Thermophiles

Biochemistry, 1996 Jul 30, 35(30), 9880 - 91
Metal binding sites of H(+)-ATPase from chloroplast and Bacillus PS3 studied by EPR and pulsed EPR spectroscopy of bound manganese(II); Buy C et al.; The metal binding sites of isolated F1 ATPase from spinach chloroplasts (CF1) and from the thermophilic bacterium Bacillus PS3 (TF1) have been studied by EPR and pulsed EPR spectroscopy using Mn(II) as a paramagnetic probe . After dialysis in the presence of EDTA, purified CF1 retains 0.14 +/- 0.07 Mg(II) and approximately 0.75 +/- 0.25 ADP . TF1 retains 0.31 +/- 0.03 Mg(II) and 0.08 +/- 0.01 nucleotide (ADP + ATP) after the same treatment . Supplementing known quantities of Mn(II) to metal-depleted CF1 allowed a spectroscopic characterization of the bound Mn(II) cations, for which the EPR spectra at X- and Q-band are reported . The zero field splitting parameters of Mn(II) are derived from the simulation of the EPR signal recorded at Q-band for a sample supplemented with 0.3 Mn/CF1 . The values, magnitude of D approximately 200 x 10(-4) cm-1 and magnitude of E approximately 40 x 10(-4) cm-1 suggest that the Mn(II) binds to CF1 in a slightly distorted environment . The ESEEM spectra of complexes of Mn(II) with CF1 were also recorded for different Mn/CF1 ratios . For a complex with 0.8 Mn/CF1, the ESEEM spectrum shows two frequencies at 3.7 and 8.6 MHz that are attributed to the magnetic coupling with 31P with a hyperfine coupling constant of magnitude of A approximately 5.3 MHz, reflecting the interaction with a phosphate group from the endogenous ADP molecule . This demonstrates close proximity of the strong affinity metal site M1 and the endogenous ADP binding site N1, and binding of the ADP beta-phosphate to the divalent metal cation . For Mn(II) complexes with higher Mn/CF1 ratios, new frequency components below approximately 5 MHz are resolved in the spectra in addition to the peaks from 31P . From a comparison of the CF1 spectra and their magnetic field dependence across the Mn(II) EPR line shape with those of Mn(II) complexes with imidazole, glycine, poly-L-lysine, and nucleotide ligands, it is concluded that additional metal binding sites are filled at higher Mn contents and that these involve 14N donors . It is suggested that the most probable set of ligands of the divalent metal(s) for these additional metal sites in CF1 includes a lysine residue, in line with a previous proposal {Houseman, A . L . P., Morgan, L., LoBrutto, R., & Frasch, W . D . (1994) Biochemistry 33, 4910-4917} . Similar experiments for a Mn(II) complex with TF1 (0.4 Mn/TF1) showed no interaction with 31P; instead modulations are detected in the ESEEM below approximately 5 MHz that are attributed to a 14N ligand . This is tentatively attributed to the deprotonated amine of Lys-162 from a beta subunit, on the basis of the structural data available for the mitochondrial F1 complex . Addition of the substrate ATP to this Mn.TF1 complex leads to the formation of a ternary Mn.TF1.ATP complex with coordination of the Mn(II) by a phosphate group from the ATP as judged from the ESEEM results (magnitude of A(31P) approximately 4.5 MHz) . An increase in the hyperfine coupling constant of 31P of the phosphate bound to Mn(II) to magnitude of A(31P) approximately 5.1 MHz is observed after incubation of the ternary complex at room temperature . This is interpreted as a significant rearrangement of the coordination sphere of the Mn(II) in the M1 site of the Mn.TF1.ATP complex and may reflect conformational changes of catalytic significance that occur in the nucleotide binding site during unisite hydrolysis of ATP to ADP by this complex.

Biochemistry, 1996 Jul 30, 35(30), 9792 - 6
Stereospecificity of thermostable ornithine 5-aminotransferase for the hydrogen transfer in the L- and D-ornithine transamination; Jhee KH et al.; The thermostable ornithine 5-aminotransferase of a thermophile, Bacillus sp . YM-2, is unique in acting on both enantiomers of ornithine, although less effectively on the D-enantiomer . We studied the stereospecificity of the enzyme for the hydrogen abstraction from C-5 of the substrate moiety and the addition and removal of the hydrogen at C-4' of the cofactor (pyridoxal phosphate and pyridoxamine phosphate) moiety of the external Schiff base intermediate in the transamination of L- and D-ornithine . L- and D-{5-3H}ornithines were prepared by incubation of L- and D-ornithines with the enzyme in 3H2O, respectively . When the L-{5-3H}ornithine was incubated with L-ornithine 5-aminotransferase of a mesophile, Bacillus sphaericus, which catalyzes the stereospecific abstraction of pro-S hydrogen from C-5 of L-ornithine, most of the tritium was released into the solvent . The D-{5-3H}ornithine also reacted with the enzyme of B . sphaericus in the presence or absence of the amino acid racemase of Pseudomonas putida . Tritium was released only in the presence of the racemase, which catalyzes the racemization of ornithine but does not act on C-5 of ornithine . These results show that the Bacillus sp . YM-2 ornithine 5-aminotransferase stereospecifically abstracts the pro-S hydrogen from C-5 of L- and D-ornithine . When the apo form of the enzyme was incubated with pyridoxamine 5'-phosphate that was stereospecifically tritiated at C-4' and 2-oxoglutarate in the presence of L-ornithine or D-ornithine, tritium was released exclusively from (4'S)-{4'-3H}pyridoxamine . Therefore, addition and abstraction of hydrogen at C-4' of the cofactor moiety stereospecifically occur on the si face of the external Schiff base intermediate in the overall transamination catalyzed by Bacillus sp . YM-2 ornithine 5-aminotransferase irrespective of the C-2 configuration of the amino donor.

J Biol Chem, 1996 Jul 26, 271(30), 17687 - 91
Catalytic properties of human manganese superoxide dismutase; Hsu JL et al.; The depletion of superoxide catalyzed by human manganese superoxide dismutase (MnSOD) was observed spectrophotometrically by measuring the absorbance of superoxide at 250-280 nm following pulse radiolysis and by stopped-flow spectrophotometry . Catalysis showed an initial burst of activity lasting approximately 1 ms followed by the rapid emergence of a greatly inhibited catalysis of zero-order rate . These catalytic properties of human MnSOD are qualitatively similar to those reported for MnSOD from Thermus thermophilus (Bull, C., Niederhoffer, E . C., Yoshida, T., and Fee, J . A.(1991) J . Am . Chem . Soc . 113, 4069-4076) . However, there are significant quantitative differences; the emergence of the inhibited form is approximately 30-fold more rapid for human MnSOD . The turnover number for human MnSOD at pH 9.4 and 20 degrees C was kcat = 4 x 10(4) s-1 and kcat/Km = 8 x 10(8) M-1 s-1, determined by a simulated fit of the model of Bull et al . (1991) to the pulse radiolysis data . We also report that the maximum of the visible absorption spectrum of human MnSOD (epsilon480 = 525 M-1 cm-1) showed a strong dependence on pH that could be described by an ionization of pKa 9.4 +/- 0.1 with a maximum at low pH.

