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