophilic starter and a thermophilic starter of high aminopeptidase activity . Addition of the bacteriocin producer promoted early lysis of mesophilic and thermophilic starter bacteria . Extracellular aminopeptidase activity in 7-day-old cheese made using mesophilic and thermophilic starters plus bacteriocin producer was 3.0-fold the level reached in cheese made without the bacteriocin producer . Proteolysis in cheese made with mesophilic and thermophilic starters plus bacteriocin-producing adjunct culture after 25 days of ripening was 1.5-fold the level reached in cheese made without the bacteriocin producer, and the level of total free amino acids was 2.9-fold the level found in cheese made without the bacteriocin producer . Cheese made with mesophilic and thermophilic starters plus bacteriocin producer received the highest scores for flavor quality and flavor intensity and reached in 25 days the flavor intensity score of a 75-day-old cheese made without the bacteriocin producer. J Ind Microbiol Biotechnol, 2002 Jun, 28(6), 344 - 8 Production and properties of an alkaline, thermophilic lipase from Pseudomonas fluorescens NS2W; Kulkarni N et al.; Eighteen bacterial strains were isolated from soil samples and screened for alkaline, thermophilic lipase production . Pseudomonas fluorescens NS2W was selected and its production of lipase was optimized in shake flasks using a statistical experimental design . Cell growth and lipase production were studied in shake flasks and in a 1-l fermenter in the optimized medium . Maximum lipase yields were 69.7 and 68.7 U ml(-1), respectively . The optimized medium resulted in about a five-fold increase in the enzyme production, compared to that obtained in the basal medium . The lipase had an optimal activity at pH 9.0 and was stable over a wide pH range of 3-11 with more than 70% activity retention . The lipase had an optimal activity at 55 degrees C and was stable up to 60 degrees C with more than 70% activity retention for at least 2 h. Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7514 - 7 Importance of compartment formation for a self-encoding system; Matsuura T et al.; A self-encoding system designed to have strict "compartition" of the molecules, i.e., to contain only a single molecule of DNA in each compartment, was established, and its evolutionary fate was analyzed . The system comprised the Thermus thermophilus DNA polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule . Imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system ceased to work after the third generation when loose compartition initiated with 100 molecules was imposed . These results provide experimental evidence on the importance of compartition for the maintenance of a self-encoding system . In addition, the extent of diversity in self-replication activity of the compartments was found to be another vital difference in the evolutionary dynamics between the strict and loose compartitions . Although the system with strict compartition provides widely diversified activity of the compartments at each generation, the values of the activity diverge only within a small range in the system with loose compartition . When the variety in the activity of a compartment is small, functional selection becomes weak, and to conform Darwinian evolution may become unfeasible . Therefore, strict compartition is essential for the evolvability of a self-encoding system. Comp Biochem Physiol B Biochem Mol Biol, 2002 Jun, 132(2), 415 - 22 The highly stable alcohol dehydrogenase of Thermomicrobium roseum: purification and molecular characterization; Yoon SY et al.; An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum . The native enzyme was found to be a homo-dimer of 43-kDa subunits . The pI of the enzyme was determined to be 6.2, while its optimum pH is 10.0 . The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol, n-propanol and crotyl alcohol . The highest reaction rate was observed when ethanol was used as substrate and the K(m) value of the enzyme for ethanol was 24.2 mM . Pyrazole notably inhibited the enzymatic activity . The enzyme had the optimal temperature of 70 degrees C and was highly stable against high temperature. J Food Prot, 2002 May, 65(5), 799 - 805 Behavior of Listeria monocytogenes and Staphylococcus aureus in yogurt fermented with a bacteriocin-producing thermophilic starter; Benkerroum N et al.; Streptococcus salivarius subsp . thermophilus B producing a bacteriocin active against Listeria monocytogenes ATCC 7644 and Staphylococcus aureus SAD 30 was isolated from bakery yeast . The bacteriocin was partially purified by an adsorption/desorption technique, and its spectrum of action was compared to that of a neutralized cell-free supernatant (CFS) . Although the CFS inhibited a number of gram-positive and -negative bacteria of health and spoilage significance, the spectrum of action of the partially purified bacteriocin was limited to gram-positive bacteria . L . monocytogenes was the most sensitive to both preparations . The bacteriocin-producing streptococcal strain was used in combination with a Bac- Lactobacillus delbrueckii subsp . bulgaricus CY strain isolated from commercial yogurt to assess the effectiveness of the resulting thermophilic starter in controlling L . monocytogenes and S . aureus in yogurt during fermentation and storage at refrigeration (ca . 7 degrees C) or abuse (ca . 22 degrees C) temperature . Yogurt samples were contaminated with L . monocytogenes or S . aureus to the approximate levels of 10(3) and 10(6) CFU/ml of milk, respectively . The results showed that in situ bacteriocin production was more active against L . monocytogenes than against S . aureus in vitro and in contaminated samples . While L . monocytogenes leveled off below the detectable limit in a 1-ml sample of yogurt within 24 h of processing, S . aureus survived in Bac+ and Bac- samples during 10 days of storage at room temperature (ca . 22 degrees C) . Use of a Bac+ starter resulted in a 5-day extension of the shelf life. Curr Microbiol, 2002 Jul, 45(1), 54 - 62 Transcriptional analysis of the xylose ABC transport operons in the thermophilic anaerobe Thermoanaerobacter ethanolicus; Jones CR et al.; Thermoanaerobacter ethanolicus is a Gram-positive thermophile that converts xylose to ethanol . A portion of the T . ethanolicus xylose transport permease gene ( xylH) was cloned, and the deduced protein exhibited greater than 60% similarity to homologs in enterobacteria . Xylose-binding protein ( xylF) and ( xylH) transcripts were quantitated and compared from cells grown in batch or continuous cultures grown on xylose, glucose, or a mixture of both sugars . In contrast to the strong repression of xyl operons by glucose in other bacteria, both xylF and xylH expression were detected in the presence of this hexose sugar . Expression of xylF and xylH generally increased with dilution rate (3- and 1.5-fold, respectively) and seemed to be growth rate rather than substrate dependent . Overall, these unusual sugar utilization patterns in batch and continuous culture seem to result from a basal expression level of xyl genes in the absence of xylose . T . ethanolicus is unique in possessing a triumvirate of xylose transport and catabolism operons and, given its extensive hemicellulolytic capabilities, may have evolved to constitutively express xyl genes. J Bacteriol, 2002 Jun, 184(12), 3385 - 91 Thermoadaptation of alpha-galactosidase AgaB1 in Thermus thermophilus; Fridjonsson O et al.; The evolutionary potential of a thermostable alpha-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria . For this purpose, hybrid alpha-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD (DeltaagaT) by transformation . This selector strain is unable to metabolize melibiose (alpha-galactoside) without recombinant alpha-galactosidases, because the native alpha-galactosidase gene, agaT, has been deleted . Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene . With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67 degrees C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated . The mutant alpha-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced . A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes . The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability . To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell. J Bacteriol, 2002 Jun, 184(12), 3305 - 12 Unique presence of a manganese catalase in a hyperthermophilic archaeon, Pyrobaculum calidifontis VA1; Amo T et al.; We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1 . Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity . The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70 degrees C . The enzyme exhibited a K(m) value of 170 mM toward H(2)O(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees C . Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da . The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes . In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide . Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese . Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea . Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese . Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat(Pc)) . The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp . YS 8-13 . Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene . Moreover, a homology search with the sequence of kat(Pc) revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii . Therefore, Kat(Pc) can be considered a rare example of a manganese catalase from archaea. Microb Ecol, 2001 Aug, 42(2), 117 - 125 Species Composition of Cultivated and Noncultivated Bacteria from Short Filaments in an Icelandic Hot Spring at 88 degrees C; Hjorleifsdottir S et al.; Samples of short pink-grayish filaments were collected from a hot spring in the Hengill area in southwestern Iceland at 85-88 degrees C, pH 6.9 and 1.7 mg/L sulfide . The species composition was studied by cloning and sequencing small subunit rRNA genes obtained by PCR amplifications from mat DNA . Using 98% sequence similarity as a cutoff value, a total of 5 bacterial operational taxonomic units (OTUs) and 6 archaeal OTUs were detected among 68 bacterial clones and 97 archaeal clones . Database matching showed that 80.5% of the archaeal sequences were 99% similar to Pyrobaculum islandicum and 14.5% were closest to the Korarchaeota clone sequence SRI306 . About 87% of the bacterial sequences had the closest database match (99%) to the clone sequence SRI48 but were also found to be 99% identical with hydrogen-oxidizing strains previously isolated in this laboratory from hot springs in the same region . Out of 7 Thermus sequences, 4 were 100% identical to T . scotoductus NMX2 A.1 but 3 represented a new uncultivated Thermus species . Four different media, varying in organic nutrients and phosphate composition were used to isolate 81 aerobic thermophilic heterotrophs . Four isolates were Bacillus spp; but out of 77 Thermus isolates, 42 belonged to T . scotoductus and 35 to T . brockianus . T . scotoductus seemed to be preferably isolated on media low in nutrients and phosphate, whereas for T . brockianus it was the opposite . The T . scotoductus clones and isolates had 99-100% sequence similarity to each other . No T . brockianus sequences were found in the bacterial clone library. J Dairy Sci, 2002 Apr, 85(4), 721 - 9 Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products; Vinderola CG et al.; Interactions among lactic acid starter and probiotic bacteria were investigated to establish adequate combinations of strains to manufacture probiotic dairy products . For this aim, a total of 48 strains of Streptococcus thermophilus, Lactobacillus delbrueckii subsp . bulgaricus, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium spp . (eight of each) were used . The detection of bacterial interactions was carried out using the well-diffusion agar assay, and the interactions found were further characterized by growth kinetics . A variety of interactions was demonstrated . Lb . delbrueckii subsp . bulgaricus was found to be able to inhibit S . thermophilus strains . Among probiotic cultures, Lb . acidophilus was the sole species that was inhibited by the others (Lb . casei and Bifidobacterium) . In general, probiotic bacteria proved to be more inhibitory towards lactic acid bacteria than vice versa since the latter did not exert any effect on the growth of the former, with some exceptions . The study of interactions by growth kinetics allowed the setting of four different kinds of behaviors between species of lactic acid starter and probiotic bacteria (stimulation, delay, complete inhibition of growth, and no effects among them) . The possible interactions among the strains selected to manufacture a probiotic fermented dairy product should be taken into account when choosing the best combination/s to optimize their performance in the process and their survival in the products during cold storage. J Dairy Sci, 2002 Apr, 85(4), 716 - 20 Capsule formation by nonropy starter cultures affects the viscoelastic properties of yogurt during structure formation; Hassan AN et al.; The aim of this work was to study the structure formation of yogurt made with cultures containing ropy Lactobacillus delbrueckii ssp . bulgaricus (R), capsule-forming nonropy Streptococcus thermophilus (CNR), and noncapsule-forming nonropy cultures (NCNR) . Similar gelation profiles were shown for milk fermented by ropy and nonropy lactic cultures . The gelation point occurred at lower pH values in milk fermented with R or NCNR compared to that of milk fermented with CNR culture . Differences between capsule forming ropy and nonropy cultures were observed in the aggregation behavior of the caseins . Gels made with R culture had the highest maximum in loss tangent (tan delta) . However, this maximum occurred at the highest pH value when CNR culture was used . The earlier gelation of milk fermented by the encapsulated nonropy strain of Strep . thermophilus resulted in increased structure rearrangement as the pH dropped, interfering with the formation of a more compact structure. Appl Biochem Biotechnol, 2002 Spring, 98-100, 765 - 73 Thermophilic fermentation of hydrolysates: the effect of inhibitors on growth of thermophilic bacteria; Thomasser C et al.; Lignocellulosic biomass has great potential as a cheap feedstock in biological processes to produce biofuels or chemicals; however, dilute acid pretreatment at high temperatures produces undesirable compounds . Toxicity tests were done with inhibitors in standard media, to predict the growth-limiting effects on thermophilic strains . The 22 inhibitors included furfural, levulinic acid, acetic acid, and cinnamaldehyde . Neutralizing reagents and additional treatment steps have been tested. Appl Biochem Biotechnol, 2002 Spring, 98-100, 301 - 9 Isolation and characterization of thermophilic benzothiophene-degrading Mycobacterium sp; Watanapokasin Y et al.; A bacterial strain, SWU-4, capable of using benzothiophene (BT) as a sole carbon and energy source was isolated from a petroleum-contaminated site in Thailand and identified by 16S rRNA gene sequence analysis to be in the genus of Mycobacterium . The strain was Gram-positive, nonspore former, and grew at 50 degrees C . Colonies of the strain on nutrient agar were rod-shaped, smooth with a convex surface, slightly mucoid, and yellow pigmented . The thermophilic Mycobacterium sp . strain SWU-4 rapidly degraded 2% (w/v) BT at 50 degrees C . Interestingly, this strain was able to degrade a wide variety of organosulfur compounds including thiophene, bromo(alpha)thiophene, and 3-methylthiophene in liquid minimum medium at 50 degrees C, which will be beneficial for industrial applications. Appl Biochem Biotechnol, 2002 Spring, 98-100, 177 - 89 Hydrogen production by the thermophilic bacterium Thermotoga neapolitana; Van Ooteghem SA et al.; Virtually all members of the order Thermotogales have demonstrated the ability to produce hydrogen; however, some members of this order produce considerably greater quantities than others . With one representative of this order, Thermotoga neapolitana, we have consistently obtained accumulation of 25-30% hydrogen with 12-15% carbon dioxide as the only other prominent product in the batch reaction . In contradistinction to information widely disseminated in the literature, we have also found that most members of this order tolerate and appear to utilize the moderate amounts of oxygen present in the gaseous phase of batch reactors (6-12%), with no apparent decrease in hydrogen production . Hydrogen accumulation has been widely reported to inhibit growth of Thermotogales . While this may be true at very high hydrogen tensions, we have observed log phase bacterial morphology (rods) even in the presence of 25-35% hydrogen concentrations . To maximize hydrogen production and minimize production of hydrogen sulfide, inorganic sulfur donors are avoided and the cysteine concentration in the medium is increased . We and others have demonstrated that different members of the order Thermotogales utilize a wide variety of feedstocks, including complex carbohydrates and proteins . Thus, it appears that organisms within this order have the potential to utilize a variety of organic wastes and to cost-effectively generate hydrogen. Appl Biochem Biotechnol, 2002 Spring, 98-100, 165 - 76 Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts; Bergquist PL et al.; Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical . We have developed two systems for the bulk production of bleaching enzymes from thermophiles . Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium . Our second system involves the filamentous fungus Trichoderma reesei . Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter . The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins. Extremophiles, 2002 Apr, 6(2), 167 - 74 Aminoacyl-tRNA formation in the extreme thermophile Thermus thermophilus; Feng L et al.; Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive . Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization . We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine . We show that the T . thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS . The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes . The heterotrimeric T . thermophilus aspartyl-tRNA(Asn) amidotransferase can form Gln-tRNA in addition to Asn-tRNA: however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates . A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles. Extremophiles, 2002 Apr, 6(2), 143 - 50 Thermodynamic activation properties of elongation factor 2 (EF-2) proteins from psychrotolerant and thermophilic Archaea; Siddiqui KS et al.; In this study, the thermodynamic activation parameters of cold-adapted proteins from Archaeaa are described for the first time for the irreversible protein unfolding and ribosome-dependent GTPase activity of elongation factor 2 (EF-2) from the psychrotolerant Methanococcoides burtonii and the thermophilic Methanosarcina thermophila . Thermolability of Methanococcoides burtonii EF-2 was demonstrated by a low activation free-energy of unfolding as a result of low activation-enthalpy . Although structural data for EF-2 are presently limited to protein homology modeling, the observed thermodynamic properties are consistent with a low number of noncovvalent bonds or an altered solvent interaction, causing a loss of entropy during the unfolding process . A physiological concentration of potassium aspartate or potassium glutamate was shown to stabilize both proteins against irreversible denaturation by strengthening noncovalent interactions, as indicated by increased activation enthalpies . The transition state of GTPase activity for Methanococcoides burtonii EF-2 was characterized by a lower activation enthalpy than for Methanosarcina thermophila EF-2 . The relative entropy changes could be explained by differential displacement of water molecules during catalysis, resulting in similar activation free energies for both proteins . The presence of solutes was shown to facilitate the breaking of enthalpy-driven interactions and structuring of more water molecules during the reaction . By studying the thermodynamic activation parameters of both GTPase activity and unfolding and examining the effects of intracellular solutes and partner proteins (ribosomes), we were able to identify enthalpic and entropic properties that have evolved in the archaeal EF-2 proteins to enable Methanococcoides burtonii and Methanosarcina thermophila to adapt to their respective thermal environments. Extremophiles, 2002 Apr, 6(2), 123 - 9 ATP generation during reduced inorganic sulfur compound oxidation by Acidithiobacillus caldus is exclusively due to electron transport phosphorylation; Dopson M et al.; The synthesis of adenosine 5-triphosphate (ATP) (increase in phosphorylation potential) during the oxidation of reduced inorganic sulfur compounds was studied in the moderately thermophilic acidophileAcidithiobacillus caldus (strain KU) (formerly Thiohacillus caldus) . The phosphorylation potential increased during the oxidation of all reduced inorganic sulfur compounds tested compared with resting cells . The generation of ATP in whole cells was inhibited by the F0F1 ATPase inhibitor oligomycin, electron transport chain inhibitors, valinomycin and potassium ions . There was no increase in the phosphorylation potential, nor synthesis of ATP . in the absence of electron transport . An apparent lack of substrate-level phosphorylation was indicated by the lack of adenosine 5-phosphosulfate reductase in tetrathionate-grown At . caldus . Studies were also performed on the synthesis of ATP by membrane vesicles of At . caldus when presented with an artificial proton gradient . Complete inhibition of ATP synthesis in these vesicles occurred when they were loaded with N,N-dicyclohexylcarbodiimide (DCCD), but not when they were loaded with oligomycin, vanadate or electron transport chain inhibitors . The data presented here suggest that during the oxidation of reduced inorganic sulfur compounds by At . caldus, all ATP is synthesized by oxidative phosphorylation via a membrane-bound F0F1 ATPase driven by a proton gradient. J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Apr 25, 770(1-2), 129 - 35 Isolation of chimaeric forms of elongation factor EF-Tu by affinity chromatography; Tomincova H et al.; Six different recombinant chimaeric forms of a three-domain protein, proteosynthetic elongation factor Tu (EF-Tu), composed of domains of EF-Tu of mesophilic (Escherichia coli) and thermophilic (Bacillus stearothermophilus) origin as well as free N-terminal domains of EF-Tu, and the whole recombinant EF-Tus of both organisms were prepared and isolated by the GST (glutathione S-transferase) fusion technology . Several modifications in the standard isolation and purification procedures are described that proved necessary to obtain the proteins in a purified and undegraded form. J Am Chem Soc, 2002 May 22, 124(20), 5684 - 91 Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119; Koo LS et al.; CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase . This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations . The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group . A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme . This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme . As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids. Biochim Biophys Acta, 2002 May 20, 1597(1), 22 - 7 Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity; Sedlak E et al.; The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry . The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li(+), Na(+), K(+) and NH(4)(+) in the chloride form . The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tu(f)) in the presence of kirromycin are comparable with those of the EF-Tu x guanosine-5'-{beta,gamma-imido}triphosphate (GppNHp) form, indicating similar conformational states . Increased concentrations of Na(+) and K(+) stabilized EF-Tu(f) in a manner similar to GppNHp . NH(4)(+) decreased the transition temperature of EF-Tu(f) and Li(+) decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tu(f) . In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tu(f) . Correlation between the GTPase activity and thermodynamic characteristics of EF-Tu(f) induced by kirromycin in the absence or presence of the cations is discussed. Biol Trace Elem Res, 2002 May, 86(2), 159 - 65 Effect of Al(III) on surface properties of Bifidobacterium thermophilum as a function of temperature; Kot E et al.; The effects of Al(III) on surface properties and lactate accumulation by Bifidobacterium thermophilum were investigated . Bacteria were treated with Al(III) at 37 degrees C and 4 degrees C, then exposed to free radicals or nisin . When exposed to Al(III) at 37 degrees C, the organism exhibited spreading on hydrophobic surfaces and showed high susceptibility to free-radical alteration as indicated by Fe(III) binding, but showed little effect on lactate production in the presence or absence of nisin, even after washing with 2 mM EDTA . At 4 degrees C, there was no increased surface spreading or binding of Fe(III), but protection against nisin action was present . This, however, was abolished after washing with EDTA . It was concluded that membrane fluidity is required to affect membrane lipid rearrangement, resulting in surface spreading and increased susceptibility to peroxidation, whereas only loose binding of Al(III) to membrane surfaces is sufficient to prevent transmembrane channel formation by nisin. FEMS Microbiol Lett, 2002 Apr 9, 209(2), 289 - 93 Activity of the enzymes involved in the synthesis of exopolysaccharide precursors in an overproducing mutant ropy strain of Streptococcus thermophilus; Escalante A et al.; The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus . These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production . The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain . The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors. Biochim Biophys Acta, 2002 Apr 29, 1596(2), 357 - 65 Microcalorimetric study of elongation factor Tu from Thermus thermophilus in nucleotide-free, GDP and GTP forms and in the presence of elongation factor Ts; Sedlak E et al.; Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle . The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry . Thermal transitions of the EF-Tu.GDP and EF-Tu.guanosine-5'-{beta,gamma-imido}triphosphate have a cooperative two-state character . Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu . Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein . Despite the fact that strong interactions between elongation factors Tu and Ts from T . thermophilus at 20 degrees C exist, the thermal transition of neither protein in the complex was significantly affected. Biosci Biotechnol Biochem, 2002 Mar, 66(3), 588 - 97 Purification and characterization of two alpha-keto ester reductases from Streptomyces thermocyaneoviolaceus IFO 14271; Yamaguchi H et al.; Two NADPH-dependent alpha-keto ester reductases (Streptomyces thermocyaneoviolaceus keto ester reductase, STKER-II and -III) were purified from S . thermocyaneoviolaceus IFO 14271, one of thermophilic actinomycetes . The molecular masses of native STKER-II and -III were estimated to be 60 kDa and 70 kDa by gel filtration chromatography, respectively . These enzymes were both homodimers, with 29-kDa and 30-kDa subunit molecular masses based on SDS polyacrylamide gel electrophoresis . STKER-II and -III were stable from pH 7.0 to 10.0 and pH 5.5 to 9.0, respectively . Ethyl 3-methyl-2-oxobutanoate was reduced by both enzymes isolated to the corresponding (R)-hydroxy ester with excellent enantiomeric excess . STKER-III showed high stereoselectivity for the reduction of bulky substrates, while the selectivity of the STKER-II-catalyzed reduction was low except for ethyl 3-methyl-2-hydrox-ybutanoate . Both enzymes had small Km values toward aliphatic keto esters having a long alkyl chain. Biosci Biotechnol Biochem, 2002 Mar, 66(3), 549 - 57 Cysteine synthase of an extremely thermophilic bacterium, Thermus thermophilus HB8; Mizuno Y et al.; O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source . Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography) . The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism . Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure . The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol . The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min) . It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein . The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine . The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor . The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8 . Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme . The meaning of a very high enzyme content in the cell is discussed. Science, 2002 May 10, 296(5570), 1077 - 82 Merging genomes with geochemistry in hydrothermal ecosystems; Reysenbach AL et al.; Thermophilic microbial inhabitants of active seafloor and continental hot springs populate the deepest branches of the universal phylogenetic tree, making hydrothermal ecosystems the most ancient continuously inhabited ecosystems on Earth . Geochemical consequences of hot water-rock interactions render these environments habitable and supply a diverse array of energy sources . Clues to the strategies for how life thrives in these dynamic ecosystems are beginning to be elucidated through a confluence of biogeochemistry, microbiology, ecology, molecular biology, and genomics . These efforts have the potential to reveal how ecosystems originate, the extent of the subsurface biosphere, and the driving forces of evolution. Bioresour Technol, 2002 Apr, 82(2), 157 - 64 Enhancement of proteolytic enzyme activity excreted from Bacillus stearothermophilus for a thermophilic aerobic digestion process; Kim YK et al.; Proteolysis is one of the main enzymatic reactions involved in waste activated sludge (WAS) digestion . In this study, proteases excreted from Bacillus stearothermophilus (ATCC 31197) were classified, and an enhancement of protease activity was achieved using economical chemical additives for WAS digestion . Proteases excreted from B . stearothermophilus were classified into two families: serine and metallo-proteases . Various metal ions were investigated as additives which could potentially enhance protease activity . It was observed that Ca2+ and Fe2+ could markedly activate these enzymes . These results were applied to thermophilic aerobic digestion (TAD) of industrial WAS using B . stearothermophilus . The addition of these divalent ions enhanced the degradation performance of the TAD process in terms of reducing the total suspended solids (TSSs), the dissolved organic carbon (DOC) content, and the intracellular and extracellular protein concentrations . The best result, with respect to protein reduction in a digestion experiment, was obtained by the addition of 2 mM Ca2+ . Therefore, a proposed TAD process activated by calcium addition can be successfully used for industrial and municipal WAS digestion to the upgrading of TAD process performance. Microbiol Res, 2002, 157(2), 149 - 56 Clostridium thermobutyricum: growth studies and stimulation of butyrate formation by acetate supplementation; Canganella F et al.; Clostridium thermobutyricum produces butyrate as the main fermentation product from glucose, and from yeast extract, which is required for substantial growth . After sequential transfer in the presence of increasing butyrate concentrations, strain JW 171 K grew in the presence of up to 350 mM butyrate either at pH 5.5 or at pH 8.0 and at 40 degrees C as well as at 60 degrees C . This result indicated that butyrate-dependent growth inhibition was independent from the concentration of undissociated butyric acid . Increased butyrate concentration decreased the level of tolerated glucose from above 15% to below 10% . At 0.05 and 2.0% (wt/vol) yeast extract, the Y(Glucose) was 30 and 55 g dry weight cells per mole glucose, respectively . Y(ATP) values between 18 and 21 g weight cells per mole ATP, obtained after growth in the presence of 2% yeast extract, indicate that the butyrate fermentation under thermophilic growth conditions is as energy efficient as it is under mesophilic conditions . Externally added acetate stimulated the production of butyrate . Supplemented 14C-acetate was converted to butyrate, resulting in the formation of 44% labeled butyrate (i.e . formed from 14C-acetate) and 56% unlabeled butyrate (formed from glucose and yeast extract) . Continuous removal of H2 in batch cultures led to a shift in the fermentation products from more butyrate to the more oxidized and more energy yielding acetate. Nature, 2002 Jun 13, 417(6890), 712 - 9 Epub 2002 May 08. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution; Vassylyev DG et al.; In bacteria, the binding of a single protein, the initiation factor sigma, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation . Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 A resolution . In the structure, two amino-terminal domains of the sigma subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft . The carboxy-terminal domain of sigma is near the outlet of the RNA-exit channel, about 57 A from the N-terminal domains . The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains . The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation. Nucleic Acids Res, 2002 May 15, 30(10), 2097 - 102 A novel type of uracil-DNA glycosylase mediating repair of hydrolytic DNA damage in the extremely thermophilic eubacterium Thermus thermophilus; Starkuviene V et al.; Spontaneous hydrolytic deamination of DNA cytosine and 5-methyl-cytosine residues is an abundant source of C/G (5-meC/G) to T/A transition mutations . As a result of this pressure, at least six different families of enzymes have evolved that initiate repair at U/G (T/G) mispairs, the relevant pre-mutagenic intermediates . The necessarily higher rate of the process at elevated temperatures must pose a correspondingly accentuated problem to contemporary thermophilic organisms and may have been a serious bottleneck in early evolution when life passed through a phase of very high ambient temperatures . Here we show that Thermus thermophilus, an aerobic, Gram-negative eubacterium thriving at up to 85 degrees C, harbors two uracil-DNA glycosylases (UDGs), termed TTUDGA and TTUDGB . According to both amino acid sequence and enzymatic properties, TTUDGA clearly belongs to the family of 'thermostable UDGs' . TTUDGB shares with TTUDGA 23% sequence identity, but differs from it in profound functional aspects . TTUDGB, unlike TTUDGA, does not act upon uracil residues in the context of single-stranded DNA whereas both enzymes process various double-stranded substrates, albeit with different preferences . TTUDGB shows a number of sequence features characteristic of the UDG superfamily, but surprisingly lacks any polar residue within its so-called motif 1 (GLAPG-X(10)-F) . This finding is in conflict with a previously assumed crucial catalytic role of motif 1 in water activation and supports a more recently suggested alternative of a dissociative ('S(N)1-type') reaction mechanism . Together, the characteristics of TTUDGB and its homologs in other organisms define a novel family of UDG repair enzymes. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6585 - 90 Epub 2002 May 07. Telomerase recognizes its template by using an adjacent RNA motif; Miller MC et al.; Telomerase adds telomeric repeats to chromosome 3' ends, forestalling the cellular senescence, apoptosis, and genomic instability that result from telomere loss caused by incomplete DNA replication . The telomerase ribonucleoprotein is dedicated to synthesis of tandem, simple-sequence repeats by virtue of its specialization for copying only a specific template region within the integral RNA . Here, using circularly permuted variants of Tetrahymena thermophila telomerase RNA, we identify the features that allow recognition of the template region within the RNA . We engineered a template-less telomerase ribonucleoprotein that can position and reverse transcribe an exchangeable RNA oligonucleotide template accurately . Only a short "template-recognition" element sequence tag is required to direct efficient use of adjacent 5' residues as a template for telomeric repeat synthesis . Our findings reveal molecular requirements for template selection by telomerase and physically resolve templating from other RNA functions in catalysis. Genome Res, 2002 May, 12(5), 689 - 700 A complete sequence of the T . tengcongensis genome; Bao Q et al.; Thermoanaerobacter tengcongensis is a rod-shaped, gram-negative, anaerobic eubacterium that was isolated from a freshwater hot spring in Tengchong, China . Using a whole-genome-shotgun method, we sequenced its 2,689,445-bp genome from an isolate, MB4(T) (Genbank accession no . AE008691) . The genome encodes 2588 predicted coding sequences (CDS) . Among them, 1764 (68.2%) are classified according to homology to other documented proteins, and the rest, 824 CDS (31.8%), are functionally unknown . One of the interesting features of the T . tengcongensis genome is that 86.7% of its genes are encoded on the leading strand of DNA replication . Based on protein sequence similarity, the T . tengcongensis genome is most similar to that of Bacillus halodurans, a mesophilic eubacterium, among all fully sequenced prokaryotic genomes up to date . Computational analysis on genes involved in basic metabolic pathways supports the experimental discovery that T . tengcongensis metabolizes sugars as principal energy and carbon source and utilizes thiosulfate and element sulfur, but not sulfate, as electron acceptors . T . tengcongensis, as a gram-negative rod by empirical definitions (such as staining), shares many genes that are characteristics of gram-positive bacteria whereas it is missing molecular components unique to gram-negative bacteria . A strong correlation between the G + C content of tDNA and rDNA genes and the optimal growth temperature is found among the sequenced thermophiles . It is concluded that thermophiles are a biologically and phylogenetically divergent group of prokaryotes that have converged to sustain extreme environmental conditions over evolutionary timescale. FEBS Lett, 2002 May 8, 518(1-3), 139 - 43 Leucyl-tRNA synthetase from the extreme thermophile Aquifex aeolicus has a heterodimeric quaternary structure; Gouda M et al.; Class I aminoacyl-tRNA synthetases have been thought to be single polypeptide enzymes . However, the complete genome sequence of a hyper thermophile Aquifex aeolicus suggests that the gene for leucyl-tRNA synthetases (LeuRS) is probably split into two pieces (leuS and leuS') . In this research, each gene was separately cloned and overexpressed in Escherichia coli and the protein products were examined for LeuRS activity . Leucylation activity was detected only when both gene products coexisted . Gel filtration analysis showed that the active form of A . aeolicus LeuRS has a heterodimeric (alpha/beta type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases. Biochemistry, 2002 May 14, 41(19), 6008 - 18 An insight into the mechanism of inhibition and reactivation of the F(1)-ATPases by tentoxin; Santolini J et al.; The mechanism of inhibition and reactivation of chloroplast ATP-synthase by the fungal cyclotetrapeptide tentoxin was investigated by photolabeling experiments, binding studies, and kinetic analysis using synthetic analogues of tentoxin . The alpha-subunit of chloroplast F(1)-ATPase (CF(1)) was specifically labeled by a photoactivatable tentoxin derivative, providing the first direct evidence of tentoxin binding to the alpha-subunit, and 3D homology modeling was used to locate tentoxin in its putative binding site at the alpha/beta interface . The non-photosynthetic F(1)-ATPase from thermophilic bacterium (TF(1)) proved to be also tentoxin-sensitive, and enzyme turnover dramatically increased the rate of tentoxin binding to its inhibitory site, contrary to what was previously observed with epsilon-depleted CF(1) {Santolini, J., Haraux, F., Sigalat, C., Moal, G., and Andre, F . (1999) J . Biol . Chem . 274, 849-858} . We propose that tentoxin preferentially binds to an ADP-loaded alpha beta pair, and mechanically blocks the catalytic cycle, perhaps by the impossibility of converting this alpha beta pair into an ATP-loaded alpha beta pair . Using (14)C-tentoxin and selected synthetic analogues, we found that toxin binding to the tight inhibitory site of CF(1) exerts some cooperative effect on the loose reactivatory site, but that no reciprocal effect exists . When the two tentoxin-binding sites are filled in reactivated F(1)-ATPase, they do not exchange their role during catalytic turnover, indicating an impairment between nucleotide occupancy and the shape of tentoxin-binding pocket . This analysis provides a mechanical interpretation of the inhibition of F(1)-ATPase by tentoxin and a clue for understanding the reactivation process. Biochemistry, 2002 May 14, 41(19), 5990 - 7 GXXXG and AXXXA: common alpha-helical interaction motifs in proteins, particularly in extremophiles; Kleiger G et al.; The GXXXG motif is a frequently occurring sequence of residues that is known to favor helix-helix interactions in membrane proteins . Here we show that the GXXXG motif is also prevalent in soluble proteins whose structures have been determined . Some 152 proteins from a non-redundant PDB set contain at least one alpha-helix with the GXXXG motif, 41 +/- 9% more than expected if glycine residues were uniformly distributed in those alpha-helices . More than 50% of the GXXXG-containing alpha-helices participate in helix-helix interactions . In fact, 26 of those helix-helix interactions are structurally similar to the helix-helix interaction of the glycophorin A dimer, where two transmembrane helices associate to form a dimer stabilized by the GXXXG motif . As for the glycophorin A structure, we find backbone-to-backbone atomic contacts of the C alpha-H...O type in each of these 26 helix-helix interactions that display the stereochemical hallmarks of hydrogen bond formation . These glycophorin A-like helix-helix interactions are enriched in the general set of helix-helix interactions containing the GXXXG motif, suggesting that the inferred C alpha-H...O hydrogen bonds stabilize the helix-helix interactions . In addition to the GXXXG motif, some 808 proteins from the non-redundant PDB set contain at least one alpha-helix with the AXXXA motif (30 +/- 3% greater than expected) . Both the GXXXG and AXXXA motifs occur frequently in predicted alpha-helices from 24 fully sequenced genomes . Occurrence of the AXXXA motif is enhanced to a greater extent in thermophiles than in mesophiles, suggesting that helical interaction based on the AXXXA motif may be a common mechanism of thermostability in protein structures . We conclude that the GXXXG sequence motif stabilizes helix-helix interactions in proteins, and that the AXXXA sequence motif also stabilizes the folded state of proteins. Ir J Med Sci, 2002 Jan-Mar, 171(1), 33 - 6 Molecular characterisation of human campylobacteriosis in Northern Ireland: evidence of clonal stability; Matsui T et al.; BACKGROUND: No study has compared Campylobacter isolates from a human source in Northern Ireland over an extended period of time . AIM: To investigate the clonal stability of thermophilic campylobacters isolated from acute bacterial enteritis in Northern Ireland from 1992 to 1999 . METHODS: Human isolates (n=272), originating from faeces, were characterised at the sub-species level using enterobacterial repetitive intergenic consensus sequence (ERIC2)--random amplification of polymorphic DNA (RAPD) typing . RESULTS: Thirteen genotypes were identified where three types, namely ERIC A, ERIC C and ERIC I, accounted for 28.3%, 14.3% and 13.6%, respectively . There were no significant associations (p>0.05) between sex, age groupings and year of isolation and ERIC2 genotype, with the exception of ERIC D, which showed a significant decline in isolation with time (p=0.019) . CONCLUSIONS: ERIC genotypes were stable over this period, except genotype C which was lost during this time . Previous molecular typing methods showed campylobacters to be heterogeneous but this study suggests that the local human Campylobacter population consists of several common and stable genotypes . This study has established a database of local ERIC2 genotypes . This methodology may allow the establishment of an all-island database of clinical campylobacters that would be valuable in reducing human campylobacteriosis in Ireland. Nature, 2002 May 2, 417(6884), 63 - 7 A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont; Huber H et al.; According to small subunit ribosomal RNA (ss rRNA) sequence comparisons all known Archaea belong to the phyla Crenarchaeota, Euryarchaeota, and--indicated only by environmental DNA sequences--to the 'Korarchaeota' . Here we report the cultivation of a new nanosized hyperthermophilic archaeon from a submarine hot vent . This archaeon cannot be attached to one of these groups and therefore must represent an unknown phylum which we name 'Nanoarchaeota' and species, which we name 'Nanoarchaeum equitans' . Cells of 'N . equitans' are spherical, and only about 400 nm in diameter . They grow attached to the surface of a specific archaeal host, a new member of the genus Ignicoccus . The distribution of the 'Nanoarchaeota' is so far unknown . Owing to their unusual ss rRNA sequence, members remained undetectable by commonly used ecological studies based on the polymerase chain reaction . 'N . equitans' harbours the smallest archaeal genome; it is only 0.5 megabases in size . This organism will provide insight into the evolution of thermophily, of tiny genomes and of interspecies communication. Biochemistry, 2002 May 7, 41(18), 5799 - 806 Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme; Uchida T et al.; We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg2+-induced folding and the urea-induced denaturation of the folded state as a function of Na+ under equilibrium folding conditions using hydroxyl radical footprinting . These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts . Monovalent ions compete with Mg2+ ions in mediating P4-P6 folding . The urea denaturation isotherms demonstrated DeltaDeltaG values of >2 kcal x mol(-1) in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl2 . However, the individual-site isotherms reported by footprinting revealed that larger than average changes in DeltaG values were localized to specific sites within the Mg2+-rich A-bulge . The competitive effects of monovalent ions were less when K+ rather than Na+ was the monovalent cation present . This result indicates the importance of the specific K+ binding sites that are associated with AA-platform structures to P4-P6 folding and stability . These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data. Biochemistry, 2002 May 7, 41(18), 5712 - 9 The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase; Simpson PJ et al.; The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus . CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft . From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146 . Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar . It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance . Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins . It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft. Arch Microbiol, 2002 May, 177(5), 401 - 9 Epub 2002 Mar 05. ATP-dependent 6-phosphofructokinase from the hyperthermophilic bacterium Thermotoga maritima: characterization of an extremely thermophilic, allosterically regulated enzyme; Hansen T et al.; The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic bacterium Thermotoga maritimawas purified 730-fold to homogeneity . The enzyme is a 140-kDa homotetramer composed of 34 kDa subunits . Kinetic constants were determined for all substrates in both reaction directions at pH 7 and at 75 degrees C . Rate dependence (forward reaction) on fructose 6-phosphate (F-6-P) showed sigmoidal kinetics with a half-maximal saturation constant ( S(0.5)) of 0.7 mM and a Hill coefficient of 2.2 . The apparent K(m) for ATP was 0.2 mM and the apparent V(max) value was about 360 U/mg . The enzyme also catalyzed in vitro the reverse reaction with an apparent K(m) for fructose 1,6-bisphosphate and ADP of 7.6 mM and 1.4 mM, respectively, and an apparent V(max) of about 13 U/mg . Divalent cations were required for maximal activity; Mg(2+), which was most effective, could partially be replaced by Mn(2+) and Fe(2+) . Enzyme activity was allosterically regulated by classical effectors of ATP-PFKs of Eukarya and Bacteria; it was activated by ADP and inhibited by PEP . The enzyme had a temperature optimum of 93 degrees C and showed a significant thermostability up to 100 degrees C . Using the N-terminal amino acid sequence of the subunit, the pfk gene coding for ATP-PFK was identified and functionally overexpressed in Escherichia coli . The purified recombinant ATP-PFK had identical kinetic and allosteric properties as the native enzyme purified from T . maritima . The deduced amino acid sequence showed high sequence similarity to members of the PFK-A family . In accordance with its allosteric properties, ATP-PFK of T . maritima contained the conserved allosteric effector-binding sites for ADP and PEP. Acta Crystallogr D Biol Crystallogr, 2002 May, 58(Pt 5), 867 - 9 Epub 2002 Apr 26. Towards the crystal structure of glycerol dehydrogenase from Thermotoga maritima; Srinivasan V et al.; The NAD(+)-dependent glycerol dehydrogenase (EC 1.1.1.6) from the extremely thermophilic bacterium Thermotoga maritima has been crystallized in the presence of glycerol by the hanging-drop vapour-diffusion method using 2-methyl-2,4-pentanediol (MPD) as the precipitating agent . Crystals of the enzyme complexed with NAD(+) have also been obtained . The crystals belong to the tetragonal system with space group I422 and unit-cell parameters a = 105.3, c = 134.5 A . They diffract to a maximum resolution of 1.4 A using synchrotron radiation (lambda = 0.838 A) . Crystals of the enzyme-NAD(+) complex diffract to 2.5 A resolution using in-house Cu Kalpha radiation. J Bacteriol, 2002 May, 184(10), 2821 - 6 Effects of rodA and pbp2b disruption on cell morphology and oxidative stress response of Streptococcus thermophilus CNRZ368; Thibessard A et al.; Insertional mutagenesis was used to isolate clones from Streptococcus thermophilus CNRZ368 that were modified in their abilities to tolerate oxidative stress . During this process, two menadione-sensitive clones (6G4 and 18C3) were found to display abnormal cell morphologies and distorted chain topologies and were further studied . Molecular characterization of both 6G4 and 18C3 mutants indicated that they were disrupted in open reading frames homologous to rodA and pbp2b, respectively . Both genes encoded proteins in Escherichia coli that were described as being implicated in peptidoglycan synthesis during the process of cell elongation and to function in determining the rod shape of the cell . This work reports a possible connection between peptidoglycan biosynthesis and oxidative stress defense in S . thermophilus CNRZ368. Genetics, 2002 Apr, 160(4), 1469 - 79 Polymorphism and selection at the SerH immobilization antigen locus in natural populations of Tetrahymena thermophila; Gerber CA et al.; The SerH locus of Tetrahymena thermophila is one of several paralogous loci with genes encoding variants of the major cell surface protein known as the immobilization antigen (i-ag) . The locus is highly polymorphic, raising questions concerning functional equivalency and selective forces acting on its multiple alleles . Here, we compare the sequences and expression of SerH1, SerH3, SerH4, SerH5, and SerH6 . The precursor i-ags are highly similar . They are rich in alanine, serine, threonine, and cysteine and they share nearly identical ER translocation and GPI addition signals . The locations of the 39 cysteines are highly conserved, particularly in the 3.5 central, imperfect tandem repeats in which 8 periodic cysteines punctuate alternating short and long stretches of amino acids . Hydrophobicity patterns are also conserved . Nevertheless, amino acid sequence identity is low, ranging from 60.7 to 82.9% . At the nucleotide level, from 9.7 to 26.7% of nucleotide sites are polymorphic in pairwise comparisons . Expression of each allele is regulated by temperature-sensitive mRNA stability . H mRNAs are stable at <36 degrees but are unstable at >36 degrees . The H5 mRNA, which is less affected by temperature, has a different arrangement of the putative mRNA destabilization motif AUUUA . Statistical analysis of SerH genes indicates that the multiple alleles are neutral . Significantly low ratios of the rates of nonsynonymous to synonymous amino acid substitutions suggest that the multiple alleles are subject to purifying (negative) selection enforcing constraints on structure. Mol Microbiol, 2002 Apr, 44(2), 549 - 59 RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions; Sharples GJ et al.; The RusA protein of Escherichia coli is a DNA structure-specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5' to CC dinucleotides . It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin . We show that rusA-like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species . They are also found in many bacteriophages, including Lactococcus lactis phage r1t . However, rusA is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus . In this case, there is no obvious association of rusA with prophage-like sequences . Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that RusA may be of bacterial origin . The RusA proteins of A . aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions . The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase . Both enzymes cleave junctions in a sequence-dependent manner, but the A . aeolicus RusA shows a different sequence preference (3' to TG) from the E . coli protein (5' to CC), and the r1t RusA has relaxed sequence dependence, requiring only a single cytosine. J Appl Microbiol, 2002, 92(5), 812 - 20 Autolytic phenotype of Lactococcus lactis strains isolated from traditional Tunisian dairy products; Ouzari H et al.; AIMS: To evaluate the autolytic properties of Lactococcus lactis strains isolated from artisan Tunisian dairy products, their peptidoglycan hydrolase content and their activity spectrum . METHODS AND RESULTS: The autolytic phenotype of Lactococcus strains was evaluated under starvation conditions in potassium phosphate buffer . The results obtained highlighted a high degree of diversity among the strains analysed, allowing the identification of high and low autolytic Lactococcus lactis strains . Peptidoglycan hydrolase content was evaluated by renaturing SDS-PAGE using cells of Micrococcus lysodeikticus as a target for the enzymatic activity . A major activity band migrating at about 45 kDa was observed . The lytic activity, evaluated in the presence of different chemicals, was retained in 8% NaCl, 15 mmol l(-1) CaCl2, and in a pH range between 5 and 9.5 . The substrate specificity of peptidoglycan hydrolase from Lactococcus strains was evaluated in renaturing SDS-PAGE incorporating cells of different bacterial species . The major autolysin of Lactococcus lactis was active against cells of Lactococcus lactis subsp . lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp . bulgaricus, Lactobacillus helveticus and Listeria monocytogenes . CONCLUSIONS: Autolytic activity is widely distributed in Lactococcus lactis and the rate of autolysis is strain-dependent . The major peptidoglycan hydrolase showed a wide spectrum of activity against several lactic acid bacteria and bacterial species involved in food-related infection . SIGNIFICANCE AND IMPACT OF THE STUDY: The autolytic phenotype of Lactococcus lactis strains isolated from Tunisian artisan dairy products has been determined, and the data obtained should allow the selection of strains of technological interest in the cheese-ripening process. Biochemistry, 2002 Apr 30, 41(17), 5359 - 74 Maximal stabilities of reversible two-state proteins; Kumar S et al.; The hydrophobic effect is the major force driving protein folding . Around room temperature, small organic solutes and hydrophobic amino acids have low solubilities in water and the hydrophobic effect is the strongest . These facts suggest that globular proteins should be maximally stable around room temperature . While this fundamental paradigm has been expected, it has not actually been shown to hold . Toward this goal, we have collected and analyzed experimental thermodynamic data for 31 proteins that show reversible two-state folding <--> unfolding transitions at or near neutral pH . Twenty-six of these are unique, and 20 of the 26 are maximally stable around room temperature irrespective of their structural properties, the melting temperature, or the living temperatures of their source organisms . Their average temperature of maximal stability is 293 +/- 8 K (20 +/- 8 degrees C) . These proteins differ in size, fold, and number of domains, hydrophobic folding units, and oligomeric states . They derive from the cold-loving psychrophiles, from mesophiles, and from thermophiles . Analysis of the single-domain proteins present in this set shows that the variations in their thermodynamic parameters are correlated in a way which may explain the adaptation of the proteins to the living temperatures of the organisms from which they derive . The average energetic contribution of the individual amino acids toward protein stability decreases with an increase in protein size, suggesting that there may be an upper limit for protein maximal thermodynamic stability . For the remaining proteins, deviation of the maximal stability temperatures from room temperature may be due to greater uncertainties in their heat capacity change (DeltaC(p)) values, a weaker hydrophobic effect, and/or a stronger electrostatic contribution. Environ Microbiol, 2002 Jan, 4(1), 58 - 64 Carbon isotopic fractionations associated with thermophilic bacteria Thermotoga maritima and Persephonella marina; Zhang CL et al.; Stable carbon isotopes can provide insight into carbon cycling pathways in natural environments . We examined carbon isotope fractionations associated with a hyperthermophilic fermentative bacterium, Thermotoga maritima, and a thermophilic chemolithoautotrophic bacterium Persephonella marina . In T . maritima, phospholipid fatty acids (PLFA) are slightly enriched in 13C relative to biomass (epsilon = 0.1-0.8 per thousand) . However, PLFA and biomass are depleted in 13C relative to the substrate glucose by approximately 8 per thousand . In P . marina, PLFA are 1.8-14.5 per thousand enriched in 13C relative to biomass, which suggests that the reversed tricarboxylic acid (TCA) cycle or the 3-hydroxypropionate pathway may be used for CO2 fixation . This is supported by small fractionation between biomass and CO2 (epsilon = -3.8 per thousand to -5.0 per thousand), which is similar to fractionations reported for other organisms using similar CO2 fixation pathways . Identification of the exact pathway will require biochemical assay for specific enzymes associated with the reversed TCA cycle or the 3-hydroxypropionate pathway. J Mol Evol, 2002 May, 54(5), 595 - 613 The chemical basis of membrane bioenergetics; Berry S; All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation . This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life . For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures . In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way . It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis . The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron-sulfur chemistry . This perspective can help to explain the prominent role of iron-sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Biochem J, 2002 May 1, 363(Pt 3), 553 - 61 The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold; Woestenenk EA et al.; We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus . L18 is a 12.5 kDa protein of the large subunit of the ribosome and binds to both 5 S and 23 S rRNA . In the uncomplexed state L18 folds to a mixed alpha/beta globular structure with a long disordered N-terminal region . We compared our high-resolution structure with RNA-complexed L18 from Haloarcula marismortui and T . thermophilus to examine RNA-induced as well as species-dependent structural differences . We also identified T . thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved . Important features, for instance a bulge in the RNA-contacting beta-sheet, are conserved in both proteins . We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA-protein-recognition module is tolerant to variations in sequence. Appl Biochem Biotechnol, 2001 Spring, 91-93, 597 - 603 Cobalt activation of Bacillus BR449 thermostable nitrile hydratase expressed in Escherichia coli; Kim SH et al.; Expression of nitrile hydratase enzymes utilized in a new "green" process for acrylamide production has proven difficult in Escherichia coli owing to lack of a cobalt transport system to introduce the required cobalt ion into this host . We describe the expression of a thermostable nitrile hydratase from a moderate thermophile Bacillus sp . BR449 in E . coliin which the cobalt required for enzyme activation is introduced by incubation of the apoenzyme in the presence of Co++ ion at 50 degrees C, yielding active and thermostable enzyme. Nutr Cancer, 2001, 40(2), 185 - 96 Apoptotic effects of selected strains of lactic acid bacteria on a human T leukemia cell line are associated with bacterial arginine deiminase and/or sphingomyelinase activities; Di Marzio L et al.; The aim of the present work was, first, to analyze the apoptotic effect in vitro of sonicated preparations of selected strains of lactic acid bacteria on normal and tumor human lymphocytes . Incubation with bacterial samples led to a relevant time-dependent apoptotic cell death of Jurkat cells but not normal human peripheral blood lymphocytes . Lactobacillus brevis (CD2) samples were more efficient in inducing apoptosis of Jurkat cells than were samples of Streptococcus thermophilus (S244) . In an attempt to characterize the mechanisms underlying these effects, we found that the apoptotic death-inducing ability of S244 preparations could be attributed to the ability of high levels of neutral sphingomyelinase activity to generate relevant amounts of ceramide, a known apoptotic death messenger, in Jurkat cells . On the other hand, our results indicate that apoptosis induced by CD2 samples could also be associated with high levels of arginine deiminase activity, which in turn was able to downregulate polyamine synthesis in Jurkat cells. Digestion, 2002, 65(1), 16 - 20 Lack of therapeutic effect of a specially designed yogurt for the eradication of Helicobacter pylori infection; Wendakoon CN et al.; BACKGROUND: Current antibiotic treatment for Helicobacter pylori infection is often associated with frequent adverse effects and resistance to antibiotics . Alternative treatment methods to control H . pylori infection are needed . Some specific strains of lactic acid bacteria (probiotics) in dairy products are known to inhibit the growth of H . pylori in vitro . A clinical trial was conducted to see the efficacy of a specially designed yogurt product containing specific probiotics on the eradication of H . pylori . METHOD: The yogurt was prepared using three Lactobacillus spp . (L . acidophilus and L . casei) and one commercial starter culture (L . acidophilus, L . bulgaricus and Streptococcus thermophilus) . All these cultures were previously evaluated, and found to have strong in vitro inhibitory effects on the growth of H . pylori . Twenty-seven asymptomatic women positive for H . pylori on gastric biopsy and 13C urea breath test were recruited, and administered 175 ml of the yogurt three times a day for 30 days . The 13C urea breath test was administered again, one month after stopping the yogurt treatment to detect the presence of H . pylori . RESULTS: In 26 of 27 subjects, the urea breath test values remained positive, indicating that the consumption of the yogurt had no effect on the eradication of H . pylori . CONCLUSION: Although the designed fermented milk containing lactobacilli is very effective in the inhibition of H . pylori growth in vitro, eradication of this infection in humans is difficult to achieve by consuming this product . J Biol Chem, 2002 Jun 21, 277(25), 22547 - 52 Epub 2002 Apr 17. Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila; Miles RD et al.; Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions . Planar AlF(3) or AlF(4)(-) molecules are proposed to mimic the phosphoryl group in the catalytic transition state . Acetate kinase catalyzes phosphoryl transfer of the ATP gamma-phosphate to acetate . Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl(2), ADP, AlCl(3), NaF, and acetate . Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme . Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl(2), AlCl(3), NaF, and acetate, inhibit acetate kinase activity . Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl(3), NaF, and acetate . These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site . The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase. FEMS Microbiol Lett, 2002 Mar 5, 208(2), 275 - 9 Active and energy-dependent rapid formation of cell aggregates in the thermophilic photosynthetic bacterium Chloroflexus aggregans; Hanada S et al.; The thermophilic filamentous phototroph Chloroflexus aggregans was able to form a bacterial mat-like dense cell aggregate rapidly . The aggregate formation, which was observed in growing cells in a liquid medium in a bottle, occurred every time within 20-30 min after the cells were dispersed by shaking . The aggregation depended on the energy supplied by photosynthesis or respiration . Cells aggregated most rapidly under temperature and pH conditions that support maximum growth . The aggregation was also accelerated by the addition of 3-isobutyl-1-methylxanthine that inhibits cyclic 3',5'-AMP phosphodiesterase . Microscopic observation revealed that the bacterium has a fast gliding mobility (1-3 microm s(-1)) . The distinctive cell aggregation of C . aggregans was due to this rapid gliding movement. J Food Prot, 2002 Apr, 65(4), 609 - 15 Behavior of Escherichia coli O157:H7 during the manufacture and ripening of feta and telemes cheeses; Govaris A et al.; Pasteurized whole ewe's and cow's milk was used in the manufacture of Feta end Telemes cheeses, respectively, according to standard procedures . In both cases, the milk had been inoculated with Escherichia coli O157:H7 at a concentration of ca . 5.1 log CFU/ml and with thermophilic or mesophilic starter cultures at a concentration of ca . 5.3 to 5.6 log CFU/ml . In the first 10 h of cheesemaking, the pathogen increased by 1.18 and 0.82 log CFU/g in Feta cheese and by 1.56 and 1.35 log CFU/ g in Telemes cheese for the trials with thermophilic and mesophilic starters, respectively . After 24 h of fermentation, a decrease in E . coli O157:H7 was observed for all trials . At that time, the pH was reduced to 4.81 to 5.10 for all trials . Fresh cheeses were salted and held at 16 degrees C for ripening until the pH was reduced to 4.60 . Cheeses were then moved into storage at 4 degrees C to complete ripening . During ripening, the E . coli O157:H7 population decreased significantly (P < or = 0.001) and finally was not detectable in Feta cheese after 44 and 36 days and in Telemes cheese after 40 and 30 days for the trials with thermophilic and mesophilic starters, respectively . The estimated times required for one decimal reduction of the population of E . coli O157:H7 after the first day of processing were 9.71 and 9.26 days for Feta cheese and 9.09 and 7.69 days for Telemes cheese for the trials with thermophilic and mesophilic starters, respectively. Anal Biochem, 2002 Apr 15, 303(2), 138 - 44 A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus; D'Auria S et al.; We describe the use of a thermostable glucokinase in a novel competitive fluorescence assay for glucose . Glucokinase from Bacillus stearothermophilus (BSGK) was found to retain enzymatic activity in solution for over 20 days . The single cysteine residue in BSGK, which is near the active site, was labeled with a fluorescent probe, 2-(4-iodoacetamidoanilino)naphthalene-6-sulfonic acid . The ANS-labeled BSGK displayed a modest 25% decrease in the emission intensity upon binding glucose but no change in lifetime . To obtain a larger spectral change we developed a competitive assay for glucose using the intrinsic tryptophan fluorescence from BSGK and a resonance energy transfer (RET) acceptor-labeled sugar . The sugar-labeled acceptor quenched the BSGK tryptophan emission, and the quenching was reversed upon addition of glucose . The use of RET as the sensing mechanism can be easily extended to longer wavelengths for a more practical glucose sensor . (C)2002 Elsevier Science (USA) Mikrobiol Z, 2002 Jan-Feb, 64(1), 48 - 56 {Ethanol formation by methane-utilizing bacteria at ethane co-metabolism}; Romanovskaia VA et al.; It was established, that EDTA (1.0 mM) and formamide (100 mM) are inhibitors of methanol dehydrogenase in Methylobacter luteus 12b, Methylomonas rubra 15sh and Methylococcus thermophilus 111p . The investigated strains co-metabolised ethane with the use of formate as the co-substrate . The application of formamide (or EDTA) as inhibitors of methanol dehydrogenase prevented from further transformation of ethanol and resulted in accumulation of extracellular ethanol . It was shown, that M . rubra 15sh accumulated extracellular ethanol under cultivation in a chemostate . The carried out researches have shown a regulation path of co-metabolism process of hydrocarbons by methane utilizing bacteria . Using the specific inhibitors of methanol dehydrogenase and a source of reducing agent (energy) for methane monooxygenase with the help of the cells of methane-oxidizing bacteria it is possible to obtain from ethane or other hydrocarbons the products of their monooxygenation--alcohols. J Biol Chem, 2002 Jun 7, 277(23), 20117 - 9 Epub 2002 Apr 09. Substitution of a single amino acid switches the tentoxin-resistant thermophilic F1-ATPase into a tentoxin-sensitive enzyme; Groth G et al.; In contrast to the homologous bacterial and mitochondrial enzymes the chloroplast F(1)-ATPase (CF(1)) is strongly affected by the phytopathogenic inhibitor tentoxin . Based on structural information obtained from crystals of a CF(1)-tentoxin co-complex (Groth, G . (2002) Proc . Natl . Acad . Sci . U . S . A . 99, 3464-3468) we have replaced residues betaSer(66) and alphaArg(132) in the alpha(3)beta(3)gamma subcomplex of the thermophilic F(1)-ATPase from Bacillus PS3 by the corresponding residues of the chloroplast ATPase to confer tentoxin sensitivity to the thermophilic enzyme . The mutation alphaArg(132) --> Pro, proposed to relieve steric constraints on tentoxin binding, did not have any significant effect . However, mutation betaSer(66) --> Ala, predicted to provide a crucial hydrogen bond with the inhibitor, resulted in tentoxin inhibition of ATP hydrolysis comparable with the situation found with the chloroplast enzyme. J Biol Inorg Chem, 2002 Apr, 7(4-5), 445 - 50 Epub 2002 Jan 08. Magnetization studies of the active and fluoride-inhibited derivatives of the reduced catalase of Lactobacillus plantarum: toward a general picture of the anion-inhibited and active forms of the reduced dimanganese catalases; Le Pape L et al.; The magnetic properties of the reduced catalase from Lactobacillus plantarum have been studied for the active enzyme and its fluoride complex through variable field/variable temperature magnetization measurements . The magnetic exchange interaction deduced from these experiments {fluoride complex: - J=1.3(1) cm(-1); active enzyme: - J=5.6(5) cm(-1); H=-2 J S(1) S(2)} are similar to those presently obtained in a re-analysis of the data for the corresponding forms of the Thermus thermophilus enzyme (previously published in 1997, Angew Chem Int Ed Engl 36:1626-1628): phosphate complex: - J=2.1(2) cm(-1); active enzyme - J=5.0(3) cm(-1) . These results concur to a unified picture for the two enzymes, consistent with the presence of a hydroxide bridge in the reduced active catalases and its replacement by an aqua bridge in the anion-inhibited enzymes as the main mediators of the magnetic exchange. J Biol Inorg Chem, 2002 Apr, 7(4-5), 357 - 62 Epub 2001 Oct 30. Formation of a linear {3Fe-4S} cluster in a seven-iron ferredoxin triggered by polypeptide unfolding; Jones K et al.; Cubic iron-sulfur ({Fe-S}) clusters are common inorganic cofactors in proteins . The presence of a linear {3Fe-4S} cluster in a protein was first observed in beef-heart aconitase at high pH, where the protein structure was perturbed . Not long ago, the same linear cluster was discovered upon unfolding of a thermophilic di-cluster seven-iron ferredoxin, suggesting a more general relevance for this type of linear clusters in Nature . Since structure-induced cluster rearrangements may be important regulatory, on-going processes in living systems, we decided to further characterize the formation of the linear iron-sulfur cluster observed upon ferredoxin unfolding . Here we present a kinetic investigation of parameters that affect the linear-cluster formation and disassembly in the Sulfolobus acidocaldarius seven-iron ferredoxin . We find the linear cluster to be an intermediate on the protein-mediated cluster-degradation pathway under a wide range of pH and denaturant conditions . The linear species forms in parallel with secondary-structure disappearance . In contrast, the disassembly rate constant for the linear cluster is independent of denaturant concentration but depends strongly on solution pH . At high pH, the disassembly rate is slower and the linear iron-sulfur species has a longer lifetime, than at low pH. Lett Appl Microbiol, 2002, 34(4), 287 - 9 Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC); Miyajima M et al.; AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC) . METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR . About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing . CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp . coli, 97-98% to Camp . jejuni and 97-98% to Camp . lari . SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed . The sequence of ISR was almost identical among the four strains of UPTC . It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms. Biochemistry, 2002 Apr 16, 41(15), 4760 - 70 Identification of a novel archaebacterial thioredoxin: determination of function through structure; Bhattacharyya S et al.; As part of a high-throughput, structural proteomic project we have used NMR spectroscopy to determine the solution structure and ascertain the function of a previously unknown, conserved protein (MtH895) from the thermophilic archeon Methanobacterium thermoautotrophicum . Our findings indicate that MtH895 contains a central four-stranded beta-sheet core surrounded by two helices on one side and a third on the other . It has an overall fold superficially similar to that of a glutaredoxin . However, detailed analysis of its three-dimensional structure along with molecular docking simulations of its interaction with T7 DNA polymerase (a thioredoxin-specific substrate) and comparisons with other known members of the thioredoxin/glutaredoxin family of proteins strongly suggest that MtH895 is more akin to a thioredoxin . Furthermore, measurement of the pK(a) values of its active site thiols along with direct measurements of the thioredoxin/glutaredoxin activity has confirmed that MtH895 is, indeed, a thioredoxin and exhibits no glutaredoxin activity . We have also identified a group of previously unknown proteins from several other archaebacteria that have significant (34-44%) sequence identity with MtH895 . These proteins have unusual active site -CXXC- motifs not found in any known thioredoxin or glutaredoxin . On the basis of the results presented here, we predict that these small proteins are all members of a new class of truncated thioredoxins. J Ind Microbiol Biotechnol, 2002 Jan, 28(1), 1 - 6 Lactic acid bacteria and yeasts in kefir grains and kefir made from them; Simova E et al.; In an investigation of the changes in the microflora along the pathway: kefir grains (A)-->kefir made from kefir grains (B)-->kefir made from kefir as inoculum (C), the following species of lactic acid bacteria (83-90%) of the microbial count in the grains) were identified: Lactococcus lactis subsp . lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp . bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp . pseudoplantarum and Lactobacillus brevis . Yeasts (10-17%) identified were Kluyveromyces marxianus var . lactis, Saccharomyces cerevisiae, Candida inconspicua and Candida maris . In the microbial population of kefir grains and kefir made from them the homofermentative lactic streptococci (52-65% and 79-86%, respectively) predominated . Within the group of lactobacilli, the homofermentative thermophilic species L . delbrueckii subsp . bulgaricus and L . helveticus (70-87% of the isolated bacilli) predominated . Along the pathway A-->B-->C, the streptococcal proportion in the total kefir microflora increased by 26-30% whereas the lactobacilli decreased by 13-23% . K . marxianus var . lactis was permanently present in kefir grains and kefirs, whereas the dominant lactose-negative yeast in the total yeast flora of the kefir grains dramatically decreased in kefir C. J Biol Inorg Chem, 2002 Mar, 7(3), 260 - 72 Epub 2001 Sep 08. Characterization of Hydrogenobacter thermophilus cytochromes c(552 )expressed in the cytoplasm and periplasm of Escherichia coli; Karan EF et al.; Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family . Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli . This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment . Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E . coli is reported . The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies . The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix . These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors . The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation . These results support the hypothesis that proper heme attachment can occur spontaneously in the E . coli cytoplasm for Ht cyt c(552). Biotechnol Prog, 2002 Mar-Apr, 18(2), 276 - 81 Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures; Bokhari SA et al.; We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp . with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis . It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents . The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan . The modified enzyme was also stabilized against irreversible thermal denaturation . Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min . We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively. Int J Food Microbiol, 2002 Mar, 73(2-3), 415 - 25 Simulation modelling of bacterial growth in yoghurt; Ginovart M et al.; INDISIM, an individual-based simulator, was used to specifically study the influence of the shape and size of Streptococcus salivarius subsp . thermophilus and Lactobacillus delbrueckii subsp . bulgaricus in yoghurt processing . To this effect, two different sets of simulations were carried out . In the first set of simulations, it was assumed that the initial acidity of the medium has the same value as the acidity of the cytoplasm of the microorganisms . Hence, the differences in bacterial growth by the two species are only attributable to differences in their geometry . It was found that, in an optimum culture, the growth in biomass of S . salivarius subsp . thermophilus is larger than that of L . delbrueckii subsp . bulgaricus . An important factor for understanding this difference might be the larger mass-to-surface ratio of the former . In the second set of simulations, a simplified model of yoghurt, the parametrisation differs both in the geometry and the metabolism of the two species . The results of these simulations are in very good qualitative agreement with the experimental data of {Lait 69 (1989) 519} . Finally, by inhibiting the uptake of amino acids by S . salivarius subsp . thermophilus, the large relative importance of lactic acid in yoghurt processing was highlighted. Int J Food Microbiol, 2002 Mar, 73(2-3), 251 - 9 A novel area of predictive modelling: describing the functionality of beneficial microorganisms in foods; Leroy F et al.; Predictive microbiology generally focuses on the potential outgrowth of spoilage bacteria and foodborne pathogens in foods . Little attention has been paid to the biokinetics of beneficial foodgrade microorganisms, such as lactic acid bacteria . The latter is commonly used in the food fermentation industry, mainly for the in situ production of the antimicrobial lactic acid to extend the shelf life of the food . Furthermore, many strains show additional industrial potential as novel starter cultures since they produce functional metabolites, such as bacteriocins and exopolysaccharides . The production of these functional metabolites has been demonstrated during in vitro experiments, but in many cases these novel starter cultures seem to be less efficient when applied in a food system . A modelling approach may contribute to a better understanding of the tight relation between the food environment and bacterial functionality . Primary modelling can be applied to fit the experimental data concerning cell growth, sugar metabolism, and the production of functional metabolites for a given set of environmental conditions . This led to conclusions concerning the growth-associated production of bacteriocin and exopolysaccharides, the inactivation of these molecules when cell growth levels off, and a minimum cell concentration to trigger on bacteriocin production . Examples deal with the production of the bacteriocin sakacin K by the natural fermented sausage isolate Lactobacillus sakei CTC 494, and the production of heteropolysaccharides by the yoghurt starter culture Streptococcus thermophilus LY03 . Secondary modelling of biokinetic parameters quantifies the production of bacteriocin and exopolysaccharides in function of environmental factors . As an example, the specific bacteriocin production by Lb . sakei CTC 494 decreases with increasing sodium chloride concentrations . Furthermore, since the assessment of functionality is frequently hampered by the nature of the food system, mathematical modelling techniques may help to predict the functional behaviour of novel lactic acid bacteria starter cultures in a food matrix, and hence quantify in situ production . For example, a model may simulate cell growth and exopolysaccharide production of S . thermophilus LY03 in a milk environment, where direct measurements are difficult to perform. Proteins, 2002 May 1, 47(2), 236 - 49 Comparative structural analysis of psychrophilic and meso- and thermophilic enzymes; Gianese G et al.; Enzymes adapted to cold display structures comparable with those of their meso- and thermophilic homologs but are characterized by a higher catalytic efficiency at low temperatures and by thermolability at moderate temperatures . To identify the structural factors responsible of such features, we undertook a systematic comparative analysis of several structural properties in a data set consisting of 7 cold active enzymes belonging to different structural families and 28 related structures from meso/thermophiles representing most of the structural information now available . Only high-resolution and high-quality structures were considered . Properties were calculated and then compared for each pair of 3D structures displaying different temperatures of adaptation using a temperature-weighting scheme . The significance of the resulting differences was evaluated with a statistical method . Results reveal that each protein family adopts different structural strategies to adapt to low temperatures . However, some common trends are observed: the number of ion pairs, the side-chain contribution to the exposed surface, and the apolar fraction of the buried surface show a consistent decrease with decreasing optimal temperatures . Heredity, 2002 Feb, 88(2), 125 - 41 Origins of the machinery of recombination and sex; Cavalier-Smith T; Mutation plays the primary role in evolution that Weismann mistakenly attributed to sex . Homologous recombination, as in sex, is important for population genetics--shuffling of minor variants, but relatively insignificant for large-scale evolution . Major evolutionary innovations depend much more on illegitimate recombination, which makes novel genes by gene duplication and by gene chimaerisation--essentially mutational forces . The machinery of recombination and sex evolved in two distinct bouts of quantum evolution separated by nearly 3 Gy of stasis; I discuss their nature and causes . The dominant selective force in the evolution of recombination and sex has been selection for replicational fidelity and viability; without the recombination machinery, accurate reproduction, stasis, resistance to radical deleterious evolutionary change and preservation of evolutionary innovations would be impossible . Recombination proteins betray in their phylogeny and domain structure a key role for gene duplication and chimaerisation in their own origin . They arose about 3.8 Gy ago to enable faithful replication and segregation of the first circular DNA genomes in precellular ancestors of Gram-negative eubacteria . Then they were recruited and modified by selfish genetic parasites (viruses; transposons) to help them spread from host to host . Bacteria differ fundamentally from eukaryotes in that gene transfer between cells, whether incidental to their absorptive feeding on DNA and virus infection or directly by plasmids, involves only genomic fragments . This was radically changed by the neomuran revolution about 850 million years ago when a posibacterium evolved into the thermophilic cenancestor of eukaryotes and archaebacteria (jointly called neomurans), radically modifying or substituting its DNA-handling enzymes (those responsible for transcription as well as for replication, repair and recombination) as a coadaptive consequence of the origin of core histones to stabilise its chromosome . Substitution of glycoprotein for peptidoglycan walls in the neomuran ancestor and the evolution of an endoskeleton and endomembrane system in eukaryotes alone required the origin of nuclei, mitosis and novel cell cycle controls and enabled them to evolve cell fusion and thereby the combination of whole genomes from different cells . Meiosis evolved because of resulting selection for periodic ploidy reduction, with incidental consequences for intrapopulation genetic exchange . Little modification was needed to recombination enzymes or to the ancient bacterial catalysts of homology search by spontaneous base pairing to mediate chromosome pairing . The key innovation was the origin of meiotic cohesins delaying centromere splitting to allow two successive divisions before reversion to vegetative growth and replication, necessarily yielding two-step meiosis . Also significant was the evolution of synaptonemal complexes to stabilise bivalents and of monopolins to orient sister centromeres to one spindle pole . The primary significance of sex was not to promote evolutionary change but to limit it by facilitating ploidy cycles to balance the conflicting selective forces acting on rapidly growing phagotrophic protozoa and starved dormant cysts subject to radiation and other damage. Protein Eng, 2002 Mar, 15(3), 215 - 23 A low-resolution 3D model of the tetrameric alcohol dehydrogenase from Sulfolobus solfataricus; Casadio R et al.; We describe the computation of a model of the thermophilic NAD-dependent homotetrameric alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) . Modeling is based on the knowledge that each monomer contains two Zn ions with catalytic and structural function, respectively . In the database of known structures, proteins with similar functions are either dimers containing two zinc ions per monomer or tetramers with one zinc ion per monomer . In any case, the sequence identity of the target to the possible templates is low . A threading procedure is therefore developed which includes constraints taking into account residue conservation both at the zinc ion binding and at the monomer-monomer interaction sites in the tetrameric unit . The model is consistent with previously reported data . Furthermore, cross-linking experiments are described which support the computed tetrameric model. Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 401 - 7 Gelria glutamica gen . nov., sp . nov., a thermophilic, obligately syntrophic, glutamate-degrading anaerobe; Plugge CM et al.; A novel anaerobic, gram-positive, thermophilic, spore-forming, obligately syntrophic, glutamate-degrading bacterium, strain TGO(T), was isolated from a propionate-oxidizing methanogenic enrichment culture . The axenic culture was obtained by growing the bacterium on pyruvate . Cells were rod-shaped and non-motile . The optimal temperature for growth was 50-55 degrees C and growth occurred between 37 and 60 degrees C . The pH range for growth was 5.5-8 with optimum growth at pH 7 . In pure culture, strain TGO(T) could grow on pyruvate, lactate, glycerol and several sugars . In co-culture with the hydrogenotrophic methanogen Methanobacterium thermautotrophicum strain Z-245, strain TGO(T) could grow on glutamate, proline and Casamino acids . Glutamate was converted to H2, CO2, propionate and traces of succinate . Strain TGO(T) was not able to utilize sulphate, sulphite, thiosulphate, nitrate or fumarate as electron acceptors . The G+C content was 33.8 mol% . Sequence analysis of the 16S rDNA revealed that strain TGO(T) belongs to the thermophilic, endospore-forming anaerobes, though no close relations were found . Its closest relations were Moorella glycerini (92%) and Moorella thermoacetica (90%) . Strain TGOT had an unusually long 16S rDNA of more than 1700 bp . The additional base pairs were found as long loops in the V1, V7 and V9 regions of the 16S rDNA . However, the loops were not found in the 16S rRNA . The name Gelria glutamica gen . nov., sp . nov . is proposed for strain TGO(T). Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 391 - 9 Desulfotomaculum thermobenzoicum subsp . thermosyntrophicum subsp . nov., a thermophilic, syntrophic, propionate-oxidizing, spore-forming bacterium; Plugge CM et al.; From granular sludge from a laboratory-scale upflow anaerobic sludge bed reactor operated at 55 degrees C with a mixture of volatile fatty acids as feed, a novel anaerobic, moderately thermophilic, syntrophic, spore-forming bacterium, strain TPO, was enriched on propionate in co-culture with Methanobacterium thermoautotrophicum Z245 . The axenic culture was obtained by using pyruvate as the sole source of carbon and energy . The cells were straight rods with pointed ends and became lens-shaped when sporulation started . The cells were slightly motile . The optimum growth temperature was 55 degrees C and growth was possible between 45 and 62 degrees C . The pH range for growth of strain TPO was 6-8, with an optimum at pH 7-7.5 . Propionate was converted to acetate, CO2 and CH4 by a co-culture of strain TPO with Methanobacterium thermoautotrophicum Z245 . In pure culture, strain TPO could grow fermentatively on benzoate, fumarate, H2/CO2, pyruvate and lactate . Sulphate could serve as inorganic electron acceptor when strain TPO was grown on propionate, lactate, pyruvate and H2/CO2 . The G+C content was 53.7 mol% . Comparison of 16S rDNA sequences revealed that strain TPO is related to Desulfotomaculum thermobenzoicum (98%) and Desulfotomaculum thermoacetoxidans (98%) . DNA-DNA hybridization revealed 88.2% reassociation between strain TPO and D . thermobenzoicum and 83.8% between strain TPO and D . thermoacetoxidans . However, both organisms differ physiologically from strain TPO and are not capable of syntrophic propionate oxidation . It is proposed that strain TPO should be classified as new subspecies of D . thermobenzoicum as D . thermobenzoicum subsp . thermosyntrophicum. Int J Food Microbiol, 2002 Mar 25, 74(1-2), 13 - 25 Prediction of the adherence, growth and release of microorganisms in production chains; de Jong P et al.; The aim of this study was to develop a mathematical model that describes the bacterial contamination of food as a result of adherence, growth and release of bacteria in process equipment . The model developed can be applied to control the bacterial quality of food products produced in process chains in which the final contamination of the product is governed by growth and heat-induced destruction of bacteria . To set up the model, experiments were carried out with a plate heat exchanger using milk inoculated with Streptococcus thermophilus . The growth rate of S . thermophilus in milk could be described accurately by the modified expanded model of Ratkowsky . The observed increase in the concentration of S . thermophilus in milk at the outlet of the plate heat exchanger could be described quantitatively by the model . To predict the contamination of the product, the model was integrated into NIZO-PCS (Process Chain Simulator) . The results of computer simulations were validated by a number of measurements in a cheese factory . It turned out that the agreement between the measured and calculated concentrations of S . thermophilus was sufficient for the model to be used for predictions in industrial production chains. J Basic Microbiol, 2002, 42(1), 55 - 66 Extracellular beta-D-glucosidase from Chaetomium thermophilum var . coprophilum: production, purification and some biochemical properties; Venturi LL et al.; The thermophilic fungus Chaetomium thermophilum var . coprophilum produced large amounts of extracellular and intracellular beta-glucosidase activity when grown on cellulose or cellobiose as carbon sources . The presence of glucose in the culture medium drastically decreased the level of beta-glucosidase activity, while cycloheximide prevented the induction of the extracellular enzyme activity by cellobiose . An extracellular beta-glucosidase induced by avicel was purified by a procedure involving acetone precipitation and chromatography on two DEAE-cellulose columns . The purified enzyme was a basic protein, with a carbohydrate content of 73% . The deglycosylated enzyme exhibited a molecular mass of 43 kDa, with pH and temperature optima of 5.5 and 65 degrees C respectively . The beta-glucosidase hydrolysed only cellobiose and p-nitrophenyl-beta-D-glucopyranoside, exhibiting apparent Km values of 3.13 mM and 0.76 mM, respectively . The native purified enzyme was stable up to 2 hours at 60 degrees C, and its thermal stability was directly dependent on glycosylation. Med Pr, 2001, 52(6), 417 - 22 {Assessment of exposure to bioaerosols in workplace ambient air during municipal waste collection and disposal}; Krajewski JA et al.; The assessment of the exposure to bioaerosols among workers engaged in the collection and disposal of municipal waste is presented . The workers were divided into the following groups, depending on the job performed: waste collectors (loaders, drivers), composting plant workers (heavy equipment operators, waste site workers), sorters (heavy equipment operators, waste sorters) and landfill site workers (heavy equipment operators, weighers) . Air samples were also collected on the city streets and in flats . They were reference points to the results obtained . Air samples were collected at the breathing zone . The concentrations of dust, viable microorganisms (mesophilic and thermophilic bacteria, and fungi), endotoxins and the total number of microbiol cells were determined . The highest, individual exposure to dust was found in composting plant workers and waste collectors . The values exceeded considerably maximum allowable concentrations (by ten times at composting plant) . The mean values for the above listed groups also exceeded the concentration of 4 mg/m3 . Dust concentrations at other workposts were substantially lower . The concentrations in the city streets and in flats maintained at the level of 0.1 mg/m3 . Endotoxin concentration at a protecting upper airway inflammation level (10 ng/m3) was exceeded in the majority (above 50%) of samples . That is why the average concentration in individual groups was also higher than 10 ng/m3 . Street and flat samples included endotoxins at the level of 1 ng/m3 . Taking as a criterion the obligatory total dust and endotoxin MAC value of 10 ng/m3 for assessing work hygiene conditions, it should be stated that waste collectors and composting plant workers were employed in conditions of poor hygiene. Environ Technol, 2002 Jan, 23(1), 15 - 26 Pig manure as a co-composting material for biodegradation of PAH-contaminated soil; Wong JW et al.; Pig manure at three different ratios of 12.5%, 25% and 50% (w/w dry weight basis) was amended with a soil spiked with 100 mg kg(-1) each of three polycyclic aromatic hydrocarbons (phenanthrene, anthracene, and pyrene) to investigate its effect on the biodegradation of these PAHs in a bench-scale composting system . An increase in pig manure amendment was effective in enhancing the amounts of soluble organic carbon, ammoniacal nitrogen, and soluble phosphorous in the composting mass . It could also increase the populations of total thermophilic and mesophilic bacteria as well as PAH-degrading bacteria, but this pattern was restricted only to the early stage of the composting process . High amounts of pig manure in the composting mass reduced the seed germination or root growth of cress seeds, but the composting process was effective in reducing the phytotoxic effects of the compost . Amendment of pig manure was beneficial to PAH removal during composting treatment and maximum removal rate at the end of composting accounted for 90% of the initial concentrations of PAHs . A pig manure application rate of 25% showed the most efficient removal of 3-ringed PAHs (phenanthrene and anthracene), while no significant difference in pyrene removal for those receiving 25 or 50% pig manure amendment . Taking into consideration the effects of pig manure on seed germination and available nutrients in the composting mass, this study suggested that a pig manure amendment of 25%, i.e., 3:1 ratio of contaminated soil: pig manure, is recommended to co-compost with PAH-contaminated soil. Chemosphere, 2002 Feb, 46(6), 879 - 85 Biodegradation of polylactide in aerobic and anaerobic thermophilic conditions; Itavaara M et al.; Biodegradable polymers are designed to resist a number of environmental factors during use, but to be biodegradable under disposal conditions . The biodegradation of polylactide (PLLA) was studied at different elevated temperatures in both aerobic and anaerobic, aquatic and solid state conditions . In the aerobic aquatic headspace test the mineralisation of PLLA was very slow at room temperature, but faster under thermophilic conditions . The clear effect of temperature on the biodegradability of PLLA in the aquatic tests indicates that its polymer structure has to be hydrolysed before microorganisms can utilise it as a nutrient source . At similar elevated temperatures, the biodegradation of PLLA was much faster in anaerobic solid state conditions than in aerobic aquatic conditions . The behaviour of PLLA in the natural composting process was similar to that in the aquatic biodegradation tests, biodegradation starting only after the beginning of the thermophilic phase . These results indicate that PLLA can be considered as a compostable material, being stable during use at mesophilic temperatures, but degrading rapidly during waste disposal in compost or anaerobic treatment facilities. Genes Cells, 2002 Mar, 7(3), 259 - 72 Identification and characterization of tRNA (Gm18) methyltransferase from Thermus thermophilus HB8: domain structure and conserved amino acid sequence motifs; Hori H et al.; BACKGROUND: Transfer RNAs from an extreme thermophile, Thermus thermophilus, commonly possess 2'-O-methylguanosine at position 18 (Gm18) in the D-loop . This modification is post-transcriptionally introduced by tRNA (Gm18) methyltransferase . RESULTS: Partial amino acid sequence data were obtained from purified T . thermophilus tRNA (Gm18) methyltransferase by peptide sequencing and mass spectrometry . The sequence data were used to screen the T . thermophilus genome database currently in progress, resulting in the identification of the corresponding gene . Purified recombinant enzyme showed a strict specificity for methylation at the 2'-OH of G18 in tRNA . Sequence alignment with other known or putative methyltransferases elucidates that tRNA (Gm18) methyltransferases have specific conserved region as well as three consensus motifs found in RNA ribose 2'-O-methyltransferases . The enzyme truncated at its N and C termini by limited tryptic digestion still retained binding activity for S-adenosyl-l-homocysteine, but lost the catalytic activity . CONCLUSION: This is the first report describing the identification of a methyltransferase gene of the trmH family through the analysis of a purified protein . Further, our results indicate that a restricted region(s) in the terminal amino acid residues of T . thermophilus tRNA (Gm18) methyltransferase are responsible for tRNA recognition and a main part of the enzyme is allocated for a catalytic core. Environ Sci Technol, 2002 Mar 1, 36(5), 1113 - 23 Mathematical model for meso- and thermophilic anaerobic sewage sludge digestion; Siegrist H et al.; A mathematical model is developed to describe the dynamic behavior of mesophilic (35 +/- 5 degrees C) and thermophilic digestion (55 +/- 5 degrees C) . Special emphasis is given to acetotrophic methanogenesis and propionate degradation, as the steps that determine the stability of anaerobic digestion, as well as to hydrolysis rate, which determines the degradation efficiency of particulate degradable organic carbon . Within the range of 6-20 (mesophilic) and 2-8 d (thermophilic) hydraulic retention time (HRT), the observed maximum growth rates for acetotrophic methanogens are 0.33 and 1.3 d(-1), respectively, with a 15% decay rate . Temperature and pH dependence as well as ammonia inhibition of acetate and propionate conversion are determined and included in the model, which allows us to simulate the effect of protein- and nitrogen-rich waste addition and the consequences of temporarily increased free ammonia at high pH . No inhibition of hydrogen conversion was observed in the same free ammonia range . The pH optimum is between 6.6 and 7.3 . Acetotrophic methanogenesis is strongly inhibited below pH 6.2, whereas above pH 7.4 it can be inhibited by free ammonia . For digesters fed with ordinary municipal sewage sludge, free ammonia inhibition of acetate conversion leads to an increase in acetate at about 35 and 140 mg of N/L for mesophilic (HRT = 20 d) and thermophilic (HRT = 6 d) conditions, respectively . The hydrolysis rate constant is 0.25 and 0.4 d(-1) respectively for these two conditions . The model is validated with load variation experiments in laboratory and full-scale digesters for step and shock loads. Nucleic Acids Res . 2002 Apr 1;30(7):e33. TspGWI, a thermophilic class-IIS restriction endonuclease from Thermus sp., recognizes novel asymmetric sequence 5'-ACGGA(N11/9)-3'; Zylicz-Stachula A et al.; A novel prototype class-IIS restriction endonuclease, TspGWI, was isolated from the thermophilic bacterium Thermus sp . GW . The recognition sequence and cleavage positions have been established: TspGWI recognizes the non-palindromic 5-bp sequence 5'-ACGGA-3' and cleaves the DNA 11 and 9 nt downstream in the top and bottom strand, respectively . In addition, an accompanying endonuclease, TspGWII, an isoschizomer of Pst I, was found in Thermus sp . GW cells. Nucleic Acids Res, 2002 Apr 1, 30(7), 1606 - 12 Efficient trans-cleavage by the Schistosoma mansoni SMalpha1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus; Vazquez-Tello A et al.; The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo . The cellular role of these repetitive elements and their transcripts is unknown . Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo . We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni . The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step . The optimal temperature for trans-cleavage was 70 degrees C . This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus . Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability . Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo. Appl Environ Microbiol, 2002 Apr, 68(4), 2081 - 4 Organophosphonate utilization by the thermophile Geobacillus caldoxylosilyticus T20; Obojska A et al.; A strain of Geobacillus caldoxylosilyticus from central heating system water could utilize a number of organophosphonates as the sole phosphorus source for growth at 60 degrees C . During growth on glyphosate, aminomethylphosphonate release to the medium was observed, and in cell extracts, a glyphosate oxidoreductase-type activity, producing stoichiometric amounts of aminomethylphosphonate and glyoxylate from glyphosate, was detectable. Appl Environ Microbiol, 2002 Apr, 68(4), 2061 - 5 Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment; Pelandakis M et al.; A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment . Multiplex PCR was also capable of identifying N . fowleri isolates with internal transcribed spacers of different sizes . In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. Appl Environ Microbiol, 2002 Apr, 68(4), 1914 - 8 Physiological function of alcohol dehydrogenases and long-chain (C(30)) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus; Burdette DS et al.; A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% {vol/vol}) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% {vol/vol}) . The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C(30) fatty acids) in response to increasing levels of ethanol . The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production . These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities). Appl Environ Microbiol, 2002 Apr, 68(4), 1882 - 92 Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis; Randazzo CL et al.; The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches . The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA) . Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively . DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure . Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation . Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening . Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected . Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation . The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology. Appl Environ Microbiol, 2002 Apr, 68(4), 1735 - 42 Use of Fe(III) as an electron acceptor to recover previously uncultured hyperthermophiles: isolation and characterization of Geothermobacterium ferrireducens gen . nov., sp . nov; Kashefi K et al.; It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms . This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor . As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor . A pure culture was recovered on solidified, Fe(III) oxide medium . The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide . Organic carbon is not required for growth . Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated . The cells are rod shaped, about 0.5 microm by 1.0 to 1.2 microm, and motile and have a single flagellum . Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100 degrees C with an optimum of 85 to 90 degrees C . To our knowledge this is the highest temperature optimum of any organism in the BACTERIA: Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the Bacteria, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland . While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers . These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences . Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the Bacteria . The name Geothermobacterium ferrireducens gen . nov., sp . nov., is proposed (ATCC BAA-426). Appl Environ Microbiol, 2002 Apr, 68(4), 1639 - 46 Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans; Takao M et al.; A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated . It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan . The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies . The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5 . The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C . The enzyme had optimal activity at 70 degrees C and pH 6.0 . The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively . The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134 . The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose . The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature. Acta Crystallogr D Biol Crystallogr, 2002 Apr, 58(Pt 4), 645 - 52 Epub 2002 Mar 22. Comparative analysis of space-grown and earth-grown crystals of an aminoacyl-tRNA synthetase: space-grown crystals are more useful for structural determination; Ng JD et al.; Protein crystallization under microgravity aims at benefiting from the quasi-absence of convection and sedimentation to favor well ordered crystal nucleation and growth . The dimeric multidomain enzyme aspartyl-tRNA synthetase from Thermus thermophilus has been crystallized within dialysis reactors of the Advanced Protein Crystallization Facility in the laboratory on earth and under microgravity aboard the US Space Shuttle . A strictly comparative crystallographic analysis reveals that the crystals grown in space are superior in every respect to control crystals prepared in otherwise identical conditions on earth . They diffract X-rays more intensely and have a lower mosaicity, facilitating the process of protein structure determination . Indeed, the electron-density map calculated from diffraction data of space-grown crystals contains considerably more detail . The resulting three-dimensional structure model at 2.0 A resolution is more accurate than that produced in parallel using the data originating from earth-grown crystals . The major differences between the structures, including the better defined amino-acid side chains and the higher order of bound water molecules, are emphasized. DNA Seq, 2001 Dec, 12(5-6), 413 - 7 Molecular cloning and sequence analysis of the manganese catalase gene from Thermoleophilum album NM; Phucharoen K et al.; The manganese catalase gene (mnct) from Thermoleophilum album NM, a thermophilic bacterium, was cloned and its nucleotide sequence was analyzed . The gene consists of 885 bp (65.4% GC content) encoding 294 amino acids with a molecular mass of 32,500 Da . The deduced amino acid sequence shows similarities to those of Thermus species strain YS 8-13 (a thermophilic bacterium) and Bacillus halodurans (an alkaliphilic bacterium) with 61 and 54% identities, respectively. J Biotechnol, 2002 May 9, 95(2), 109 - 31 The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships; Sapag A et al.; Eighty-two amino acid sequences of the catalytic domains of mature endoxylanases belonging to family 11 have been aligned using the programs MATCHBOX and CLUSTAL . The sequences range in length from 175 to 233 residues . The two glutamates acting as catalytic residues are conserved in all sequences . A very good correlation is found between the presence (at position 100) of an asparagine in the so-called 'alkaline' xylanases, or an aspartic acid in those with a more acidic pH optimum . Four boxes defining segments of highest similarity were detected; they correspond to regions of defined secondary structure: B5, B6, B8 and the carboxyl end of the alpha helix, respectively . Cysteine residues are not common in these sequences (0.7% of all residues), and disulfide bridges are not important in explaining the stability of several thermophilic xylanases . The alignment allows the classification of the enzymes in groups according to sequence similarity . Fungal and bacterial enzymes were found to form mostly separate clusters of higher similarity. Pharmacogenomics J, 2001, 1(2), 115 - 25 Global perspectives on proteins: comparing genomes in terms of folds, pathways and beyond; Das R et al.; The sequencing of complete genomes provides us with a global view of all the proteins in an organism . Proteomic analysis can be done on a purely sequence-based level, with a focus on finding homologues and grouping them into families and clusters of orthologs . However, incorporating protein structure into this analysis provides valuable simplification; it allows one to collect together very distantly related sequences, thus condensing the proteome into a minimal number of 'parts.' We describe issues related to surveying proteomes in terms of structural parts, including methods for fold assignment and formats for comparisons (eg top-10 lists and whole-genome trees), and show how biases in the databases and in sampling can affect these surveys . We illustrate our main points through a case study on the unique protein properties evident in many thermophile genomes (eg more salt bridges) . Finally, we discuss metabolic pathways as an even greater simplification of genomes . In comparison to folds these allow the organization of many more genes into coherent systems, yet can nevertheless be understood in many of the same terms. Gastroenterology, 2002 Apr, 122(4), 1070 - 87 Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium: role of iNOS and COX-2; Resta-Lenert S et al.; BACKGROUND & AIMS: Various invasive pathogens cause diarrhea, but the mechanism(s) are poorly understood . We hypothesized that nitric oxide and prostaglandins might modulate chloride secretory and barrier properties of the infected intestinal epithelium and that diarrhea is caused, in part, by altered expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) . METHODS: Studies were conducted in human intestinal epithelial cell lines (HT29/cl.19A, Caco-2, and T84) . Cells were infected with enteroinvasive Escherichia coli (EIEC 029:NM) or Salmonella dublin (SD), or nonpathogenic, noninvasive bacteria (Streptococcus thermophilus {ST} and Lactobacillus acidophilus {LA}) . Infected cells and controls were tested for transepithelial resistance, chloride secretion, prostaglandin E2, guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate, and protein expression . RESULTS: Cells infected with EIEC or SD, but not uninfected controls or ST/LA-exposed monolayers, showed a progressive reduction in transepithelial resistance starting at 6-12 hours . Infected HT29/cl.19A and Caco-2 cells, but not T84 cells, also showed an increase in total nitrite . Expression of iNOS, and consequently COX-2, was also increased, followed by increased production of prostaglandins and cyclic nucleotides . Furthermore, basal and stimulated chloride secretory responses to various agonists were enhanced in HT29/cl.19A and Caco-2 cells after infection with enteroinvasive bacteria, and this effect was reversed for some agonists by iNOS or COX-2 inhibitors . Increased expression of cystic fibrosis transmembrane conductance regulator and NKCC1 was also observed in EIEC or SD-infected cells vs . controls, secondary to NO synthase activity . CONCLUSIONS: Up-regulation of iNOS and COX-2 by enteroinvasive bacteria can modulate chloride secretion and barrier function in intestinal epithelial cells . Thus, these enzymes represent possible therapeutic targets in infectious diarrhea. FEBS Lett, 2002 Mar 6, 514(1), 90 - 5 RNA aptamers directed against release factor 1 from Thermus thermophilus; Szkaradkiewicz K et al.; An in vitro selection/amplification (SELEX) was used to generate RNA aptamers that specifically bind Thermus thermophilus release factor 1 (RF1) . From 31 isolated clones, two groups of aptamers with invariable sequences 5'-ACCU-3' and 5'-GAAAGC-3' were isolated . Chemical and enzymatic probing of the structure indicate that in both groups the invariable sequences are located in single-stranded regions of hairpin structures . Complex formations between RF1 and aptamers of both groups were identified by electrophoretic shift assay and chemical footprinting . Deletion of the invariable sequences did not effect the secondary structure of the aptamers but abolished their binding to RF1 . RNA motifs matching the invariable sequences of the aptamers are present as consensus sequences in the peptidyl transferase center of 23S rRNAs . T . thermophilus RF1 recognizes UAG stop codons in an Escherichia coli in vitro translation system . Aptamers from both groups inhibited this RF1 activity. Res Microbiol, 2002 Mar, 153(2), 61 - 7 Sugar transport in (hyper)thermophilic archaea; Koning SM et al.; Hyperthermophilic archaea show important metabolic adaptations for growth on carbohydrates under hostile conditions . For carbohydrate uptake so far only ABC-type transporters have been described that are equipped with a uniquely high affinity as compared to mesophilic bacterial systems . This allows these organisms to efficiently scavenge all available carbohydrates from the extreme environment. Cell Biochem Biophys, 2001, 34(3), 321 - 47 Mechanosensitive channel of Thermoplasma, the cell wall-less archaea: cloning and molecular characterization; Kloda A et al.; By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium . Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance . 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum . The protein encoded by the T . acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes . When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol . In analogy to the nomenclature used for bacterial MS channels, the MS channel of T acidophilum was termed MscTA . Secondary structural analysis indicated that similar to MscL, the T . acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions . When the mscTA gene was expressed in the mscL- knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was characterized by very brief openings of variable conductance . In contrast, when the mscTA gene was expressed in the wild-type mscL+ strain of E . coli, the gating properties of the channel resembled MscL . However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity . Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels. J Cell Biochem, 2002, 85(1), 146 - 57 Interaction of the ADP-ribosylating enzyme from the hyperthermophilic archaeon S . solfataricus with DNA and ss-oligo deoxy ribonucleotides; Faraone-Mennella MR et al.; The DNA-binding ability of the poly-ADPribose polymerase-like enzyme from the extremely thermophilic archaeon Sulfolobus solfataricus was determined in the presence of genomic DNA or single stranded oligodeoxyribonucleotides . The thermozyme protected homologous DNA against thermal denaturation by lowering the amount of melted DNA and increasing melting temperature . The archaeal protein induced structural changes of the nucleic acid by modifying the dichroic spectra towards a shape typical of condensing DNA . However, enzyme activity was slightly increased by DNA . Competition assays demonstrated that the protein interacted also with heterologous DNA . In order to characterize further the DNA binding properties of the archaeal enzyme, various ss-oligodeoxyribonucleotides of different base composition, lengths (12-mer to 24-mer) and structure (linear and circular) were used for fluorescence titration measurements . Intrinsic fluorescence of the archaeal protein due to tryptophan (excitation at 295 nm) was measured in the presence of each oligomer at 60 degrees C . Changes of tryptophan fluorescence were induced by all compounds in the same range of base number per enzyme molecule, but independently from the structural features of oligonucleotides, although the protein exhibited a slight preference for those adenine-rich and circular . The binding affinities were comparable for all oligomers, with intrinsic association constants of the same order of magnitude (K=10(6) M(-1)) in 0.01 M Na-phosphate buffer, pH 8.0, and accounted for a "non-specific" binding protein . Circular dichroism analysis showed that at 60 degrees C the native protein was better organized in a secondary structure than at 20 degrees C . Upon addition of oligonucleotides, enzyme structure was further stabilized and changed towards a beta-conformation . This effect was more marked with the circular oligomer . The analysed oligodeoxyribonucleotides slightly enhanced enzyme activity with the maximal increase of 50% as compared to the control . No activation was observed with the circular oligomer. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3569 - 74 Epub 2002 Mar 12. A Gaussian-chain model for treating residual charge-charge interactions in the unfolded state of proteins; Zhou HX; Characterization of the unfolded state is essential for understanding the protein folding problem . In the unfolded state, a protein molecule samples vastly different conformations . Here I present a simple theoretical method for treating residual charge-charge interactions in the unfolded state . The method is based on modeling an unfolded protein as a Gaussian chain . After sampling over all conformations, the electrostatic interaction energy between two charged residues (separated by l peptide bonds) is given by W = 332(6/pi)(1/2){1 - pi(1/2)xexp(x(2))erfc(x)}/epsilond, where d = bl(1/2) + s and x = kappad/6(1/2) . In unfolded barnase, the residual interactions lead to downward pK(a) shifts of approximately 0.33 unit, in agreement with experiment . pK(a) shifts in the unfolded state significantly affect pH dependence of protein folding stability, and the predicted effects agree very well with experimental results on barnase and four other proteins . For T4 lysozyme, the charge reversal mutation K147E is found to stabilize the unfolded state even more than the folded state (1.39 vs . 0.46 kcal/mol), leading to the experimentally observed result that the mutation is net destabilizing for the folding . The Gaussian-chain model provides a quantitative characterization of the unfolded state and may prove valuable for elucidating the energetic contributions to the stability of thermophilic proteins and the energy landscape of protein folding. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3499 - 504 Epub 2002 Mar 12. The tRNA specificity of Thermus thermophilus EF-Tu; Asahara H et al.; By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)*GTP by using a ribonuclease protection assay . The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated . The E . coli elongator tRNAs exhibit a wide range of binding affinities that varied from -11.7 kcal/mol for Val-tRNA(Glu) to -8.1 kcal/mol for Val-tRNA(Tyr), clearly establishing EF-Tu*GTP as a sequence-specific RNA-binding protein . Because the ionic strength dependence of k(off) varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein . Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3734 - 9 Epub 2002 Mar 12. A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila; Shang Y et al.; The Cd(2+)-inducible metallothionein (MTT1) gene was cloned from Tetrahymena thermophila . Northern blot analysis showed that MTT1 mRNA is not detectable in the absence of Cd(2+), is induced within 10 min of its addition, is expressed in proportion to its concentration, and rapidly disappears upon its withdrawal . Similarly, when the neo1 gene coding region flanked by the MTT1 gene noncoding sequences was used to disrupt the MTT1 locus, no transformants were observed in the absence of Cd(2+), and the number of transformants was proportional to increased Cd(2+) concentration . The neo3 cassette, in which the MTT1 promoter replaced the histone gene HHF1 promoter of the previously used neo2 cassette, transformed cells at much higher frequencies than neo2 and produced germ-line knockouts where neo2 had failed . Rescuing the progeny of a mating of gamma-tubulin gene, GTU1, knockout heterokaryons with a GTU1 gene inserted into the MTT1 locus yielded >75 times more transformants than rescuing with the wild-type GTU1 gene itself . When cells rescued with the MTT1-GTU1 chimeric gene were transferred to medium lacking Cd(2+), they stopped growing and had phenotypic changes indistinguishable from cells containing only disrupted GTU1 genes . Thus, it is now possible to create conditional lethal mutants and study the terminal phenotypes of null mutations for essential genes by replacing the endogenous gene with one under the control of the MTT1 promoter . The MTT1 promoter also resulted in approximately 30 times more overexpression of the IAG48{G1} surface antigen gene of the ciliate fish parasite Ichthyophthirius multifiliis than the highly expressed BTU1 promoter, accounting for approximately 1% of the total cell protein . Thus, the MTT1 promoter should enable routine over-expression of endogenous and foreign genes in Tetrahymena. Biochem Biophys Res Commun, 2002 Mar 22, 292(1), 280 - 6 A novel five-subunit-type 2-oxoglutalate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus TK-6; Yun NR et al.; A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive TCA cycle . 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) of this strain is one of the key enzymes of the pathway . OGOR of strain TK-6 has been reported to be a two-subunit-type OGOR and encoded by korAB . A gene cluster, forDABGEF, encoding another OGOR was found 148 bp upstream of korAB in the opposite orientation . Five of the for genes (forDABGE) were required for the expression of the active recombinant enzyme in Escherichia coli . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed five polypeptides corresponding to the forDABGE gene products, suggesting that the enzyme had a novel five-subunit structure . The recombinant enzyme had high substrate specificity toward 2-oxoglutarate as in the case of the gene products of korAB . Primer extension analysis showed that the korA and forD genes were transcribed from one and two transcriptional initiation sites, respectively . The results also suggested that both gene clusters were expressed in the cells of strain TK-6 . (C)2002 Elsevier Science (USA). Arch Microbiol, 2002 Apr, 177(4), 339 - 44 Epub 2002 Feb 07. Thermaerobacter nagasakiensis sp . nov., a novel aerobic and extremely thermophilic marine bacterium; Nunoura T et al.; A novel, extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent at depth of 22 m in Tachibana Bay, Nagasaki Prefecture, Japan . Cells were gram-negative, non-spore-forming, motile rods . Growth was observed between 52 and 78 degrees C (optimum 70 degrees C), pH 5 and 8 (optimum pH 7) and 0-4.5% NaCl (optimum 1.0%) . The isolate was a strictly aerobic heterotroph utilizing yeast extract and trypticase peptone . The G+C content of the genomic DNA is 69 mol% . Analysis of 16S rDNA sequences indicated that strain Ts1a is closely related to Thermaerobacter marianensis . The differences in physiology and DNA-DNA similarity between strain Ts1a and T . marianensis showed that strain Ts1a represents a new species of Thermaerobacter . The type strain of T . nagasakiensis is strain Ts1a (=JCM11223, DSM 14512). J Bacteriol, 2002 Apr, 184(7), 2030 - 3 Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii; Chopin MC et al.; We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium . Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames . The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria . The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively . B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria. J Bacteriol, 2002 Apr, 184(7), 1952 - 7 The pyrimidine nucleotide reductase step in riboflavin and F(420) biosynthesis in archaea proceeds by the eukaryotic route to riboflavin; Graupner M et al.; The Methanococcus jannaschii gene MJ0671 was cloned and overexpressed in Escherichia coli, and its gene product was tested for its ability to catalyze the pyridine nucleotide-dependent reduction of either 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (compound 3) to 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate (compound 4) or 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 7) to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 5) . Only compound 3 was found to serve as a substrate for the enzyme . NADPH and NADH functioned equally well as the reductants . This specificity for the reduction of compound 3 was also confirmed by using cell extracts of M . jannaschii and Methanosarcina thermophila . Thus, this step in riboflavin biosynthesis in these archaea is the same as that found in yeasts . The absence of the other genes in the biosynthesis of riboflavin in Archaea is discussed. Cryobiology, 2001 Nov, 43(3), 189 - 98 Operating conditions that affect the resistance of lactic acid bacteria to freezing and frozen storage; Fonseca F et al.; Thermophilic lactic acid bacteria exhibit different survival rates during freezing and frozen storage, depending on the processing conditions . We used a Plackett and Burman experimental design to study the effects of 13 experimental factors, at two levels, on the resistance of Streptococcus thermophilus and Lactobacillus delbrueckii subsp . bulgaricus to freezing and frozen storage . The resistance was evaluated by quantifying the decrease of acidification activity during freezing and throughout 8 weeks of storage . Acidification activity after freezing and frozen storage was affected by 12 experimental factors . Only the thawing temperature did not show any significant effect . S . thermophilus was more resistant than L . bulgaricus and the cryoprotective effect of glycerol during freezing and storage was confirmed . The temperature and duration of the cryoprotection step influenced acidification activity following the freezing step: the lower the temperature and the shorter the duration, the higher the activity . Acidification activity after storage was affected by several experimental factors involved in the fermentation stage: use of NaOH instead of NH4OH for pH control, addition of Tween 80 in the culture medium, and faster cooling led to better cryotolerance . Resistance to freezing and frozen storage was improved by using a high freezing rate and a low storage temperature . Finally, this study revealed that the conditions under which lactic acid bacteria are prepared should be well controlled to improve their preservation and to limit the variability between batches and between species . Int J Food Microbiol, 2002 Feb 25, 73(1), 11 - 21 An emission pattern of a thermophilic bacteria attached to or imbedded in porous supports; Yoo JA et al.; There are many problems with thermophilic bacteria contamination of milk in the dairy industry . This is, in part, a result of fouling by milk components on stainless steel surfaces, which provide good harboring facilities for these bacteria to attach, imbed and grow . The interactions between milk fouling and bacteria deposited in or on the fouling deposit therefore become important issues . There have been a number of previous studies on the biofilm development in dairy processing plants . Here, a different approach to investigate the bacteria emission from a porous layer has been taken . In this approach, various process fluids were flushed over the top of a model milk foulant layer that contains high percentages of milk proteins, fat and some bacteria cells, in order to investigate the behavior of the 'resident' microorganisms and how they are 'released' into the flushing liquids . Definitive results were obtained, which have created sufficient interest for a different approach taken later, where fabric layers were used as the support for the bacteria cells to explore the 'generic' behavior of the porous layer-bacteria system . This study has shown that Bacillus stearothermophilus could multiply on or within a porous layer and 'migrate' from the layer into the fluid during processing . This "migration" is somewhat peculiar in terms of its time-responses but these are reproducible in all the tests performed . The phenomena observed may have an impact on future microbial safety practice in food factories. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2678 - 83 Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation; Min B et al.; Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate . Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway {Becker, H . D . & Kern, D . (1998) Proc . Natl . Acad . Sci . USA 95, 12832-12837; and Curnow, A . W., Tumbula, D . L., Pelaschier, J . T., Min, B . & Soll, D . (1998) Proc . Natl . Acad . Sci . USA 95, 12838-12843} . A genetic knockout disrupting this pathway deprives D . radiodurans of the ability to synthesize asparagine and confers asparagine auxotrophy . The organism's capacity to make asparagine could be restored by transformation with Escherichia coli asnB . This result demonstrates that in Deinococcus, the only route to asparagine is via asparaginyl-tRNA . Analysis of the completed genomes of many bacteria reveal that, barring the existence of an unknown pathway of asparagine biosynthesis, a wide spectrum of bacteria rely on the tRNA-dependent transamidation pathway as the sole route to asparagine. J Biol Chem, 2002 Jun 14, 277(24), 21643 - 9 Epub 2002 Mar 05. F1-ATPase changes its conformations upon phosphate release; Masaike T et al.; Motor proteins, myosin, and kinesin have gamma-phosphate sensors in the switch II loop that play key roles in conformational changes that support motility . Here we report that a rotary motor, F1-ATPase, also changes its conformations upon phosphate release . The tryptophan mutation was introduced into Arg-333 in the beta subunit of F1-ATPase from thermophilic Bacillus PS3 as a probe of conformational changes . This residue interacts with the switch II loop (residues 308-315) of the beta subunit in a nucleotide-bound conformation . The addition of ATP to the mutant F1 subcomplex alpha3beta(R333W)3gamma caused transient increase and subsequent decay of the Trp fluorescence . The increase was caused by conformational changes on ATP binding . The rate of decay agreed well with that of phosphate release monitored by phosphate-binding protein assays . This is the first evidence that the beta subunit changes its conformation upon phosphate release, which may share a common mechanism of exerting motility with other motor proteins. Virology, 2001 Dec 20, 291(2), 226 - 34 Sequences and replication of genomes of the archaeal rudiviruses SIRV1 and SIRV2: relationships to the archaeal lipothrixvirus SIFV and some eukaryal viruses; Peng X et al.; The double-stranded DNA genomes of the viruses SIRV1 and SIRV2, which infect the extremely thermophilic archaeon Sulfolobus and belong to the family Rudiviridae, were sequenced . They are linear, covalently closed at the ends, and 32,312 and 35,502 bp long, respectively, with an A+T content of 75% . The genomes of SIRV1 and SIRV2 carry inverted terminal repeats of 2029 and 1628 bp, respectively, which contain multiple direct repeats . SIRV1 and SIRV2 genomes contain 45 and 54 ORFs, respectively, of which 44 are homologous to one another . Their predicted functions include a DNA polymerase, a Holliday junction resolvase, and a dUTPase . The genomes consist of blocks with well-conserved sequences separated by nonconserved sequences . Recombination, gene duplication, horizontal gene transfer, and substitution of viral genes by homologous host genes have contributed to their evolution . The finding of head-to-head and tail-to-tail linked replicative intermediates suggests that the linear genomes replicate by the same mechanism as the similarly organized linear genomes of the eukaryal poxviruses, African swine fever virus and Chlorella viruses . SIRV1 and SIRV2 both contain motifs that resemble the binding sites for Holliday junction resolvases of eukaryal viruses and may use common mechanisms for resolution of replicative intermediates . The results suggest a common origin of the replication machineries of the archaeal rudiviruses and the above-mentioned eukaryal viruses . About 1/3 of the ORFs of each rudivirus have homologs in the Sulfolobus virus SIFV of the family Lipothrixviridae, indicating that the two viral families form a superfamily . The finding of inverted repeats of at least 0.8 kb at the termini of the linear genome of SIFV supports this inference . (C)2001 Elsevier Science. Extremophiles, 2002 Feb, 6(1), 57 - 64 Characterization of Symbiobacterium toebii, an obligate commensal thermophile isolated from compost; Rhee SK et al.; A symbiotic thermophilic bacterium, strain SC-1, was isolated from hay compost (toebi) in Korea . The new isolate exhibited an obligate commensal interaction with a thermophilic Geobacillus strain and required crude extracts and/or culture supernatant from Geobacillus sp . SK-1 for axenic growth . The growth factors from Geobacillus sp . SK-1 were irreversibly inactivated by phenol or protease treatment, suggesting that they might be proteins . The cells of strain SC-1 were non-spore forming, nonmotile rods that were stained Gram-negatively . The isolate was a microaerophilic heterotroph . Growth was observed between 45 degrees and 70 degrees C (optimum: 60 degrees C; 2.4-h doubling time) and pH 6.0 and 9.0 (optimum: pH 7.5) . The G+C content of the genomic DNA was 65 mol%, and the major quinones were MK-6 and MK-7 . A phylogenetic analysis of its 16S rDNA sequence indicated that strain SC-1 is closely related to Symbiobacterium thermophilum and so was named Symbiobacterium toebii on the basis of its physiological and molecular properties. Extremophiles, 2002 Feb, 6(1), 51 - 6 Rhodothermus marinus: a thermophilic bacterium producing dimeric and hexameric citrate synthase isoenzymes; Nordberg KE et al.; Two separate citrate synthases from the extremely thermophilic bacterium Rhodothermus marinus have been identified and purified . One of the enzymes is a hexameric protein and is the first thermostable, hexameric citrate synthase to be isolated . The other is a dimeric enzyme, which is also thermostable but possesses both citrate synthase and 2-methyl citrate synthase activities . 2-Methyl citrate synthase uses propionyl-coenzyme A as one of its substrates and in Escherichia coli, for example, it has been implicated in the metabolism of propionate . However, no growth of R . marinus was observed using minimal medium with propionate as the sole carbon source, and both hexameric and dimeric enzymes were produced irrespective of whether propionate was included in the growth medium . The data are discussed with respect to the evolutionary relationships between the known hexameric and dimeric citrate synthases and 2-methyl citrate synthase. Extremophiles, 2002 Feb, 6(1), 21 - 31 The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria; Christodoulou E et al.; Based on primary structure comparison between four highly homologous DNA-binding proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level . The contribution of the 11 amino acids that differ between the thermophilic HUBst from Bacillus stearothermophilus (Tm = 61.6 degrees C) and the mesophilic HUBsu from Bacillus subtilis (Tm = 39.7 degrees C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts . Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the thermophilic HUs, have been found to be responsible for the thermostability of HUBst . These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the protein (Tm = 45.1 degrees C) at the level of its mesophilic homologue HUBsu . By replacing these amino acids in HUBsu with their thermophilic counterparts, the HUBsu-E15G/D34E/142V mutant was generated with thermostability (Tm = 57.8 degrees C) at the level of thermophilic HUBst . Employing the same strategy, we generated several mutants in the extremely thermophilic HUTmar from Thermotoga maritima (Tm = 80.5 degrees C), and obtained data consistent with the previous results . The triplet mutant HUTmar-G15E/E34D/V421 (Tm = 35.9 degrees C) converted the extremely thermophilic protein HUTmar to mesophilic . The various forms of HU proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy . The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level . The results clearly show that three individual local interactions located at the helix-turn-helix part of the protein are responsible for the stability of HU proteins by acting cooperatively in a common mechanism for thermostability. J Biol Chem, 2002 May 10, 277(19), 16936 - 40 Epub 2002 Feb 27. An iron-sulfur cluster in the family 4 uracil-DNA glycosylases; Hinks JA et al.; The 25-kDa Family 4 uracil-DNA glycosylase (UDG) from Pyrobaculum aerophilum has been expressed and purified in large quantities for structural analysis . In the process we observed it to be colored and subsequently found that it contained iron . Here we demonstrate that P . aerophilum UDG has an iron-sulfur center with the EPR characteristics typical of a 4Fe4S high potential iron protein . Interestingly, it does not share any sequence similarity with the classic iron-sulfur proteins, although four cysteines (which are strongly conserved in the thermophilic members of Family 4 UDGs) may represent the metal coordinating residues . The conservation of these residues in other members of the family suggest that 4Fe4S clusters are a common feature . Although 4Fe4S clusters have been observed previously in Nth/MutY DNA repair enzymes, this is the first observation of such a feature in the UDG structural superfamily . Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role. Appl Microbiol Biotechnol, 2002 Feb, 58(2), 237 - 40 Thermophilic biodesulfurization of naphthothiophene and 2-ethylnaphthothiophene by a dibenzothiophene-desulfurizing bacterium, Mycobacterium phlei WU-F1; Furuya T et al.; Naphtho{2,1-b}thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives . Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degrees C), could also grow at 50 degrees C in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur . At 50 degrees C, the resting cells of WU-Fl degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h . By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds . Therefore, M . phlei WU-Fl can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds. Syst Appl Microbiol, 2001 Dec, 24(4), 572 - 87 A polyphasic taxonomic study of thermophilic bacilli from shallow, marine vents; Maugeri TL et al.; Eighty-seven thermophilic, aerobic, spore-forming bacteria were isolated from shallow, marine, thermal vents of the Eolian Islands (Italy) and tested for a broad spectrum of phenotypic characteristics . A numerical taxonomy study was performed on these isolates and 8 thermophilic Bacillus and Geobacillus reference strains by 89 selected features . Results from cluster analysis showed the formation of nine clusters . Most of the isolates (83%) fell into several phenetically well distinguished clusters, loosely related to Geobacillus thermodenitrificans . The remaining isolates grouped together with different reference strains . Eighteen isolates, representative of the different clusters, were selected for subsequent genotypic characterisation, including partial 16S rDNA sequence analysis of 18 strains and almost complete 16S rDNA sequences of 9 strains . Subsequent DNA/DNA reassociation studies and determination of the base composition of DNA identified seven isolates as Geobacillus thermodenitrificans, two isolates as G . thermoleovorans and one isolate as Bacillus pallidus . Four isolates represented two novel species of Bacillus . The remaining four represented novel Geobacillus species, one of which has recently been described as Bacillus vulcani DSMZ 13174 T. Syst Appl Microbiol, 2001 Dec, 24(4), 539 - 48 Functional grouping of thermophilic Bacillus strains using amplification profiles of the 16S-23S internal spacer region; Flint SH et al.; Molecular and biochemical assays were used to determine the identification of thermophilic bacilli isolated from New Zealand milk powder . One hundred and forty one isolates of thermophilic bacilli were classified into six species using biochemical profiles . Geobacillus stearothermophilus represented 56% of the isolates . All isolates were also analysed by randomly amplified polymorphic DNA (RAPD) analysis, with 45 types identified . Amplification of the 16S-23S rDNA internal spacer region produced two to eight amplification products per strain . The patterns from gel electrophoresis of the internal spacer region amplicons formed two major groupings suggesting the possibility of two distinct species . Partial sequences of 16S rDNA from representatives from each group were compared with sequences in GeneBank and were found to match the 16S rDNA sequences of B . flavothermus and G . thermoleovorans . Primers were designed for these species and used to screen an arbitrary selection of 59 of the dairy isolates . This enabled the identification of 28 isolates as B . flavothermus and 31 isolates as Geobacillus species and these appear to be the predominant isolates in the New Zealand milk powder samples examined . Comparison of the fragment pattern generated by amplification of the 16S-23S rDNA internal spacer region is a simple method to differentiate thermophilic Bacillus species associated with the dairy industry. J Biol Chem, 2002 May 10, 277(19), 16758 - 67 Epub 2002 Mar 01. Cleavage properties of an archaeal site-specific recombinase, the SSV1 integrase; Serre MC et al.; SSV1 is a virus infecting the extremely thermophilic archaeon Sulfolobus shibatae . The viral-encoded integrase is responsible for site-specific integration of SSV1 into its host genome . The recombinant enzyme was expressed in Escherichia coli, purified to homogeneity, and its biochemical properties investigated in vitro . We show that the SSV1 integrase belongs to the tyrosine recombinases family and that Tyr(314) is involved in the formation of a 3'-phosphotyrosine intermediate . The integrase cleaves both strands of a synthetic substrate in a temperature-dependent reaction, the cleavage efficiency increasing with temperature . A discontinuity was observed in the Arrhenius plot above 50 degrees C, suggesting that a conformational transition may occur in the integrase at this temperature . Analysis of cleavage time course suggested that noncovalent binding of the integrase to its substrate is rate-limiting in the cleavage reaction . The cleavage positions were localized on each side of the anticodon loop of the tRNA gene where SSV1 integration takes place . Finally, the SSV1 integrase is able to cut substrates harboring mismatches in the binding site . For the cleavage step, the chemical nature of the base in position -1 of cleavage seems to be more important than its pairing to the opposite strand. J Biol Chem, 2002 May 10, 277(19), 16624 - 31 Epub 2002 Feb 28. Enzymatic properties of S-adenosylmethionine synthetase from the archaeon Methanococcus jannaschii; Lu ZJ et al.; S-Adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, MAT) catalyzes a unique enzymatic reaction that leads to formation of the primary biological alkylating agent . MAT from the hyperthermophilic archaeon Methanococcus jannaschii (MjMAT) is a prototype of the newly discovered archaeal class of MAT proteins that are nearly unrecognizable in sequence when compared with the class that encompasses both the eucaryal and bacterial enzymes . In this study the functional properties of purified recombinant MjMAT have been evaluated . The products of the reaction are AdoMet, PP(i), and P(i); >90% of the P(i) originates from the gamma-phosphoryl group of ATP . The circular dichroism spectrum of the dimeric MjMAT indicates that the secondary structure is more helical than the Escherichia coli counterpart (EcMAT), suggesting a different protein topology . The steady state kinetic mechanism is sequential, with random addition of ATP and methionine; AdoMet is the first product released, followed by release of PP(i) and P(i) . The substrate specificity differs remarkably from the previously characterized MATs; the nucleotide binding site has a very broad tolerance of alterations in the adenosine moiety . MjMAT has activity at 70 degrees C comparable with that of EcMAT at 37 degrees C, consistent with the higher temperature habitat of M . jannaschii . The activation energy for AdoMet formation is larger than that for the E . coli MAT-catalyzed reaction, in accord with the notion that enzymes from thermophilic organisms are often more rigid than their mesophilic counterparts . The broad substrate tolerance of this enzyme proffers routes to preparation of novel AdoMet analogs. Appl Environ Microbiol, 2002 Mar, 68(3), 1458 - 63 Rupture of the cell envelope by decompression of the deep-sea methanogen Methanococcus jannaschii; Park CB et al.; The effect of decompression on the structure of Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen, was studied in a novel high-pressure, high-temperature bioreactor . The cell envelope of M . jannaschii appeared to rupture upon rapid decompression (ca . 1 s) from 260 atm of hyperbaric pressure . When decompression from 260 atm was performed over 5 min, the proportion of ruptured cells decreased significantly . In contrast to the effect produced by decompression from hyperbaric pressure, decompression from a hydrostatic pressure of 260 atm did not induce cell lysis. Appl Environ Microbiol, 2002 Mar, 68(3), 1173 - 9 Differential expression of methanogenesis genes of Methanothermobacter thermoautotrophicus (formerly Methanobacterium thermoautotrophicum) in pure culture and in cocultures with fatty acid-oxidizing syntrophs; Luo HW et al.; The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen, Methanothermobacter thermoautotrophicus strain TM, was investigated both in a pure culture sufficiently supplied with H(2) plus CO(2) and in a coculture with an acetate-oxidizing hydrogen-producing bacterium, Thermacetogenium phaeum strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa) . Northern blot analysis indicated that only the mcr gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas mcr and mrt, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture . In contrast to these two genes, two isofunctional genes (mtd and mth) for N(5),N(10)-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (frh and mvh) were expressed both in a pure culture and in a coculture at the early and late stages of growth . The same expression pattern was observed for Methanothermobacter thermoautotrophicus strain DeltaH cocultured with a thermophilic butyrate-oxidizing syntroph, Syntrophothermus lipocalidus strain TGB-C1 . Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of M . thermoautotrophicus strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture . These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII . Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems. J Appl Microbiol, 2002, 92(3), 433 - 42 Culturability, injury and morphological dynamics of thermophilic Campylobacter spp . within a laboratory-based aquatic model system; Thomas C et al.; AIMS: To study the survival processes of thermophilic Campylobacter spp . within a modelled aquatic system and particularly the involvement and survival potential of viable but non-culturable forms . METHODS AND RESULTS: The survival and morphological characteristics of populations of thermophilic Campylobacter species exposed to simulated aquatic conditions were examined using a combination of cultural and microscopic techniques . Populations underwent progressive decay when exposed to simulated aquatic conditions . The rates of population decay were observed to be significantly greater at the higher temperature (20 degrees C) with a rapid transition of the dominant sub-populations from non-stressed to dead cells occurred within 3 days . At 10 degrees C the rate of culturability loss was much reduced with substantial development (approx . 80% of total population) of viable but non-culturable (VBNC) populations by all species within 3 days, declining to represent approximately 5-25% of the total population at day 60 . Significant differences (P < 0.001) were identified between decay rates as a consequence of different species, sub-populations and temperature but not between sub-populations of different species . Morphological variants including spiral, elongated spirals and rods, short rods and coccoid forms were identified . The endpoints of morphological transition were temperature-independent and isolate-specific yet the rate of morphological transition was directly related to temperature and approximately equivalent between species . CONCLUSION: The VBNC state is a transitory stage in the degeneration of Campylobacter population within the aquatic environments simulated during this study . SIGNIFICANCE AND IMPACT OF THE STUDY: VBNC cells form the most persistent, viable, potentially pathogenic sub-population of Campylobacter populations exposed to aquatic stress conditions. Plant Cell Physiol, 2002 Feb, 43(2), 159 - 69 Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties; Chen LM et al.; A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe . The S . vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa . SvPEPC was expressed in E . coli BL21 Codonplus (DE3), using pET32a as a vector . The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE . The enzyme forms homotetramer as judged by gel filtration . SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)) . SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C . The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln . Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity . SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs . I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold . The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models. Gene, 2002 Jan 23, 283(1-2), 107 - 15 A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein; Morana A et al.; A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression . One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate . The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S . solfataricus and a full-length esterase coding sequence could be identified . Expression of the active thermostable esterase in E . coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences . The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene . The same protein was revealed in S . solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested . The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0 . The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase. J Mol Biol, 2002 Feb 22, 316(3), 725 - 68 Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA; Brodersen DE et al.; We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution . With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions . In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure . Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA . Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later . We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit . Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 769 - 74 Pyrococcus prefoldin stabilizes protein-folding intermediates and transfers them to chaperonins for correct folding; Okochi M et al.; A molecular chaperone prefoldin/GimC from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 was characterized . Pyrococcus prefoldin protected porcine heart citrate synthase from thermal aggregation whereas each subunit alone afforded little protection . It also arrested the spontaneous refolding of acid-denatured green fluorescent protein and then transferred it not only to a group II chaperonin from the hyperthermophilic archaeum Thermococcus sp . strain KS-1, but also to a group I chaperonin from the thermophilic bacterium Thermus thermophilus HB8 for subsequent ATP dependent refolding. Curr Biol, 2002 Feb 19, 12(4), 313 - 6 Both carboxy-terminal tails of alpha- and beta-tubulin are essential, but either one will suffice; Duan J et al.; Microtubules (MTs) are organized into distinct systems essential for cell shape, movement, intracellular transport, and division . Electron crystallographic analyses provide little information about how MTs produce diverse structures and functions, perhaps because they failed to visualize the last 10 residues of the alpha- and the last 18 of the beta-tubulin C-terminal tails (CTTs), which likely play a role in MT diversity . CTTs define conserved, nonallelic isotypes in mammals, are major sites of posttranslational modifications (PTMs), are binding sites for microtubule-associated proteins (MAPs), and determine MT motor processivity . Using mutagenesis and homologous gene replacement in Tetrahymena thermophila, we analyzed mutations, deletions, tail switches, and tail duplications of alpha- and beta-tubulin CTTs . We demonstrate that a tail is required for the essential function of both alpha- and beta-tubulin . However, the two tails are interchangeable, and cells grow normally with either an alpha or a beta tail on both tubulins . In addition, an alpha gene containing a duplicated alpha C terminus rescues a lethal mutant lacking all known posttranslational modification sites on the beta C terminus but cannot rescue deletion of the beta tail . Thus, tubulin tails have a second essential function that is not associated with posttranslational modification. Biochemistry, 2002 Mar 5, 41(9), 3226 - 34 The (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 has altered ATPase activity after cross-linking alpha and beta subunits at noncatalytic site interfaces; Bandyopadhyay S et al.; In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites . To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2) . The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type . Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following . Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites . Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site . When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme . These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures. Mol Biol (Mosk), 2002 Jan-Feb, 36(1), 106 - 13 {Replication protein RepN encoded by the RC plasmid of thermophilic bacterium Thermoanaerobacterium saccharolyticum: mutational analysis and deletion mapping of domains responsible for its lethal effect}; Belogurova NG et al.; Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110 . Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein . Its lethal effect on bacterial cells was unusually high for a protein of the kind . Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity . On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein . The RepN fragments essential for the lethal effect were found to share a motif, which showed no appreciable homology to known conserved motifs . The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for the stable maintenance of the AT-rich plasmid in thermophilic bacterial cells. Nat Cell Biol, 2002 Mar, 4(3), 256 - 9 Polyglycylation domain of beta-tubulin maintains axonemal architecture and affects cytokinesis in Tetrahymena; Thazhath R et al.; Polyglycylation occurs through the post-translational addition of a polyglycine peptide to the gamma-carboxyl group of glutamic acids near the C terminus of alpha- and beta-tubulin, and has been found only in cells with axonemes, from protists to humans . In Tetrahymena thermophila, multiple sites of polyglycylation on alpha-tubulin are dispensable . By contrast, mutating similar sites on beta-tubulin has site-specific effects, affecting cell motility and cytokinesis, or resulting in cell death . Here, we address the lethality of a polyglycylation deficiency in T . thermophila using heterokaryons . Cells with a lethal mutation in the polyglycylation domain of beta-tubulin assembled axonemes that lack the central pair, B-subfibres and the transitional zone of outer microtubules (MTs) . Furthermore, an arrest in cytokinesis occurred, and was associated with incomplete severing of cortical MTs positioned near the cleavage furrow . Thus, tubulin polyglycylation is required for the maintenance of some stable microtubular organelles that are all known to be polyglycylated in vivo, but its effects on MTs appear to be organelle-specific. Methods, 2001 Nov, 25(3), 292 - 302 Ribosomal crystallography: from poorly diffracting microcrystals to high-resolution structures; Gluehmann M et al.; The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize . Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit . These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates . J Biol Chem, 2002 May 10, 277(19), 17334 - 48 Epub 2002 Feb 21. Analysis of a multicomponent thermostable DNA polymerase III replicase from an extreme thermophile; Bruck I et al.; This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus . Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (tau, delta, and delta') were identified . The dnaX gene produces only the full-length product, tau, and therefore differs from Escherichia coli dnaX that produces two proteins (gamma and tau) . Nonetheless, the A . aeolicus proteins form a taudeltadelta' complex . The dnaN gene encoding the beta clamp was identified, and the taudeltadelta' complex is active in loading beta onto DNA . A . aeolicus contains one dnaE homologue, encoding the alpha subunit of DNA polymerase III . Like E . coli, A . aeolicus alpha and tau interact, although the interaction is not as tight as the alpha-tau contact in E . coli . In addition, the A . aeolicus homologue to dnaQ, encoding the epsilon proofreading 3'-5'-exonuclease, interacts with alpha but does not form a stable alpha.epsilon complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system . Despite these differences to the E . coli system, the A . aeolicus proteins function to yield a robust replicase that retains significant activity at 90 degrees C . Similarities and differences between the A . aeolicus and E . coli pol III systems are discussed, as is application of thermostable pol III to biotechnology. Protein Expr Purif, 2002 Mar, 24(2), 260 - 7 Cloning and expression of human phenylalanyl-tRNA synthetase in Escherichia coli: comparative study of purified recombinant enzymes; Moor N et al.; Human phenylalanyl-tRNA synthetase (PheRS) was cloned and expressed in Escherichia coli . The cDNAs of the alpha and beta subunits were cloned into pET-21b(+) and pET-28b(+) vectors . The 6x histidine-tagged (HT) plasmids pET-21_HTbeta, pET-28_HTalpha, and pET-28_HTbeta were constructed . Three different types of (alphabeta)(2) heterodimers of human PheRS carrying HT at the N-terminus of either of two alpha or beta subunits or simultaneously on both of them were overproduced and purified . The heterodimeric protein with HT appended to the N-terminus of the beta subunit revealed no activity in the aminoacylation reaction as opposed to those with HT on the alpha subunit . It is known from the structure of the Thermus thermophilus Phe system that the N-terminal coiled-coil domain of the alpha subunit is involved in the binding of cognate tRNA(Phe) . Our data demonstrate that a histidine-tagged N-terminal extension appended to the alpha subunit does not affect the kinetic parameters of tRNA(Phe) aminoacylation . Elimination of the HT from the alpha subunit by thrombin cleavage leads to nonspecific splitting of the enzyme that occurs in parallel to the main reaction . In addition to the tagged proteins the properly assembled heterodimer containing intact alpha and beta subunits free of HT was overproduced and purified . Aminoacylation activity of the overproduced human PheRS in the crude bacterial extract is two orders of magnitude higher than the corresponding activity in human placenta and the yield of the recombinant enzyme overproduced in E . coli is five times higher . Biotechnol Bioeng, 2002 Apr 5, 78(1), 110 - 6 A biosensor for the detection of triazine and phenylurea herbicides designed using Photosystem II coupled to a screen-printed electrode; Koblizek M et al.; A biosensor for the detection of triazine- and phenylurea-type herbicides was constructed using isolated Photosystem II (PS II) complexes as a biosensing element . PSII isolated from the thermophilic cyanobacterium Synechococcus elongatus was immobilized on the surface of a screen-printed sensor composed of a graphite working electrode and Ag/AgCl reference electrode deposited on a polymeric substrate . The biosensor was mounted in a flow microcell with illumination . The principle of the detection was based on the fact that herbicides selectively block PSII electron transport activity in a concentration-dependent manner . Changes of the activity were registered amperometrically as the rate of photoreduction of an artificial electron acceptor . The setup resulted in a reusable herbicide biosensor with a good stability (half-life of 24 h) and limit of detection of approximately 10(-9) M for diuron, atrazine and simazine . Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 546 - 8 Epub 2002 Feb 21. Thermophilic alcohol dehydrogenase from the mesophile Entamoeba histolytica: crystallization and preliminary X-ray characterization; Shimon LJ et al.; The tetrameric NADP(+)-dependent secondary alcohol dehydrogenase from Entamoeba histolytica has been crystallized in its apo form . The crystals belong to space group C222(1), with unit-cell parameters a = 76.89, b = 234.24, c = 96.24 A, and diffract to 1.9 A at liquid-nitrogen temperature . Analysis of the Patterson self-rotation function shows that the crystals contain one dimer per asymmetric unit. Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 531 - 2 Epub 2002 Feb 21. Crystallization and preliminary X-ray crystallographic analysis of beta-xylosidase from Thermoanaerobacterium saccharolyticum, a thermophilic anaerobe; Yang JK et al.; beta-Xylosidases are involved in the breakdown of xylans into xylose and belong to either family 39 or 43 of the glycosyl hydrolases . At present, no structural information is available for any member of these families . beta-Xylosidase from the thermophilic anaerobe Thermoanaerobacterium saccharolyticum, a member of glycosyl hydrolase family 39, has been crystallized at 296 K using the hanging-drop vapour-diffusion method . The crystal diffracts to 2.4 A resolution with synchrotron X-rays and belongs to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 92.75, c = 241.37 A . The asymmetric unit contains two monomers of the recombinant enzyme, giving a corresponding V(M) of 2.21 A(3)Da(-1) and a solvent content of 44.3%. Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 421 - 30 Epub 2002 Feb 21. The structure of AhrC, the arginine repressor/activator protein from Bacillus subtilis; Dennis C CA et al.; In the Gram-positive bacterium Bacillus subtilis the concentration of the amino acid L-arginine is controlled by the transcriptional regulator AhrC . The hexameric AhrC protein binds in an L-arginine-dependent manner to pseudo-palindromic operators within the promoter regions of arginine biosynthetic and catabolic gene clusters . AhrC binding results in the repression of transcription of biosynthetic genes and in the activation of transcription of catabolic genes . The crystal structure of AhrC has been determined at 2.7 A resolution . Each subunit of the protein has two domains . The C-terminal domains are arranged with 32 point-group symmetry and mediate the major intersubunit interactions . The N-terminal domains are located around this core, where they lie in weakly associated pairs but do not obey strict symmetry . A structural comparison of AhrC with the arginine repressor from the thermophile B . stearothermophilus reveals close similarity in regions implicated in L-arginine binding and DNA recognition, but also reveals some striking sequence differences, especially within the C-terminal oligomerization domain, which may contribute to the different thermostabilities of the proteins . Comparison of the crystal structure of AhrC with a 30 A resolution model obtained by combining X-ray structure-factor amplitudes with phases derived from electron-microscopic analyses of AhrC crystals confirms the essential accuracy of the earlier model and suggests that such an approach may be more widely useful for obtaining low-resolution phase information. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2630 - 5 Epub 2002 Feb 19. tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase; Myers CA et al.; The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure . Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core . Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively . Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure . Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs. Prikl Biokhim Mikrobiol, 2002 Jan-Feb, 38(1), 20 - 4 {Testing and isolation of high-purity restriction endonucleases}; Puchkova LI et al.; A new method of testing restriction nucleases is proposed . This method is based on high-temperature treatment of crude cell extracts . Disrupted cells were heated at 50-60 degrees C, centrifuged, and assayed for restrictases . This method provides the opportunity for screening new enzymes in microbial strains enriched with nonspecific restrictases . High-temperature treatment of cell extracts of certain producers reduces the number of steps of the procedure used for isolating high-purity restrictases; the resulting preparations are capable of maintaining high enzymatic activity during long-term storage . It was shown that high-temperature treatment can be applied not only to thermophilic but also to mesophilic strains of microorganisms of different taxa. FEBS Lett, 2002 Feb 13, 512(1-3), 269 - 74 Characterization of a lysK gene as an argE homolog in Thermus thermophilus HB27; Miyazaki J et al.; We conducted a chromosome walk to obtain a DNA fragment downstream of lysJ and found an argE homolog in a putative operon composed of lysJ-orfC-orfD-argE homologs . A knockout mutant of the argE homolog showed significantly slow growth on a minimal medium, and the growth was markedly improved by addition of lysine . We therefore termed this gene lysK . Purified LysK protein has deacetylating activities for both N(2)-acetyllysine and N(2)-acetylornithine at almost equal efficiency . These results suggest that lysK which may share an ancestor with argE functions not only for the lysine biosynthesis, but also for arginine biosynthesis in Thermus thermophilus. J Appl Microbiol, 2001 Dec, 91(6), 1023 - 9 Development of a minimal chemically-defined medium for the exponential growth of Streptococcus thermophilus; Letort C et al.; AIMS: The present work aimed to define a minimal chemically-defined medium which could sustain the growth of most (if not all) strains of Streptococcus thermophilus . METHODS AND RESULTS: A minimal medium containing 20 components, including one carbohydrate source, six amino acids, two metallic ions, six vitamins and urea allowed for growth of 13 out of 15 Strep . thermophilus strains . Growth of the two last strains required the presence of additional amino acids, the number of which depended on the strain . Growth rates of the strains in the minimal medium ranged from 0.38 to 0.64 h(-1), and final populations were about 10(8) cfu ml(-1) . CONCLUSIONS: Streptococcus thermophilus appears much less demanding than other lactic acid bacteria . SIGNIFICANCE AND IMPACT OF THE STUDY: The definition of such a growth medium will be very useful for metabolic flux studies as well as peptide transport studies. Biochemistry, 2002 Feb 26, 41(8), 2485 - 91 Crystal structure of F65A/Y131C-methylimidazole carbonic anhydrase V reveals architectural features of an engineered proton shuttle; Jude KM et al.; The crystal structure of F65A/Y131C murine alpha-carbonic anhydrase V (CAV), covalently modified at cysteine residues with 4-chloromethylimidazole, is reported at 1.88 A resolution . This modification introduces a methylimidazole (MI) group at residue C131 in the active site with important consequences . F65A/Y131C-MI CAV exhibits an up to 3-fold enhancement of catalytic activity over that of wild-type CAV {Earnhardt, J . N., Wright, S . K., Qian, M., Tu, C., Laipis, P . J., Viola, R . E., and Silverman, D . N . (1999) Arch . Biochem . Biophys . 361, 264-270} . In this modified CAV variant, C131-MI acts as a proton shuttle, facilitating the deprotonation of a zinc-bound water molecule to regenerate the nucleophilic zinc-bound hydroxide ion . A network of three hydrogen-bonded water molecules, across which proton transfer likely proceeds, bridges the zinc-bound water molecule and the C131-MI imidazole group . The structure of F65A/Y131C-MI CAV is compared to structures of Y64H/F65A murine CAV, wild-type human alpha-carbonic anhydrase II, and the gamma-carbonic anhydrase from Methanosarcina thermophilain an effort to outline common features of catalytic proton shuttles. J Mol Biol, 2002 Feb 15, 316(2), 327 - 40 Comparison of the folding processes of T . thermophilus and E . coli ribonucleases H; Hollien J et al.; In order to examine how the stabilization of thermophilic proteins affects their folding, we have characterized the folding process of Thermus thermophilus ribonuclease H using circular dichroism, fluorescence, and pulse-labeling hydrogen exchange . Like its homolog from Escherichia coli, this thermophilic protein populates a partially folded kinetic intermediate within the first few milliseconds of folding . The structure of this intermediate is similar to that of E.coli RNase H and corresponds remarkably well to a partially folded form that is populated at low levels in the native state of the protein . Proline isomerization appears to partly limit the folding of the thermophilic but not the mesophilic protein . Lastly, unlike other thermophilic proteins, which unfold much more slowly than their mesophilic counterparts, T.thermophilus RNase H folds and unfolds with overall rates similar to those of E.coli RNase H . J Biol Chem, 2002 May 24, 277(21), 18947 - 53 Epub 2002 Feb 15. An open conformation of the Thermus thermophilus gyrase B ATP-binding domain; Lamour V et al.; DNA gyrase forms an A(2)B(2) tetramer involved in DNA replication, repair, recombination, and transcription in which the B subunit catalyzes ATP hydrolysis . The Thermus thermophilus and Escherichia coli gyrases are homologues and present the same catalytic activity . When compared with that of the E . coli 43K-5'-adenylyl-beta,gamma-imidodiphosphate complex, the crystal structure of Gyrase B 43K ATPase domain in complex with novobiocin, one of the most potent inhibitors of gyrase shows large conformational changes of the subdomains within the dimer . The stabilization of loop 98-118 closing the active site through dimeric contacts and interaction with domain 2 allows to observe novobiocin-protein interactions that could not be seen in the 24K-inhibitor complexes . Furthermore, this loop adopts a position which defines an "open" conformation of the active site in absence of ATP, in contrast with the "closed" conformation adopted upon ATP binding . All together, these results indicate how the subdomains may propagate conformational changes from the active site and provide crucial information for the design of more specific inhibitors. Mol Microbiol, 2002 Jan, 43(1), 187 - 94 Co-operative binding of triplicate carbohydrate-binding modules from a thermophilic xylanase; Boraston AB et al.; Family 6 carbohydrate-binding modules were amplified by polymerase chain reaction (PCR) from Clostridium stercorarium strain NCIB11754 genomic DNA as a triplet . Individually, these modules bound to xylooligosaccharides and cellooligosaccharides with affinities varying from approximately 3 x 10(3) M(-1) to approximately 1 x 10(5) M(-1) . Tandem and triplet combinations of these modules bound co-operatively to soluble xylan and insoluble cellulose to give approximately 20- to approximately 40-fold increases in affinity relative to the individual modules . This co-operativity was an avidity effect resulting from the modules within the tandems and triplet interacting simultaneously with proximal binding sites on the polysaccharides . This occurred by both intrachain and interchain interactions . The duplication or triplication of modules appears to be linked to the growth temperature of the organism; co-operativity in these multiplets may compensate for the loss of affinity at higher temperatures. Lett Appl Microbiol, 2002, 34(2), 119 - 23 Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var . thermoidea in Trichoderma reesei; de Faria FP et al.; AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei . METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker . The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T . reesei xylanases, resulting in an active enzyme . CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T . reesei . Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source . Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal. J Appl Microbiol, 2002, 92(2), 297 - 306 Exopolysaccharide production by Streptococcus thermophilus SY: production and preliminary characterization of the polymer; Ricciardi A et al.; AIMS: To evaluate the effect of yeast extract (YE) concentration, temperature and pH on growth and exopolysaccharide (EPS) production in a whey-based medium by Streptococcus thermophilus SY and to characterize the partially purified EPS . METHODS AND RESULTS: Factorial experiments and empirical model building were used to optimize fermentation conditions and the chemical composition, average molecular weight (MW) and rheological properties of aqueous dispersions of the EPS were determined . Exopolysaccharide production was growth associated and was higher (152 mg l(-1)) at pH 6.4 and 36 degrees C with 4 g l(-1) YE . High performance size exclusion chromatography of the partially purified EPS showed two peaks, with a weight average MW of 2 x 10(6) and 5 x 10(4), respectively . The EPS was a heteropolysaccharide, with a glucose : galactose : rhamnose ratio of 2 : 4.5 : 1 . Its water dispersions had a pseudoplastic behaviour and showed a higher viscosity of xanthan solutions . SIGNIFICANCE AND IMPACT OF THE STUDY: The fermentation conditions and some properties of an EPS produced by Strep . thermophilus, a dairy starter organism, were described. J Appl Microbiol, 2002, 92(1), 127 - 33 Successive rapid reductive dehalogenation and mineralization of pentachlorophenol by the indigenous microflora of farmyard manure compost; Jaspers CJ et al.; AIMS: To determine whether composting with animal manure can be used to effectively remediate soil from a pentachlorophenol (PCP)-contaminated site, and to establish the fate of the degraded xenobiotic . METHODS AND RESULTS: Contaminated soil from a sawmill site was mixed with farm animal manure and composted in a 0.5 m3 silo under fully aerobic conditions . The disappearance and fate of PCP was monitored by gas chromatography (GC-ECD) and extensive mineralization confirmed in experiments with 14C-radiolabelled PCP . The disappearance of PCP was rapid and virtually complete within 6 days, prior to the onset of thermophilic conditions . Dechlorination of the PCP was found to be both reductive and sequential . CONCLUSIONS: PCP removal from contaminated soil by aerobic composting with animal manure is efficient and proceeds via reductive dechlorination to virtually complete mineralization . This contrasts with other chlorophenol composting regimes in which mineralization is achieved but dechlorination intermediates do not accumulate to detectable levels . SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate that anaerobic reductive dechlorination can proceed in an aerobic composting environment and contribute to efficient pentachlorophenol removal . Farmyard manure composts may represent a rapid, low-cost, low-technology option for treatment of chlorophenol-contaminated soils. J Appl Microbiol, 2002, 92(1), 47 - 54 Validation of a polymerase chain reaction/restriction enzyme analysis method for species identification of thermophilic campylobacters isolated from domestic and wild animals; Engvall EO et al.; AIMS: To compare and evaluate a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method with standard phenotypic tests for the identification and differentiation of the thermophilic campylobacters Campylobacter jejuni, C . coli, C . lari and C . upsaliensis . METHODS AND RESULTS: One hundred and eighty-two presumptive thermophilic campylobacters from 12 different animal species were tested by a recently published PCR/REA and standard phenotypic tests . By PCR/REA, 95% of the isolates were clearly identified as either one of the four thermophilic Campylobacter species or as not belonging to this group of organisms at all . By standard phenotyping, 174 of the 182 isolates were initially identified as either C . jejuni, C . coli, C . lari or C . upsaliensis . Additional genotypic tests and phenotyping showed that 52 of these identifications were either incorrect or unreliable . Of the C . jejuni isolates, 19% were identified as C . coli by initial phenotyping and 27 sheep isolates phenotyped as C . coli or C . lari were, in fact, arcobacters . CONCLUSIONS: The PCR/REA was more reliable than standard phenotyping for the identification of thermophilic campylobacters from different animals . SIGNIFICANCE AND IMPACT OF THE STUDY: Routinely used phenotypic tests often resulted in unreliable identifications, requiring additional testing . The PCR/REA, however, gave unequivocal results and was considered useful for the routine identification of thermophilic campylobacters from different animals. Water Res, 2002 Feb, 36(4), 1067 - 75 Aerobic moving bed biofilm reactor treating thermomechanical pulping whitewater under thermophilic conditions; Jahren SJ et al.; The continuously operated laboratory scale Kaldnes moving bed biofilm reactor (MBBR) was used for thermophilic (55 degrees C) aerobic treatment of TMP whitewater . In the MBBR, the biomass is grown on carrier elements that move along with the water in the reactor . Inoculation with mesophilic activated sludge gave 60-65% SCOD removal from the first day onwards . During the 107 days of experiment, the 60-65% SCOD removals were achieved at organic loading rates of 2.5-3.5 kg SCODm(-3) d(-1), the highest loading rates applied during the run and HRT of 13-22h . Carbohydrates, which contributed to 50-60% of the influent SCOD . were removed by 90-95%, while less than 15% of the lignin-like material (30-35% of SCODin) was removed . The sludge yield was 0.23g VSSg SCOD(-1)removed . The results show that the aerobic biofilm process can be successfully operated under thermophilic conditions. J Mol Biol, 2001 Nov 30, 314(3), 507 - 18 The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus; De Simone G et al.; The crystal structure of AFEST, a novel hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus, complexed with a sulphonyl derivative, has been determined and refined to 2.2 A resolution . This enzyme, which has recently been classified as a member of the hormone- sensitive-lipase (H) group of the esterase/lipase superfamily, presents a canonical alpha/beta hydrolase core, shielded on the C-terminal side by a cap region composed of five alpha-helices . It contains the catalytic