J Biol Chem, 1996 Jul 26, 271(30), 18128 - 33
Catalytic activities of alpha3beta3gamma complexes of F1-ATPase with 1, 2, or 3 incompetent catalytic sites; Amano T et al.; In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the alpha3beta3gamma complex with 1, 2, or 3 incompetent catalytic sites was developed . Ten glutamic acids (Glu.Tag) were linked to the C terminus of the catalytically incompetent beta(E190Q) subunit . The Glu.Tag itself did not affect ATPase activity of the complexes . Two kinds of alpha3beta3gamma complexes, one containing beta(wild-type) and the other Glu.Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, and alpha3beta3gamma complexes were reconstituted . Each of the complexes containing a different number of Glu.Tag-linked beta(E190Q) was separated by anion-exchange chromatography and analyzed . The results were as follows . 1) Normal steady-state ATPase activity requires three intact catalytic sites . 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites . 3) Single-site catalysis can be mediated by a single intact catalytic site alone . Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M.(1989) J . Biochem . (Tokyo) 106, 730-734).

Arch Microbiol, 1996 Jul 24, 166(1), 64 - 7
Culturability and survival of an extreme thermophile isolated from deep-sea hydrothermal vents
González JM, Kato C, Horikoshi K.
The culturability of a strictly anaerobic, extremely thermophilic archaeon, Thermococcus peptonophilus (optimal growth temperature: 85° C), was studied during survival stages at various temperatures (98, 85, 70, and 4° C) . Total cell number (determined by DAPI staining), active cells (rhodamine-stained cells), and culturable cells (using most-probable-number) were counted over time . The number of culturable cells decreased under each condition tested . The total number of cells significantly decreased only at temperatures close to the maximum for growth (98° C); at this temperature, the cells spontaneously lysed . Our results suggested that survival at 4° C in oxygenated waters might be a mechanism for the dispersion of extreme thermophiles in the ocean . In addition, we proved the existence of T . peptonophilus cells in several physiological states: culturable cells, active non-culturable cells, inactive non-culturable cells, and dead cells . Cell death was caused by cellular lysis.

J Biol Chem, 1996 Jul 19, 271(29), 17343 - 8
A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus; Motohashi K et al.; We previously reported the isolation of T.DnaK.DnaJ chaperone complex from Thermus thermophilus . Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex . A dnaK gene cluster of T . thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB . Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins . The orf4 gene encodes a novel 78-amino acid protein . Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex . Careful reexamination of the T.DnaK.DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4 . As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK.DnaJ complex in E . coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein . The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK.DnaJ assembly factor A) . The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA . Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.

FEMS Microbiol Lett, 1996 Jul 15, 141(1), 37 - 43
Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4; Gibbs MD et al.; A gene encoding a beta-mannanase (manA) has been cloned from an obligately anaerobic extreme thermophile, Caldicellulosiruptor strain Rt8B.4, which is most closely related to Caldicellulosiruptor saccharolyticus (formerly Caldocellum saccharolyticum) . The gene codes for a multidomain enzyme with a C-terminal beta-mannanase domain which was amplified by the polymerase chain reaction and cloned into a temperature-inducible expression vector in Escherichia coli . Sequence comparisons have shown that the Man domain of Rt8B.4 ManA is related to a thermophilic Dictyoglomus mannanase and a mesophilic mannanase from a Bacillus species . It appears to be unrelated to the beta-mannanase domain of C . saccharolyticus, implying acquisition of the genes from unrelated sources by the two bacteria.

Eur J Biochem, 1996 Jul 15, 239(2), 501 - 8
Asparaginyl-tRNA synthetase from Thermus thermophilus HB8 . Sequence of the gene and crystallization of the enzyme expressed in Escherichia coli; Seignovert L et al.; The gene for the asparaginyl-tRNA synthetase, a class IIb enzyme, from the extreme thermophile Thermus thermophilus HB8 has been cloned and sequenced . Sequence analysis revealed an open reading frame that codes for a protein of 438 amino acid residues (50875 Da) . Codon usage in the asparaginyl-tRNA synthetase gene (asnS) is similar to the characteristic usage in the genes for proteins from bacteria of the genus Thermus, and the G+C content in the third position of the codons is as high as 94% . The amino acid sequence of asparaginyl-tRNA synthetase from T . thermophilus shows high similarity with other bacterial asparaginyl-tRNA synthetase sequences (30-55% identity) . By expression of the T . thermophilus asnS gene in Escherichia coli, the thermostable enzyme was overproduced and purified to homogeneity by heat treatment and two chromatography steps . The protein obtained is remarkably thermostable and retains 50% of its initial tRNA aminoacylation activity after 1 h of incubation at 90 degrees C or 21 h at 85 degrees C . Crystals of the enzyme were obtained from polyethylene glycol 6000 solutions by vapour diffusion techniques . The crystals diffract X-rays beyond 2.8 A.

Eur J Biochem, 1996 Jul 15, 239(2), 265 - 71
Limited proteolysis and amino acid replacements in the effector region of Thermus thermophilus elongation factor Tu; Zeidler W et al.; The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis . The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu . EF-Tu that was cleaved at the Arg59-Gly60 peptide bond {EF-Tu-(1-59)/EF-Tu-(60-405)} bound GDP, EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis . However, the GTPase activity of EF-Tu-(1-59)/EF-Tu-(60-405) was not stimulated by T . thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu . EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1-59)/EF-Tu-(60-405) . By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T . thermophilus EF-Tu . These amino acid substitutions did not substantially affect either the affinity of EF-Tu . GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts . Similarly the intrinsic GTPase activity is not influenced . Replacement of Glu56 by Ala led to strong reduction in the ribosome-induced GTPase activity . This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity . The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.

J Biol Chem, 1996 Jul 12, 271(28), 16559 - 66
Ribonuclease P of Tetrahymena thermophila; True HL et al.; Ribonuclease P (RNase P) is responsible for the generation of mature 5' termini of tRNA . The RNA component of this complex encodes the enzymatic activity in bacteria and is itself catalytically active under appropriate conditions in vitro . The role of the subunits in eucaryotes has not yet been established . We have partially purified RNase P activity from the ciliate protozoan Tetrahymena thermophila to learn more about the biochemical characteristics of RNase P from a lower eucaryote . The Tetrahymena RNase P displays a pH optimum and temperature optimum characteristic of RNase P enzymes isolated from other organisms . The Km of the T . thermophila enzyme for pre-tRNAGln is 1.6 x 10(-7)M, which is comparable to the values reported for other examples of RNase P . The Tetrahymena RNase P is a ribonucleoprotein complex, as supported by its sensitivity to micrococcal nuclease and proteinase K . The buoyant density of the enzyme in Cs2SO4 is 1.42 g/ml, which suggests that the RNA component of the Tetrahymena enzyme comprises a significantly greater percentage of the holoenzyme than that determined for RNase P of other Eucarya or Archaea . The holoenzyme has a requirement for divalent cations displaying characteristics that are unique for RNase P but closely resemble preferences reported for the Tetrahymena group I intron RNA . Puromycin inhibits pre-tRNA processing by the Tetrahymena complex, and implications of the similarities between recognition of tRNA by ribosomal components and RNase P are discussed.

Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 224 - 8
The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum; Kocabiyik S et al.; In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme . Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A . The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability . The mutation, however, has decreased the enzyme activity . The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.

Dtsch Tierarztl Wochenschr, 1996 Jul, 103(7), 264 - 8
{Effects of residues of anti-infective agents in animal excretions on slurry treatment and the soil}; Bohm R; Application of antibiotics and feed additives in animal husbandry generates populations of resistant bacteria in the gut which are introduced via slurry into the ecosystem soil . Nevertheless the influence to the ecosystem soil seems to be low, due to inactivation and dilution in slurry and soil . The probability that this pathway contributes to the problems of antibiotic resistance in human medicine is low . Aerobic and anaerobic treatment of slurry is more or less influenced by residuals of some feed additives and antibiotics . The aerobic-thermophilic treatment seems generally to be more sensitive than the anaerobic biogas production, except in some special cases . No evidence could be found that residuals of antibiotics in slurry have a negative influence on the ecosystem soil, due to preliminary results obtained by similar field trials with disinfectants . The application of antibiotics in aquaculture must be regarded more critically.

Eur J Cell Biol, 1996 Jul, 70(3), 243 - 9
A giant protein associated with the anterior pole of a trypanosomatid cell body skeleton; Baqui MM et al.; A megadalton protein was found to be a cytoskeleton component of the promastigote forms of the flagellate Phytomonas serpens . This protein migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as a doublet of polypeptides with a molecular mass similar to muscle beta-connectin (titin) 2500-3000 kDa . A polyclonal antibody raised against this protein reacts, by immunoblot analysis, with Phytomonas serpens and two others Phytomonas species . In addition, the Phytomonas serpens protein was immunoprecipitated after being metabolically labeled with {35S}methionine . This antibody did not cross-react with the cytoskeletal proteins of Trypanosoma cruzi, Crithidia luciliae thermophila, Crithidia fasciculata and Leptomonas samueli or with beta-connectin (titin) . Indirect immunofluorescence microscopy analysis revealed a punctate fluorescence staining at the anterior region of the parasite's body skeleton . Moreover, immunogold electron microscopy of cytoskeletal preparations and of thin sections of whole cells indicates that the giant protein appears to cap the anterior end of the cell body microtubules at the level of the junctional complex . We suggest that this giant protein may serve as a linker between the cell body skeleton and the flagellum membrane.

Vet Microbiol, 1996 Jul, 51(1-2), 77 - 84
Rapid and specific detection of Mycoplasma agalactiae by polymerase chain reaction; Tola S et al.; A polymerase chain reaction (PCR)-based test was developed for the detection of Mycoplasma agalactiae in sheep milk samples . Two oligonucleotide primers were designed to amplify a 375 bp fragment of M . agalactiae chromosomal DNA . Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization using a fluorescein labeled 528 bp probe . The primers allowed the amplification of fragment of M . agalactiae DNA and did not amplify any specific fragment of other mycoplasmal DNAs (M . capricolum, M . mycoides subsp . mycoides, M . mycoides subsp . capri, M . putrefaciens, M . arginini and M . bovis) or other bacterial DNAs (S . aureus, S . epidermidis, P . haemalytica, E . coli, S . agalactiae, S . dysgalactiae, S . uberis, B . cereus, P . aeruginosa, S . durans, L . lactis, L . lactis var . diacetilactis, L . mesenteroides, S . thermophilus, L . bulgaricus and L . casei) . The limit of detection of PCR assay was between 2.5 and 25 fg of purified DNA and 10(2) CCU ml-1 on mycoplasma cultures . These results indicate that the PCR technique can be used as a rapid and specific diagnostic method for detection of M . agalactiae.

Int J Syst Bacteriol, 1996 Jul, 46(3), 727 - 35
Phylogeny and taxonomy of mesophilic Methanococcus spp . and comparison of rRNA, DNA hybridization, and phenotypic methods; Keswani J et al.; The phylogeny and taxonomy of the mesophilic methane-producing archaea of the order Methanococcales were examined by DNA relatedness, 16S rRNA sequence analysis, cellular protein patterns, and phenotypic methods . The mesophilic species Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltaei, and "Methanococcus aeolicus" formed a deep group with 5 to 30% DNA relatedness and 92 to 96% 16S rRNA sequence similarity . Twenty-two additional isolates and Methanococcus deltae were similar to the type strain of either M . voltaei or M . maripaludis . Two isolates, strains A2 and A3, exhibited 37% DNA relatedness and 99.2% 16S rRNA sequence similarity to M . voltaei PS(T) (T = type strain) . In the absence of phenotypic differences, these organisms were assigned to M . voltaei . Similarly, four autotrophic isolates, strains C5, C6, C7, and C8, exhibited 54 to 69% DNA relatedness and 99.2% 16S rRNA sequence similarity to M . maripaludis JJT and were assigned to M . Maripaludis . While these isolates were sufficiently genetically diverse to justify classification in novel species, few differences were apparent in the phenotypic properties available for measurement . Thus, the phenotypic properties of these lithotrophic archaea were highly conserved and poor indicators of genetic diversity . Partial sequencing of about 200 bases of both the 16S and 23S rRNAs of the isolates demonstrated allelic diversity within methanococcal species . This allelic diversity did not correlate with diversity measured by DNA relatedness, cellular protein pattern, and other methods . Similarly, antisera to whole cells of the type strains did not cross-react strongly to whole cells of strains that were genetically similar, and serological cross-reactivity was not a useful taxonomic method for methanococci . Lastly, on the basis of the results of 16S rRNA sequence analyses and biochemical data, the ancestor of the mesophilic methanococci may have been an autotrophic thermophile.

Appl Environ Microbiol, 1996 Jul, 62(7), 2657 - 9
L-alanine production from glucose fermentation by hyperthermophilic members of the domains bacteria and Archaea: a remnant of an ancestral metabolism?
Ravot G, Ollivier B, Fardeau ML, Patel BK, Andrews KT, Magot M, Garcia JL.
New members of the order Thermotogales were isolated from nonvolcanically heated geothermal environments, including oil fields and waters of the Great Artesian Basin of Australia, thereby extending their known habitats, previously recognized primarily as volcanic . The hyperthermophilic and thermophilic members of Thermotogales of volcanic origin, together with the recently described nonvolcanic species of this order and three new isolates described in this paper, were all found to produce L-alanine from glucose fermentation, in addition to acetate, lactate, CO2 and H2 . L-alanine production from glucose is a trait in common with Pyrococcus furiosus and Thermococcus profundus . We propose that L-alanine production from sugar fermentation be regarded as an ancestral metabolic characteristic.

Appl Environ Microbiol, 1996 Jul, 62(7), 2482 - 8
Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp . strain CK 11-4 screened from Chungkook-Jang; Kim W et al.; Bacillus sp . strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce . The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp . strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity . The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp . This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg . The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA) . A similar result was also obtained from fibrin plate analysis.

FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 271 - 6
A novel thermostable dextranase from a Thermoanaerobacter species cultured from the geothermal waters of the Great Artesian Basin of Australia; Wynter C et al.; A Gram-negative sporulating thermophilic anaerobe, designated AB11Ad, was isolated from the heated waters of the Great Artesian Basin of Australia . It grew on a variety of carbohydrates including glucose, starch, and dextran and produced a thermostable and thermoactive extracellular endo-dextranase . The enzyme was produced more actively under pH controlled continuous culture conditions than under batch conditions . Ammonium sulfate precipitated crude dextranase exhibited a temperature optimum of 70 degrees C and a pH optimum between 5 and 6 . The half life was approximately 6.5 h at 75 degrees C and 2 h at 80 degrees C at pH 5.0 and in the absence of added dextran . 16S rRNA sequence analysis indicated that isolate AB11Ad was a member of the genus Thermoanaerobacter.

Microbiology, 1996 Jul, 142 ( Pt 7), 1651 - 7
The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA; Li Y et al.; The complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined . surA was composed of 1338 bp and encoded 445 amino acid residues . The deduced polypeptide of M(r) 51519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the 'sucrose box' residues thought to be important for catalysis of the transfer of fructose from sucrose . The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA . SurA displayed an optimum temperature for sucrose hydrolysis of 55 degrees C and high stability . The M(r) of SurA determined by gel filtration was 105,000, which suggested that the active form of the enzyme is a dimer . SurA exhibited an apparent Km of 40 mM for sucrose but, unlike the homologous B . subtilis enzyme, had no detectable sucrose phosphate hydrolase activity.