triad Ser160, His285 and Asp255, whereby the nucleophile is covalently modified and the oxyanion hole formed by Gly88, Gly89 and Ala161 . A structural comparison of AFEST with its mesophilic and thermophilic homologues, Brefeldin A esterase from Bacillus subtilis (BFAE) and EST2 from Alicyclobacillus acidocaldarius, reveals an increase in the number of intramolecular ion pairs and secondary structure content, as well as a significant reduction in loop extensions and ratio of hydrophobic to charged surface area . The variety of structural differences suggests possible strategies for thermostabilization of lipases and esterases with potential industrial applications . Anal Biochem, 2002 Mar 1, 302(1), 88 - 94 Thermus scotoductus and Rhodothermus marinus DNA ligases have higher ligation efficiencies than thermus thermophilus DNA ligase; Housby JN et al.; To mimic large numbers of nicked DNA duplexes we used a technique that produces nicked duplex DNA substrates by hybridization of complementary oligonucleotides, adjacent to an initiating primer, which are ligated together by a thermostable DNA ligase . Sequential ligation of nonanucleotides to this primary duplex results in the formation of polymers that can be analyzed by gel electrophoresis . The extent of polymerization is a measure of the efficiency of ligation . We determined the efficiency of ligation of nonanucleotides, using various length initiating primers, with three thermostable DNA ligases: Thermus thermophilus (Tth), Thermus scotoductus (Ts), and Rhodothermus marinus (Rm) . Analysis of the effect of temperature for each ligase, and for each directing primer length, revealed that at 37 and 41 degrees C there was variation between ligase efficiency in the order Rm > or = Ts > or = Tth . The higher temperature of 46 degrees C was optimal for polymerization with each of the ligases and Rm ligase was the most efficient . Analysis of directionality of the ligations reactions suggests that for each of the Thermus ligases we tested, there was a bias to polymerization of nonanucleotides in a 5'-3' direction. J Biol Chem, 2002 May 3, 277(18), 15890 - 6 Epub 2002 Feb 13. Crystal structure of argininosuccinate synthetase from Thermus thermophilus HB8 . Structural basis for the catalytic action; Goto M et al.; Argininosuccinate synthetase catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate . The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its free form, complexed with intact ATP, and complexed with an ATP analogue (adenylyl imidodiphosphate) and substrate analogues (arginine and succinate) have been determined at 2.3-, 2.3-, and 1.95-A resolution, respectively . The structure is essentially the same as that of the Escherichia coli argininosuccinate synthetase . The small domain has the same fold as that of a new family of "N-type" ATP pyrophosphatases with the P-loop specific for the pyrophosphate of ATP . However, the enzyme shows the P-loop specific for the gamma-phosphate of ATP . The structure of the complex form is quite similar to that of the native one, indicating that no conformational change occurs upon the binding of ATP and the substrate analogues . ATP and the substrate analogues are bound to the active site with their reaction sites close to one another and located in a geometrical orientation favorable to the catalytic action . The reaction mechanism so far proposed seems to be consistent with the locations of ATP and the substrate analogues . The reaction may proceed without the large conformational change of the enzyme proposed for the catalytic process. J Biol Chem, 2002 May 17, 277(20), 17978 - 86 Epub 2002 Feb 13. Slow-tight binding inhibition of xylanase by an aspartic protease inhibitor: kinetic parameters and conformational changes that determine the affinity and selectivity of the bifunctional nature of the inhibitor; Dash C et al.; The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp . is described . ATBI inhibits aspartic protease (Dash, C., and Rao, M . (2001) J . Biol . Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp . The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism . The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate . The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 +/- 1 x 10(-6) s(-1) and 5 +/- 0.5 x 10(-8) s(-1), respectively . The K(i) value for the formation of EI complex was 2.5 +/- 0.5 microm, whereas the overall inhibition constant K(i)* was 7 +/- 1 nm . The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data . Thus, the conformational alterations were correlated to the isomerization of EI to EI* . ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I . Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed. Int J Food Microbiol, 2002 Jan 30, 72(1-2), 165 - 8 Prevalence of Salmonella and Campylobacter in retail chicken meat in Spain; Dominguez C et al.; From February to November 1999, 198 samples of chicken meat for sale in retail outlets and supermarkets in nine provinces of Castilla and Leon (Spain) were analysed for the prevalence of Salmonella and Campylobacter . Salmonella was isolated from 71 (35.83%) of the samples analysed . The predominant serovars were S . enteritidis (47.88%), S . hadar (25.35%) and serotype 4,12:b:-(II) (19.71%) . Other serovars such as S . mbandaka, S . derby, S . virchow and S . paratyphi B were isolated in much lower levels . Thermophilic campylobacters were isolated in 49.50% of the samples studied. Z Naturforsch {C}, 2001 Nov-Dec, 56(11-12), 1022 - 8 Production and properties of a bacterial thermostable exo-inulinase; Uzunova K et al.; Inulinase and Invertase Activities, Thermophilic Bacilli, Enzyme Thermostability Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp . 11 strain was studied by batch cultivation in a fermentor . The achieved inulinase and invertase activities after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts . The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose . It could be used at temperatures above 65 degrees C and pH range 5.5-7.5 . The obtained crude enzyme preparation possessed high thermostability . The residual inulinase and invertase activities were 92-98% after pretreatment at 65 degrees C for 60 min in the presence of substrate inulin. Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 187 - 93 Roseiflexus castenholzii gen . nov., sp . nov., a thermophilic, filamentous, photosynthetic bacterium that lacks chlorosomes; Hanada S et al.; A novel thermophilic, photosynthetic bacterium, designated strain HLO8T, was isolated from a bacterial mat in a Japanese hot spring . Morphologically, the isolate was an unbranched multicellular filament with a cell diameter of 0.8-1.0 microm . The bacterium was red to reddish-brown in colour and formed a distinct red bacterial mat in the natural environment . It was able to grow photoheterotrophically under anaerobic light conditions and also chemoheterotrophically under aerobic dark conditions . Optimal growth occurred at 50 degrees C and pH 7.5-8.0 . The cells contained bacteriochlorophyll (Bchl) a and gamma-carotene derivatives as photosynthetic pigments, but lacked Bchl c and chlorosomes . Cellular fatty acids in the isolate were mainly C16:0, C14:0 and C15:0 . The major quinone was menaquinone-11 . The DNA G+C content was 62.0 mol% (by HPLC) . Phylogenetic analysis based on 16S rDNA sequencing suggested that the isolate belonged to the anoxygenic filamentous phototrophic bacteria represented by Chloroflexus aurantiacus, but was clearly distant from all members in this group (the sequence similarities between the isolate and its relatives were less than 83.8%) . Based on genotypic and phenotypic data, the name Roseiflexus castenholzii gen . nov., sp . nov . is proposed for this isolate; the type strain is HLO8T (= DSM 13941T = JCM 11240T). Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 157 - 64 Anaerobaculum mobile sp . nov., a novel anaerobic, moderately thermophilic, peptide-fermenting bacterium that uses crotonate as an electron acceptor, and emended description of the genus Anaerobaculum; Menes RJ et al.; A novel anaerobic, moderately thermophilic, peptide-fermenting bacterium, strain NGA(T), was isolated from an anaerobic wool-scouring wastewater treatment lagoon . The cells were gram-negative, straight rods of 0.5-1.0 x 2.0-4.0 microm, motile by means of a single flagellum . The DNA G+C content was 51.5 mol% . The optimum pH and temperature range for growth were 6.6-7.3 and 55-60 degrees C, respectively . The optimum NaCl concentration was 0.08 g l(-1) . The bacterium fermented organic acids (malate, tartrate, pyruvate, glycerol and fumarate), a few carbohydrates (starch, glucose, fructose and gluconate), Casamino acids, tryptone and yeast extract . Carbohydrates and organic acids were converted to acetate, hydrogen and CO2 . The bacterium oxidized leucine to isovalerate with crotonate as an electron acceptor, but not in co-culture with Methanothermobacter thermoautotrophicus DSM 3720T . Thiosulfate, sulfur and cystine were reduced to sulfide and crotonate was reduced to butyrate with glucose and tryptone-yeast extract as electron donors . Phylogenetic analysis of the 16S rRNA gene indicated that strain NGA(T) was related to Anaerobaculum thermoterrenum (98% similarity), the only described species of the genus . The DNA-DNA hybridization value for strain NGA(T) and A . thermoterrenum ACM 5076T was 40.8% . On the basis of these results, strain NGA(T) is proposed as a novel species of the genus Anaerobaculum, namely Anaerobaculum mobile sp . nov . The type strain is NGA(T) (= DSM 13181T =ATCC BAA-54T). Biotechnol Bioeng, 2002 Mar 30, 77(7), 752 - 7 Screening of various glycosidases for the synthesis of octyl glucoside; Ducret A et al.; Thirteen glycosidases of microbial origin and almond beta-glycosidase were assayed in octanol/DMF (80:20, v/v), using a combination of hydrolysis, transglycosylation, and condensation reactions, in order to assess their potential for the production of alkyl glucosides . The two mesophile enzymes were highly impaired by the organic media . Three of the 11 thermophile enzymes gave interesting results in the hydrolysis and transglycosylation reactions, but they were highly inhibited by glucose . This made their use in a condensation reaction less interesting than the use of almond beta-glucosidase, which has a lower activity but shows less inhibition by the glucose . Biotechnol Appl Biochem, 2002 Feb, 35(Pt 1), 35 - 43 Oligo-1,6-glucosidase from a thermophile, Bacillus thermoglucosidasius KP1006, was efficiently produced by combinatorial expression of GroEL in Escherichia coli; Watanabe K et al.; To improve the production of oligo-1,6-glucosidase from the obligately thermophilic Bacillus thermoglucosidasius KP1006 in Escherichia coli, the combined expression of oligo-1,6-glucosidase with various chaperone proteins of Hsp (heat-shock protein) 60 team proteins (GroES and GroEL) or Hsp70 team proteins (GrpE, DnaK and DnaJ) from the same thermophile was examined . This attempt was based on the facts that, (i) among glycosyl hydrolases of Family 13, bacillary oligo-1,6-glucosidases share highest homology with yeast alpha-glucosidase, and (ii) this yeast enzyme interacts with GroEL . In B . thermoglucosidasius Hsp60 team proteins, in particular, GroEL brought about a remarkable rise in expression of B . thermoglucosidasius oligo-1,6-glucosidase, while Hsp70 team proteins had no significant effect . The effect of B . thermoglucosidasius GroEL on oligo-1,6-glucosidase expression was supported by the finding that thermally inactivated B . thermoglucosidasius oligo-1,6-glucosidase was revived by B . thermoglucosidasius GroEL . Although the molecular mass of B . thermoglucosidasius oligo-1,6-glucosidase (66 kDa) exceeds the major range of substrates for GroEL proteins, the GroEL molecules probably recognized the alpha/beta motifs contained in the N-terminal domain and the subdomain of the oligo-1,6-glucosidase . Here we show that (i) the production of B . thermoglucosidasius oligo-1,6-glucosidase in E . coli was improved 3.8-fold by Hsp60 team proteins, (ii) the system can function for the expression of other glycosyl hydrolases of Family 13 that have defects in expression and (iii) the combinatorial expression of thermostable proteins with GroEL from the same thermophile in E . coli can increase the production of thermostable enzymes, preventing problems derived from differences in protein biogenesis. Water Environ Res, 2001 Nov-Dec, 73(6), 684 - 90 Measurement of microbial numbers and biomass contained in thermophilic anaerobic reactors; Solera R et al.; This paper describes the determination of the microbial population in terms of the number, biomass, and composition of single- and two-phase, laboratory-scale thermophilic (55 degrees C) anaerobic reactors under steady-state conditions . Epifluorescence microscopy with 4',6-diamidine-2-phenylindole (DAPI) as fluorochrome was used to determine the total number of microorganisms in the reactors, and autofluorescence microscopy was used to determine the total methanogenic bacteria populations . The results obtained by the direct count methods were compared to the quantity of biomass contained in the system, which was determined by volatile suspended solids . The concentration of acidogenic bacteria was estimated by subtraction of the autofluorescence results from those of the DAPI epifluorescence microscopy . The viable bacterial population was determined by plating techniques using an anaerobic chamber . The total bacterial and methanogenic populations of single-stage digesters increase when the hydraulic retention time decreases; nevertheless, the percentages of the principal bacterial groups (acidogenic and methanogenic) remain constant at 87% and 13%, respectively . In the two-stage reactors, the percentages of the acidogenic and methanogenic groups are 99% and 26% of the total population in the acidogenic and methanogenic reactors, respectively . In the single-stage reactors, biomass determinations can be used to estimate microbial concentrations, and vice versa, as there is a high positive correlation between microorganism concentration and biomass . The syntrophic relationship between the bacteria involved in the anaerobic process is a possible explanation for the low values of viable population obtained in the reactors studied . Nevertheless, there is a high correlation between direct counts by epifluorescence microscopy and viable plate counts for the combined system studied. Chembiochem, 2001 Mar 2, 2(3), 180 - 9 Structural changes of cytochrome c(552) from Thermus thermophilus adsorbed on anionic and hydrophobic surfaces probed by FTIR and 2D-FTIR spectroscopy; Lecomte S et al.; The structural changes of cytochrome c(552) bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectroscopy . Binding to the anionic surface of montmorillonite is controlled by electrostatic interactions since addition of electrolyte (0.5 mol L(-1) KCl) causes desorption of more than 2/3 of the protein molecules . Electrostatic binding occurs through the back side of the protein (i.e., remote from the heme site) and is associated only with subtle changes of the secondary structure . In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in alpha-helical structure by ca . 5% and an increase in beta-sheet structure by ca . 6% . These structural changes are attributed to a hydrophobic region on the front surface of cytochrome c(552) close to the partially exposed heme edge . This part on the protein surface is identified as the interaction domain for talc and most likely also serves for binding to the natural reaction partner, a ba(3)-oxidase . Fourier transform infrared spectra of cytochrome c(552) and the clay-cytochrome c(552) complexes have been measured as a function of time following dissolution and suspension in deuterated buffer, respectively . A two-dimensional correlation analysis was applied to these spectra to investigate the dynamics of the structural changes in the protein . For both complexes, adsorption and subsequent unfolding processes in the binding domains are faster than the time resolution of the spectroscopic experiments . Thus, the processes that could be monitored are refolding of peptide segments and side chain rearrangements following the adsorption-induced perturbation of the protein structure and the solvation of the adsorbed protein . In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed . For the cytochrome c(552)-talc complex, these changes are followed by a slow refolding of the peptide chain in the binding domain and, subsequently, a further H/D exchange of amide group protons. Water Res, 2002 Jan, 36(2), 429 - 38 Microbial community analysis of thermophilic contact oxidation process by using ribosomal RNA approaches and the quinone profile method; Kurisu F et al.; Microbial community structure of a lab scale thermophilic aerobic wastewater treatment reactor was analyzed by a combination of culture-independent methods . Quinone profile method provides for chemical analysis of respiratory quinone molecular species, which corresponds to bacterial groups . Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA partial sequences (PCR-DGGE) clarifies community changes at species level, as DGGE can separate DNA fragments of different sequences . Certain phvlogenetic groups of bacterial cells can be labeled by fluorescence in situ hybridization (FISH) . Quinone profile showed a predominant presence of MK-7 . PCR-DGGE revealed that constituents of the community were unchanged during the stable phase . FISH demonstrated the existence of the relatives of Bacillus lentus and B . thermocloacae in considerable proportions . The community was mainly composed of Bacillaceae, and obligate thermophilic and mesophilic Bacillus appeared in spite of the temperature fluctuation from 35 degrees C to 60 degrees C . The combination of these culture-independent methods revealed the community precisely enough to evaluate the reactor performance. Biosci Biotechnol Biochem, 2001 Dec, 65(12), 2695 - 700 Novel substrate specificity of designer 3-isopropylmalate dehydrogenase derived from Thermus thermophilus HB8; Fujita M et al.; Redesigning of an enzyme for a new catalytic reaction and modified substrate specificity was exploited with 3-isopropylmalate dehydrogenase (IPMDH) . Point-mutation on Gly-89, which is not in the catalytic site but near it, was done by changing it to Ala, Ser, Val, and Pro, and all the mutations changed the substrate specificity . The mutant enzymes showed higher catalytic efficiency (kcat/Km) than the native IPMDH when malate was used as a substrate instead of 3-isopropylmalate . More interestingly, an additional insertion of Gly between Gly-89 and Leu-90 significantly altered the substrate-specificity, although the overall catalytic activity was decreased . Particularly, this mutant turned out to efficiently accept D-lactic acid, which was not accepted as a substrate by wild-type IPMDH at all . These results demonstrate the opportunity for creating nove,enzymes by modification of amino acid residues that do not directly participate in catalysis, or by insertion of additional residues. J Biol Chem, 2002 Apr 19, 277(16), 13401 - 8 Epub 2002 Jan 31. DNA polymerase III holoenzyme from Thermus thermophilus identification, expression, purification of components, and use to reconstitute a processive replicase; Bullard JM et al.; DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the beta sliding clamp processivity factor, and the DnaX complex clamp-loader . We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile . The minimal holoenzyme consists of alpha (pol III catalytic subunit), beta (sliding clamp processivity factor), and the essential DnaX (tau/gamma), delta and delta' components of the DnaX complex . We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates . Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including delta-delta' interaction, deltadelta'-tau/gamma complex formation, and alpha-tau interaction, also occur within the Tth enzyme . As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner . However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 degrees C, both with regard to initiation complex formation and processive DNA synthesis . The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 degrees C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event. Appl Environ Microbiol, 2002 Feb, 68(2), 938 - 41 Streptococcus thermophilus is able to produce a beta-galactosidase active during its transit in the digestive tract of germ-free mice; Drouault S et al.; This work presents data on the application of a bacterial luciferase used to monitor gene expression of Streptococcus thermophilus in the digestive tract . The main result is that the bacterium was able to produce an active beta-galactosidase in the digestive tract, although it did not multiply during its transit . This production was enhanced when lactose (the inducer) was added to the diet. Appl Environ Microbiol, 2002 Feb, 68(2), 784 - 90 Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus; Levander F et al.; It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars . To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03 . Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source . However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose . A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain . The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain . However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain . Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains . These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism. Appl Environ Microbiol, 2002 Feb, 68(2), 745 - 55 Molecular analyses of the natural transformation machinery and identification of pilus structures in the extremely thermophilic bacterium Thermus thermophilus strain HB27; Friedrich A et al.; Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation . To identify genes of the natural transformation machinery of T . thermophilus HB27, we performed homology searches in the partially completed T . thermophilus genomic sequence for conserved competence genes . These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria . Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency . One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1 . Analysis of the piliation phenotype of T . thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures . These results suggest that pili and natural transformation in T . thermophilus HB27 are functionally linked. Appl Environ Microbiol, 2002 Feb, 68(2), 588 - 96 Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages; Sturino JM et al.; Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S . thermophilus bacteriophages and one pac-type bacteriophage . The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S . thermophilus bacteriophages of industrial importance . All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA . A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S . thermophilus parental strain . When progeny were released, the burst size was reduced . Growth curves of S . thermophilus NCK1125, in the presence of variable levels of bacteriophage kappa3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0 . When the hel3.1 antisense RNA cassette was expressed in cis from the kappa3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10(-7) and 10(-8), beyond the resistance conferred by the PER element alone (less than 10(-6)) . These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S . thermophilus bacteriophages. Can J Microbiol, 2001 Dec, 47(12), 1075 - 81 Structural analyses of the deduced amino acid sequences of a novel type heme-copper terminal oxidase, cytochrome aco3, from alkalophilic Bacillus YN-2000; Denda K et al.; Cytochrome aco3 from a facultatively alkalophilic bacterium, Bacillus YN-2000, was found to be alkaline- and heat-tolerant . To better understand the structural features of Bacillus YN-2000 cytochrome aco3, the gene encoding this enzyme was cloned and sequenced . Nucleotide sequence analyses of the region neighboring the acoI (subunit I) gene revealed that the acoII (subunit II) and acoIII (subunit III) genes were concomitantly clustered upstream and downstream of the acoI gene, respectively, forming an operon with transcriptional polarity . The deduced amino acid sequence of subunit I was highly similar to that of cytochrome caa3 from thermophilic bacterium Bacillus PS3 in which the heme a3 could be replaced with heme o . Furthermore, a marked paucity of basic amino acid residues was found in the cytochrome c-binding subunit II, which might be a result of the adaptation to a highly alkaline external milieu. Biochem Biophys Res Commun, 2002 Feb 8, 290(5), 1434 - 40 ATP binding and hydrolysis and autophosphorylation of CbbQ encoded by the gene located downstream of RubisCO genes; Hayashi NR et al.; CbbQ is encoded by the gene located downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase genes (cbbLS) in the thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus . The protein possesses two nucleotide-binding motifs in its amino acid sequence, and it posttranslationally activates RubisCO . We present ATP hydrolysis and binding of CbbQ . CbbQ releases P(i) from ATP only in the presence of Mg(2+) . CbbQ interacts with an 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate in the presence or absence of Mg(2+) . The interaction with Mg(2+) and/or a nucleotide induces a conformational change in CbbQ . Autophosphorylation of CbbQ occurs only in the absence of Mg(2+) . (c)2002 Elsevier Science (USA). Luminescence, 2002 Jan-Feb, 17(1), 15 - 8 Individual molecules of thermostable alkaline phosphatase support different catalytic rates at room temperature; Dyck AC et al.; Thermus thermophilus cells were grown at 70 degrees C, lysed and the lysate subjected to single molecule alkaline phosphatase assays, using a capillary electrophoresis laser-induced fluorescence detection-based method . The enzyme was found to be heterogeneous with respect to catalytic rate when assayed at room temperature . Turnover numbers ranged 12-fold, with an average of 400 +/- 200 reactions/min for the 80 molecules assayed .
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