DNA Cell Biol, 1996 Jul, 15(7), 589 - 94
Fidelity and predominant mutations produced by deep vent wild-type and exonuclease-deficient DNA polymerases during in vitro DNA amplification; Huang H et al.; Denaturing gradient gel electrophoresis (DGGE) was used to examine error rates and mutations induced by native (wt) and exonuclease-deficient (exo-) Deep Vent DNA polymerases during DNA amplification by polymerase chain reaction (PCR), in the presence or absence of the T4 bacteriophage gene 32 protein (gp32).gp32 was found to decrease the error rate of the wt, but not that of the exo-, Deep Vent . The average errors per base duplication for the native form were 8.0 x 10(-5) and 6.0 x 10(-5) in the absence and presence of gp32, respectively . For the exo- form, the error rates were 2.0 x 10(-4) and 2.2 x 10(-4) errors per base duplication in the absence and presence of gp32, respectively . Examination of mutations produced by native Deep Vent showed that A/T to G/C transition predominated, consistent with the results of our earlier studies with DNA polymerases derived from other thermophilic bacteria . These results indicate that PCR with high fidelity can be achieved by using wt Deep Vent in combination with gp32.

Eur J Biochem, 1996 Jul 1, 239(1), 93 - 7
Si-face stereospecificity at C5 of coenzyme F420 for F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus; Klein AR et al.; Coenzyme F420 is a 5-deazaflavin . Upon reduction, 1,5-dihydro-coenzyme F420 is formed with a prochiral center at C5 . In this study we report that the F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and the F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus are Si-face stereospecific with respect to C5 of the 5-deazaflavin . These results were obtained by following the stereochemical course of the reversible incorporation of 3H into F420 from tritium-labeled substrates . Our findings bring to eight the number of coenzyme-F420-dependent enzymes shown to be Si-face stereospecific . No F420-dependent enzyme with Re-face stereospecificity is known . This is noteworthy since coenzyme F420 is functionally similar to pyridine nucleotides for which both Si-face and Re-face specific enzymes have been found.

Eur J Biochem, 1996 Jul 1, 239(1), 150 - 5
Purification and characterisation of an alpha-glucan phosphorylase from the thermophilic bacterium Thermus thermophilus; Boeck B et al.; An alpha-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus . In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose . T . thermophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M pyridoxal-P/M subunit), subunit molecular mass (about 90 kDa) and inhibitor constants . The optimum temperature of T . thermophilus phosphorylase was observed at 70 degrees C in the pH range 5.5-6.5 . While at 25 degrees C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70 degrees C seems to be a dimer . Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other alpha-glucan phosphorylases . Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (Vmax = 18 U/mg) . Correspondingly, maltotriose functions as primer in the synthesis direction . Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenate acting as a substrate indicate that the active site structure of T . thermophilus phosphorylase differs from that of known alpha-glucan phosphorylases.

Biochem J, 1996 Jul 1, 317 ( Pt 1), 29 - 34
Molecular cloning of delta 9 fatty acid desaturase from the protozoan Tetrahymena thermophila and its mRNA expression during thermal membrane adaptation; Nakashima S et al.; In response to a decrease in its growth temperature, the protozoan Tetrahymena is known to increase the level of unsaturated fatty acids in its membrane phospholipids so as to maintain the correct physical state (fluidity) of the membranes . In this organism, synthesis of unsaturated fatty acids is initiated by delta 9 acyl-CoA desaturase . Our previous studies have shown that, during cold adaptation, the activity of microsomal palmitoyl- and stearoyl-CoA desaturase increases, reaching a maximal level at 2 h after a temperature down-shift to 15 degrees C . Two hypotheses have been proposed to explain this increase in desaturase activity: (1) self-regulation via a direct effect of reduced membrane fluidity, and (2) induction of desaturase mRNA . However, the precise mechanism is not clearly understood . In order to obtain further insight into the mechanism of regulation of the desaturase, we have isolated a gene that encodes delta 9 fatty acid desaturase from T . thermophila and examined its expression during cold adaptation . The nucleotide sequence indicates that the 1.4 kbp gene encodes a polypeptide of 292 amino acid residues which shows marked sequence similarity to delta 9 acyl-CoA desaturases from other sources, e.g . rat, mouse, Amblyomma americanum and Saccharomyces cerevisiae . This protein has three histidine-cluster motifs (one HXXXXH and two HXXHH), and two hydrophobic regions which are conserved among delta 9 acyl-CoA desaturases . The level of desaturase mRNA was sensitive to decreasing the temperature of the culture media, and was close to maximal immediately after the temperature was shifted down from 35 degrees C to 15 degrees C (0.8 degrees C/min) . Thereafter, the amount of mRNA gradually decreased with time, but remained above the control level for at least 5 h . Furthermore, during the course of the cooling process to 15 degrees C, the increased expression of desaturase mRNA became evident at 27 degrees C . Nuclear run-on analysis and actinomycin D chase experiments revealed that the elevation of the mRNA level was due to increases in both transcription and mRNA stability . These results suggest that the enhanced desaturase activity is controlled, at least in part, at the transcriptional level.

Biochem J, 1996 Jul 1, 317 ( Pt 1), 235 - 45
Tetrameric malate dehydrogenase from a thermophilic Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and characterization of the recombinant enzyme; Wynne SA et al.; The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid . The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa . Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species . Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs . MDHs and LDHs resemble each other in catalytic mechanism and several other respects . However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs . The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric . A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E . coli plasmid vector . BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E . coli strain carrying the clone . MDH purified from BI and that purified from the E . coli strain carrying the MDH gene clone appear to be identical proteins by several criteria . A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters . Whereas the MDH is a stable tetramer in the pH range 5-7, it appears to be converted into a stable dimer at pH 3.5 . This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH.

EMBO J, 1996 Jul 1, 15(13), 3286 - 95
Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila; Rusconi CP et al.; The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis . It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria . T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA) . Each of these tRNAs functions in cytosolic translation . However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well . Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs . Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented . Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import . Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.

Mol Cell Biol, 1996 Jul, 16(7), 3720 - 9
The testis-specific high-mobility-group protein, a phosphorylation-dependent DNA-packaging factor of elongating and condensing spermatids; Alami-Ouahabi N et al.; Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines . The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA . A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process . We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids . Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro . The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner . We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG . Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I . This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.

Mol Cell Biol, 1996 Jul, 16(7), 3658 - 67
Non-Mendelian, heritable blocks to DNA rearrangement are induced by loading the somatic nucleus of Tetrahymena thermophila with germ line-limited DNA; Chalker DL et al.; Site-specific DNA deletion occurs at thousands of sites within the genome during macronuclear development of Tetrahymena thermophila . These deletion elements are usually not detected in macronuclear chromosomes . We have interfered with the normal deletion of two of these elements, the adjacent M and R elements, by loading vegetative macronuclei with these elements prior to sexual conjugation . Transformed cell lines containing the exogenous M or R element, carried on high-copy-number vectors containing genes encoding rRNA within parental (old) macronuclei, consistently failed to excise chromosomal copies of the M or R element during formation of new macronuclei . Little or no interference with the deletions of adjacent elements or of unlinked elements was observed . The micronucleus (germ line)-limited region of each element was sufficient to inhibit specific DNA deletion . This interference with DNA deletion usually is manifested as a cytoplasmic dominant trait: deletion elements present in the old macronucleus of one partner of a mating pair were sufficient to inhibit deletion occurring in the other partner . Remarkably, the failure to excise these elements became a non-Mendelian, inheritable trait in the next generation and did not require the high copy number of exogenously introduced elements . The introduction of exogenous deletion elements into parental macronuclei provides us with an epigenetic means to establish a heritable pattern of DNA rearrangement.

J Biol Chem, 1996 Jun 28, 271(26), 15358 - 66
Unidirectional reconstitution into detergent-destabilized liposomes of the purified lactose transport system of Streptococcus thermophilus; Knol J et al.; The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S . thermophilus, respectively . In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter . Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein . Membranes from S . thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-beta-D-glucoside, n-dodecyl-beta-D-maltoside, or C12E8 . The LacS protein carrying a poly-histidine tag was purified in large quantities (approximately 5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography . The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E . coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization . The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured . Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-beta-D-glucoside . For reconstitutions mediated by n-dodecyl-beta-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization) . Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide . The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane . Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport.

Biochemistry, 1996 Jun 18, 35(24), 7812 - 8
Isolation and characterization of soluble electron transfer proteins from Chromatium purpuratum; Kerfeld CA et al.; Several soluble electron transfer proteins were isolated and characterized from the marine purple-sulfur bacterium Chromatium purpuratum . The C . purpuratum flavocytochrome c is similar in molecular mass (68 kDa) and isoelectric point (6.5) to flavocytochromes isolated from other phototrophs . Redox titrations of the flavocytochrome c hemes show two components with midpoint potential values of +15 and -120 mV, behavior similar to that observed with the flavocytochrome isolated from the thermophilic Chromatium tepidum . Moreover, N-terminal amino acid sequence analysis of both the flavin and the cytochrome subunit indicates substantial homology to the primary structure of the flavocytochrome c of Chromatium vinosum . In contrast, the C . purpuratum high-potential iron-sulfur protein (HiPIP) differs from those isolated from other photosynthetic bacteria in its relatively high midpoint potential (+390 mV) and the possibility that it exists as a dimer in solution . Two low molecular mass c-type cytochromes were also characterized . One appears to be a high-potential (+310 mV) c8-type cytochrome . Amino acid sequencing suggests that the second cytochrome may be a homologue of the low-potential cytochrome c-551, previously described in two species of Ectothiorhodospirillaceae.

Nucleic Acids Res, 1996 Jun 15, 24(12), 2429 - 34
Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n microsatellite repeats; Hite JM et al.; The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors . An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n x (dG-dT)n microsatellites in the polymerase chain reaction (PCR) . These products differ from the primary product by multiples of 2 nucleotides . We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n x (dG-dT)n frameshifting during the PCR . Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification . However, 3'-->5' exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3' terminal nucleotides . Most interestingly, extension temperatures of 37 degrees C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3'-->5' exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products . This method can improve the resolution of heterozygous or mutant (dC-dA)n x (dG-dT)n alleles differing in size by one or two repeat units.

J Biol Chem, 1996 Jun 14, 271(24), 13987 - 92
The caa3 terminal oxidase of Bacillus stearothermophilus . Transient spectroscopy of electron transfer and ligand binding; Giuffre A et al.; The thermophilic bacterium Bacillus stearothermophilus possesses a caa3-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R . I . R., and Konings, W . N . (1989) Eur . J . Biochem . 178, 763-770) . We have carried out extensive kinetic experiments on the purified enzyme by stopped-flow time-resolved optical spectroscopy combined with singular value decomposition analysis . The results indicate a striking similarity of behavior between this enzyme and the electrostatic complex between mammalian cytochrome c and cytochrome c oxidase . CO binding to fully reduced caa3 occurs with a second order rate constant (k = 7.8 x 10(4)M-1 s-1) and an activation energy (E* = 6.1 kcal mol-1) similar to those reported for beef heart cytochrome c oxidase . Dithionite reduces cytochrome a with bimolecular kinetics, while cytochrome a3 (and CuB) is reduced via intramolecular electron transfer . When the fully reduced enzyme is mixed with O2, cytochrome a3, and cytochrome c are rapidly oxidized, whereas cytochrome a remains largely reduced in the first few milliseconds . When cyanide-bound caa3 is mixed with ascorbate plus TMPD, cytochrome c and cytochrome a are synchronously reduced; the value of the second order rate constant (k = 3 x 10(5) M-1 s-1 at 30 degrees C) suggests that cytochrome c is the electron entry site . Steady-state experiments indicate that cytochrome a has a redox potential higher than cytochrome c . The data from the reaction with O2 reveal a remarkable similarity in the kinetic, equilibrium, and optical properties of caa3 and the electrostatic complex cytochrome c/cytochrome c oxidase.

Gene, 1996 Jun 12, 172(1), 49 - 51
BstF5I, an unusual isoschizomer of FokI; Abdurashitov MA et al.; BstF5I, a new restriction endonuclease (ENase) from Bacillus stearothermophilus F5, has been discovered . This enzyme recognizes 5'-GGATG-3' and cleaves DNA, generating a 2-base 3'extension: 5'-GGATG NN{symbol: see text}-3' 3'-CCTAC{symbol: see text}NN-5' BstF5I is an isoschizomer of FokI and seems to be evolutionarily close to other nonpalindromic-recognizing ENases from thermophilic bacilli.

Biochemistry, 1996 Jun 11, 35(23), 7447 - 58
Identity of prokaryotic and eukaryotic tRNA(Asp) for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus; Becker HD et al.; The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements . It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS . This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems . Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs . It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions . Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T . thermophilus AspRS . In the homologous systems, the mutational effects are less pronounced . However, effects in E . coli and T . thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes . Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.

EMBO J, 1996 Jun 3, 15(11), 2858 - 69
Developmentally programmed DNA deletion in Tetrahymena thermophila by a transposition-like reaction pathway; Saveliev SV et al.; We provide a molecular description of key intermediates in the deletion of two internal eliminated sequences (IES elements), the M and R regions, during macronuclear development in Tetrahymena thermophila . Using a variety of PCR-based methods in vivo, double-strand breaks are detected that are generated by hydrolytic cleavage and correspond closely to the observed chromosomal junctions left behind in the macronuclei . The breaks exhibit a temporal and structural relationship to the deletion reaction that provides strong evidence that they are intermediates in the deletion pathway . Breaks in the individual strands are staggered by 4 bp, producing a four nucleotide 5' extension . Evidence is presented that breaks do not occur simultaneously at both ends . The results are most consistent with a deletion mechanism featuring initiation by double-strand cleavage at one end of the deleted element, followed by transesterification to generate the macronuclear junction on one DNA strand . An adenosine residue is found at all the nucleophilic 3' ends used in the postulated transesterification step . Evidence for the transesterification step is provided by detection of a 3' hydroxyl that would be liberated by such a step at a deletion boundary where no other DNA strand ends are detected.

EMBO J, 1996 Jun 3, 15(11), 2843 - 9
Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase; Saks ME et al.; Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69 . The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition . Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper . Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate . This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser) . The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein . The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.

EMBO J, 1996 Jun 3, 15(11), 2834 - 42
The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site; Cusack S et al.; The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution . The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits . The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem . In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs . This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA . Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition . These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.

Cell Biol Int, 1996 Jun, 20(6), 437 - 44
Insulin produces a biphasic response in Tetrahymena thermophila by stimulating cell survival and activating proliferation in two separate concentration intervals; Christensen ST et al.; Cells of Tetrahymena may produce autocrine signal molecules with effects on survival and proliferation . Here we have tested the effects of human recombinant and bovine insulin, and the B22-B30 fragment of bovine insulin over a wide range of concentrations (10(-5)-10(-18) M) on cell survival and proliferation in a synthetic nutrient medium . The cells were grown in conical flasks at low initial cell densities (40 and 400 cells/ml) . Insulin prevented rapid cell death and/or promoted cell proliferation over two separate concentration ranges: down to nanomolar levels and again in the low pico- and femtomolar range . At an initial population density of 400 cells/ml the cells multiplied at both concentration intervals . At 40 or fewer organisms/ml the cells multiplied in the high concentration interval, whereas in the low interval they survived for about four times longer than those in the control cultures . B22-B30 added to cultures of 40 initial cells/ml produced a stimulation of cell survival in the low pico- and high femtomolar range . In the presence of hemin (50 nM) cells at 400 initial organisms/ml multiplied at insulin concentrations down to about 3 nM and again from 300 am to 10 pM . In some cases, hemin plus insulin activated cell proliferation between the two concentration intervals as well . At 40 cells/ml the cells not only survived but proliferated in the femtomolar range . Cells in cultures supplemented with both hemin and B22-B30 multiplied at the low concentration interval (from about 100 fM to 10 pM).

Biol Chem Hoppe Seyler, 1996 Jun, 377(6), 343 - 56
Glycyl-tRNA synthetase; Freist W et al.; Glycyl-tRNA synthetase, a class II aminoacyl-tRNA synthetase, catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into proteins . In a side reaction the enzyme also synthesizes dinuceloside polyphosphates, which probably participate in regulation of cell functions . Glycine is the smallest amino acid occurring in natural proteins, probably established as a protein component very early in evolution . Besides the amino and the carboxyl groups there is no functional group in the molecule . Alanine, the amino acid which is structurally most similar to glycine, possesses an additional methyl group as 'side chain' . Glycyl-tRNA synthetase is one of the few synthetases which exhibit different oligomeric structures in different organisms (alpha 2 beta 2 and alpha 2) . The alpha 2 beta 2 enzymes exhibit similarities to PheRS (also an alpha 2 beta 2 enzyme) . The alpha 2 forms belong to the subclass IIa enzymes with regard to sequence homologies . In eukaryotes the polypeptide is weakly associated with multienzyme complexes consisting of aminoacyl-tRNA synthetases . In the aminoacylation reaction a 'half-of-the-sites' mechanism as found for GlyRS from Bombyx mori is probably used by all glycyl-tRNA synthetases under in vivo conditions . Essentially, tRNAGly is recognized by GlyRS through standard identity elements in the anticodon region and in the acceptor stem . The last three facts may indicate that GlyRS is an enzyme which still possesses properties of a primordial aminoacyl-tRNA synthetase . Nine genes of glycyl-tRNA synthetases from six organisms have been sequenced . They encode synthetase subunits of chain lengths ranging from 300-700 amino acids . One crystal structure, that of the alpha 2 enzyme from Thermus thermophilus, has also been determined . The two subunits each possess three domains: the active site resembling that of aspartyl and seryl enzymes, a C-terminal anticodon recognition domain, and one domain which almost certainly interacts with the acceptor stem of tRNAGly . Antibodies against glycyl-RNA synthetase occur in the sera of patients suffering from polymyositis and interstitial lung disease.

J Dairy Sci, 1996 Jun, 79(6), 943 - 55
Performance of commercial cultures in fluid milk applications; Sanders ME et al.; Six Lactobacillus acidophilus, 5 Bifidobacterium, and 6 Streptococcus thermophilus strains were studied for characteristics that are important to activity and stability in unfermented fluid milk products . Speciation, strain relatedness, frozen concentrate stability, bile sensitivity, and lactase activity were evaluated . The microbiological stability of a culture-containing fluid milk product was also determined . Two of the bifidobacteria cultures contained > 1 strain . Some strains were shown to be closely related or identical by pulsed-field gel electrophoresis of fragmented chromosomal DNA . Selective media that distinguished among all 3 added genera were identified . All lactobacilli and most of the bifidobacteria were resistant to bile concentrations varying from 1 to 3%, and all streptococci were sensitive to bile . Lactase activities were highest for S . thermophilus strains, supporting use of this species in fluid milk and dairy products to aid in the digestion of lactose by consumers . The experimental product evaluated in this study contained 10(7) cfu/ml of both L . Acidophilus and Bifidobacterium spp . and 5 x 10(7) cfu/ml of S . thermophilus . Lactic, but not psychrotrophic, populations were fairly stable during storage . The results suggest that milk formulated with high concentrations of three different genera of probiotic bacteria can be manufactured with commercial strains.

J Biochem (Tokyo), 1996 Jun, 119(6), 1076 - 9
Pyridine-promoted factor- and energy-free peptide synthesis systems prepared from various organisms including prokaryote, eukaryote, and mitochondria; Nojima T et al.; We demonstrate here that ribosomes from not only Escherichia coli and Thermus thermophilus {Nitta et al . (1994) J . Biochem . 115, 803-807; ibid., (1995) 118, 841-849} but also yeast and bovine mitochondria catalyze peptide synthesis promoted by a high concentration of pyridine in the absence of soluble protein factors and chemical energy sources, and compare some characteristic features of the reactions among these organisms . Sensitivities against antibiotics, chloramphenicol and cycloheximide, showed the same tendency to those in the in vitro aqueous translation systems of these organisms, suggesting that the basic mechanism for peptide synthesis is the same among these organisms . The optimal concentration of pyridine was centered at 50% for all systems, although the dependencies on the pyridine concentrations and the yields of the products were different from one another . All these systems required Mg2+, and only mitochondrial system showed the extra Mn(2+)-requirement, which enhanced the yield by several fold . The optimum reaction temperatures coincided closely with the growing temperatures of the organisms except for the mitochondrial system, which showed the highest activity above 80 degrees C . The rationale for these observations remains to be solved.

Appl Environ Microbiol, 1996 Jun, 62(6), 2191 - 4
Pyrimidine biosynthesis genes (pyrE and pyrF) of an extreme thermophile, Thermus thermophilus; Yamagishi A et al.; We have isolated uracil auxotrophic mutants of an extreme thermophile, Thermus thermophilus . A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced . The pyrE gene can be a bidirectional marker for the gene manipulation system of the thermophile.

Appl Environ Microbiol, 1996 Jun, 62(6), 2066 - 73
Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues; Watanabe K et al.; To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced . In spite of a big difference in their thermostabilities, B . coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%) . The number of prolines (19 for B . cereus oligo-1,6-glucosidase, 24 for B . coagulans enzyme, and 32 for B . thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases . Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency . Judging from proline residues occurring in B . coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K . Watanabe, T . Masuda, H . Ohashi, H . Mihara, and Y . Suzuki, Eur . J . Biochem . 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B . cereus oligo-1,6-glucosidase . With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory . The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues . It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.

Curr Opin Biotechnol, 1996 Jun, 7(3), 337 - 42
Biochemistry and genetics of microbial xylanases; Jeffries TW; Xylanases are classified into two major families (10 or F and 11 or G) of glycosyl hydrolases . Both use ion pair catalytic mechanisms and both retain anomeric configuration following hydrolysis . Family 10 xylanases are larger, more complex and produce smaller oligosaccharides; Family 11 xylanases are more specific for xylan . Alkaline-active and extreme-thermophilic enzymes are of particular interest . Such xylanases are being commercialized for bleaching pulps and other applications.

Protein Sci, 1996 Jun, 5(6), 1014 - 25
An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases; Salminen T et al.; The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase . Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions . Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule . Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar . The one striking difference, however, is in the oligomeric interactions . In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity . Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces . The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces . The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16% . The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization . Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

Genetics, 1996 Jun, 143(2), 811 - 21
Identification, mapping and linkage analysis of randomly amplified DNA polymorphisms in Tetrahymena thermophila; Brickner JH et al.; Using the random amplified polymorphic DNA (RAPD) technique and exploiting the unique genetics of Tetrahymena thermophila, we have identified and characterized 40 DNA polymorphisms occurring between two inbred strains (B and C3) of this ciliated protozoan . These RAPD markers permit the PCR amplification of a DNA species using template DNA from SB1969 (B strain) but fail to do so using DNA from C3-368-5 (C3 strain) . Polymorphisms were mapped to chromosomes using a panel of monosomic strains constructed by crossing B strain-derived nullisomic strains to inbred strain C3 . They map to all five chromosomes and appear to be evenly distributed throughout the genome . Chromosomal groups were then analyzed for linkage using meiotic segregants; four linkage groups were identified in chromosomes 1R 2L, 3 and 5 . The RAPD method appears useful for the construction of a genetic map of the Tetrahymena genome based on DNA polymorphisms.

Biochem J, 1996 Jun 1, 316 ( Pt 2), 615 - 22
Thermostable chaperonin from Clostridium thermocellum; Cross SJ et al.; Homologues of the chaperonins Cpn60 and Cpn10 have been purified from the Gram-positive cellulolytic thermophile Clostridium thermocellum . The Cpn60 protein was purified by ATP-affinity chromatography and the Cpn10 protein was purified by gel-filtration, ion-exchange and hydrophobic interaction chromatographies . The identities of the proteins were confirmed by N-terminal sequence analysis and antigenic cross-reactivity . The Cpn60 homologue is a weak, thermostable ATPase (t1/2 at 70 decrees C more than 90 min) with optimum activity (Kcat 0.07 S-1) between 60 degrees C and 70 degrees C . The ATPase activity of the authentic Cpn60 was inhibited by Escherichia coli GroES . The catalytic properties of a recombinant C . thermocellum Cpn60 purified from a GST-Cpn60 fusion protein expressed in E . coli {Ciruela (1995) Ph.D . Thesis, University of Kent} were identical with those of the authentic C . thermocellum Cpn60 . Gel-filtration studies show that at room temperature the Cpn60 migrates mainly as a heptamer . Electron microscopy confirms the presence of complexes showing 7-fold rotational symmetry and also reveals a small number of particles that seem to be tetradecamers with a similar structure to E . coli GroEL complexes.

J Bacteriol, 1996 Jun, 178(12), 3654 - 7
Differential domain accessibility to monoclonal antibodies in three different morphological assemblies built up by the S-layer protein of Thermus thermophilus HB8; Caston JR et al.; A collection of 27 monoclonal antibodies (MAbs) against the S-layer protein (P100) of Thermus thermophilus HB8 has been obtained . They have been classified according to their ability to recognize S-layer regions expressed in E . coli from plasmids containing different fragments of its coding gene, slpA . The accessibility of the binding sites in hexagonal, trigonal, or tetragonal assemblies of P100 was analyzed by enzyme-linked immunosorbent assays with six of these MAbs and their respective Fab fragments . When packed hexagonally as the native S-layer (S1 assemblies), only a small region located near the amino terminus of the P1OO was accessible . However, when P1OO was assembled into trigonal (pS2 assemblies) or tetragonal (S2 assemblies) arrays, most of the protein domains analyzed were easily detected, thus suggesting that P1OO is assembled in S2 and pS2 in a similar way and that these two arrangements are quite different from the S1 assembly . Relationships between accessibility and sequence predictions are discussed.

J Bacteriol, 1996 Jun, 178(11), 3396 - 8
Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea; Ruggero D et al.; Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics . It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.

J Bacteriol, 1996 Jun, 178(11), 3365 - 8
Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6; Yoon KS et al.; 2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography . The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa) . The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H . thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen) . NAD, NADP, and ferredoxins from Chlorella spp . and Clostridium pasteurianum were ineffective . The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h . The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8 . The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.

J Bacteriol, 1996 Jun, 178(11), 3270 - 4
Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila; Alber BE et al.; The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity . The enzyme purified from E . coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M . thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E . coli or M . thermophila . Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures . No esterase activity was detected with p-nitrophenylacetate as the substrate . Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit . As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer . Western blot (immunoblot) analysis of cell extract proteins from M . thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.

Mol Cell Biol, 1996 Jun, 16(6), 2878 - 87
Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae; Liu X et al.; H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins . Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae . However, no H2A.F/Z variant has been reported in this budding yeast species . We sought to distinguish among three explanations for these observations: (i) that S . cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S . cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule . Repeated attempts to clone an S . cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2 . To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1 . Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function . The Tetrahymena H2A genes confer a cold-sensitive phenotype . Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins . Proteins from S . cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies . These studies make it likely that S . cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog . A hypothesis is presented relating the absence of H2A.F/Z in S . cerevisiae to its function in other organisms.

Biochim Biophys Acta, 1996 May 31, 1301(1-2), 105 - 14
Thermoalkalophilic lipase of Bacillus thermocatenulatus . I . molecular cloning, nucleotide sequence, purification and some properties; Schmidt-Dannert C et al.; An expression library was generated by partial Sau3A digestion of genomic DNA from the thermophile Bacillus thermocatenulatus and cloning of DNA fragments in pUC18 in Escherichia coli DH5alpha . Screening for lipase activity identified a 4.5 kb insert in pUC18 which directed the production of lipase in E . coli DH5alpha . A subclone with a 2.2 kb insert was sequenced . The lipase gene codes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 43 kDa . As in other Bacillus lipases, an Ala replaces the first Gly in the conserved pentapeptide Gly-X-Ser-X-Gly found in most lipases . The region upstream of the lipase gene contains a Bacillus promoter which directs the expression of lipase in E . coli DH5alpha . The expressed lipase was isolated and purified 312-fold to homogeneity . N-terminal sequencing of the purified lipase revealed a correct cleavage of the preprotein in E . coli DH5alpha . Maximum activity was found at pH 8.0-9.0 with tributyrin and olive oil as substrates and at 60-70 degrees C with p-NPP and olive oil as substrates . The lipase showed high stability at pH 9.0-11.0 and towards various detergents and organic solvents.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5281 - 5
Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp . 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity; Southworth MW et al.; Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli . Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein . Of the five cloned DNA polymerases, the Thermococcus sp . 9 degrees N-7 DNA polymerase was chosen for biochemical characterization . Thermococcus sp . 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase . Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp . 9 degrees N-7 DNA polymerase . Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Gene, 1996 May 24, 171(1), 103 - 6
Cloning, sequencing and expression of the uvrA gene from an extremely thermophilic bacterium, Thermus thermophilus HB8; Yamamoto N et al.; One of the most important DNA repair systems is the nucleotide (nt) excision repair system . The uvr A gene, which plays an essential role in the prokaryotic excision repair system, was cloned from an extremely thermophilic eubacterium, Thermus thermophilus (Tt) HB8, and its nt sequence was determined . In the amino acid (aa) sequence of Tt UvrA, a characteristic duplicated structure, two nt-binding consensus sequences (Walker's A-type motif) and two zinc finger DNA-binding motifs were found . The aa sequence showed 73% homology with that of Escherichia coli (Ec) . These features suggest that Tt has the same excision repair system as Ec . Upon comparison of the Tt and Ec UvrA, some characteristic aa substitutions were found . The numbers of Arg and Pro residues were increased (from 66 to 81 and from 47 to 55, respectively), and the numbers of Asn and Met residues were decreased (from 33 to 18 and from 18 to 11, respectively) in Tt . The Tt uvr A gene was expressed in Ec under control of the lac promoter . Purified UvrA was stable up to 80 degrees C (at neutral pH) and at pH 2-11 (at 25 degrees C).

J Biol Chem, 1996 May 24, 271(21), 12457 - 62
Bacillus stearothermophilus qcr operon encoding rieske FeS protein, cytochrome b6, and a novel-type cytochrome c1 of quinol-cytochrome c reductase; Sone N et al.; The gcr of Bacillus stearothermophilus K1041 encoding three subunits of the quinol-cytochrome c oxidoreductase (cytochrome reductase, b6c1 complex) was cloned and sequenced . The gene (qcrA) for a Rieske FeS protein of 19,144 Da with 169 amino acid residues, and the gene (qcrC) for cytochrome c1 of 27,342 Da with 250 amino acid residues were found at adjacent upstream and downstream sides of the previously reported qcrB (petB) for cytochrome b6 of subunit 25,425 Da with 224 residues (Sone, N., Sawa, G., Sone, T., and Noguchi, S . (1995) J . Biol . Chem . 270, 10612-10617) . The three structural genes for thermophilic Bacillus cytochrome reductase form a transcriptional unit . In the deduced amino acid sequence for the FeS protein, the domain including four cysteines and two histidines binding the 2Fe-2S cluster was conserved . Its N-terminal part more closely resembled the cyanobacteria-plastid type than the proteobacteria-mitochondria type when their sequences were compared . The amino acid sequence of cytochrome c1 was not similar to either type; the thermophilic Bacillus cytochrome c1 is composed of an N-terminal part corresponding to subunit IV with three membrane-spanning segments, and a C-terminal part of cytochrome c reminiscent of cytochrome c-551 of thermophilic Bacillus . The subunit IV in the enzyme of cyanobacteria and plastids is the counterpart of C-terminal part of cytochrome b of proteobacteria and mitochondria . These characteristics indicate that Bacillus cytochrome b6c1 complex is unique.

Biochim Biophys Acta, 1996 May 23, 1294(2), 106 - 10
Cloning, characterization and functional analysis of groEL-like gene from thermophilic cyanobacterium Synechococcus vulcanus, which does not form an operon with groES; Furuki M et al.; A gene encoding 57 102 Da polypeptide homologous to groEL of Escherichia coli but accompanying no groES, has been cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus . The amount of the gene transcript increased several folds by heat shock . The gene was expressed as a minor component of two types of HSP60, and designated as groEL2 . Although expressed and induced well upon heat shock treatment in the E . coli, introduction of the cloned groEL2 gene of S . vulcanus into an E . coli groEL-less mutant did not result in the complementation of heat sensitivity.

Arch Microbiol, 1996 May 22, 165(5), 342 - 5
Thermocryptoxanthins: novel intermediates in the carotenoid biosynthetic pathway of Thermus thermophilus
Yokoyama A, Shizuri Y, Hoshino T, Sandmann G.
Various thermozeaxanthins are the end products of the carotenoid biosynthetic pathway of the thermophilic eubacterium Thermus thermophilus . These compounds are zeaxanthin glucoside esters . Carotenoid analysis and inhibitory studies led to the identification of most of the intermediates of the pathway: beta-carotene, beta-cryptoxanthin, zeaxanthin, and several new carotenoids . The intermediates, identified by various spectroscopic methods as beta-cryptoxanthin glucoside esters carrying fatty acid moieties of different chain lengths, were designated as thermocryptoxanthins . The use of the inhibitors diphenylamine and 2-(4-chlorophenylthio)-triethylamine-HCl resulted in the accumulation of the intermediates phytoene, lycopene, and gamma-carotene derivatives, which normally are present in amounts below the detection limit . The levels of non-esterified glycosides were extremely low . The results presented were used to establish the complete carotenoid biosynthetic pathway of T . thermophilus.

FEBS Lett, 1996 May 20, 386(2-3), 260 - 2
5S rRNA binding ribosomal proteins from Thermus thermophilus: identification and some structural properties; Gongadze G et al.; An unusual acidic ribosomal protein from Thermus thermophilus, TL5, that binds to 5S rRNA specifically and strongly, has been investigated . The N-terminal sequence of TL5 does not reveal any homology with known ribosomal proteins . Two large tryptic fragments of TL5 have been isolated and characterized . 5S rRNA protected TL5 and its unstable N-terminal fragment against trypsin action . The 5S rRNA binding ability of TL5 is probably inherent in its N-terminal part . The other 5S rRNA binding ribosomal protein from T . thermophilus, TL4, has been identified as a homolog of the ribosomal protein L5 from Escherichia coli.

FEBS Lett, 1996 May 20, 386(2-3), 215 - 8
The cloning, expression and crystallisation of a thermostable arginase; Bewley MC et al.; The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox' has been cloned and sequenced . Expression of recombinant arginase at high levels has been achieved in E . coli using an inducible T7 RNA polymerase-based system . A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture . The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme . The recombinant protein has been crystallised and one crystal form is isomorphous to crystals of the native protein.

EMBO J, 1996 May 15, 15(10), 2323 - 30
A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila; Kisker C et al.; A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized . Here we present the structure of this enzyme, which adopts a left-handed parallel beta-helix fold . This fold is of particular interest since it contains only left-handed crossover connections between the parallel beta-strands, which so far have been observed very infrequently . The active form of the enzyme is a trimer with three zinc-containing active sites, each located at the interface between two monomers . While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction . Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life.

Nucleic Acids Res, 1996 May 15, 24(10), 1943 - 9
Programmed DNA rearrangement from an intron during nuclear development in Tetrahymena thermophila: molecular analysis and identification of potential cis-acting sequences; Li J et al.; During macronuclear development in the ciliate Tetrahymena thermophila, extensive rearrangement events occur as DNA deletions . We have studied a developmentally programmed deletion called mse2.9 that occurs within an intron in a gene in both genomic DNA and in an rDNA vector introduced into the cell by transformation . Extensive microheterogeneity at the deletion junctions has been found in caryonidal strains and in the rDNA in transformed cells . A transformation assay has been used to identify sequences required for proper processing of mse2.9 . Models to explain deletion site selection as well as microheterogeneity at junction sites are presented.

FEMS Microbiol Lett, 1996 May 15, 139(1), 27 - 35
A modular xylanase from mesophilic Cellulomonas fimi contains the same cellulose-binding and thermostabilizing domains as xylanases from thermophilic bacteria; Clarke JH et al.; The xynC gene from mesophilic Cellulomonas fimi encodes a large 125 kDa modular xylanase (XYLC), consisting of six distinct functional domains . In addition to a single Family 10 catalytic domain, XYLC contains a domain homologous with the nodulation protein, NodB, from nitrogen-fixing bacteria and thermostabilizing and cellulose-binding domains found previously only in xylanases from thermophilic bacteria.

Biochem J, 1996 May 15, 316 ( Pt 1), 115 - 22
Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme; Burdette DS et al.; The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Escherichia coli . The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits . The purified recombinant enzyme is optimally active above 90 degrees C with a half-life of approx . 1.7 h at 90 degrees C . An NADP(H)-dependent enzyme, the recombinant S-ADH has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation . The enzyme was inactivated by chemical modification with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis . Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, implicating the involvement of bound zinc in catalysis . Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 degrees C when the enzymes were incubated at 55 degrees C before assay . Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 degrees C . The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, catalytically significant S-ADH structural change . Hydrophobic cluster analysis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed . These comparisons predicted that specific proline residues might contribute to S-ADH thermostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S-ADHs (Cys, His, and Asp).

Biochemistry, 1996 May 14, 35(19), 6150 - 6
Purification and reconstitution of the glutamate carrier GltT of the thermophilic bacterium Bacillus stearothermophilus; Gaillard I et al.; An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus . The fusion protein was expressed in Escherichia coli and shown to transport glutamate . The highest levels of expression were observed in E . coli strain DH5 alpha grown on rich medium . The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100 . Purified GltT was reconstituted in an active state in liposomes prepared from E . coli phospholipids . The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads . Active reconstitution was realized with a wide range of Triton X100 concentrations . Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state . In B . stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions . In membrane vesicles derived from E . coli cells expressing GltT, the Na+ ion dependency seems to be lost {Tolner, B., Ubbink-Kok, T., Poolman, B., & Konings, W . N . (1995) Mol . Microbiol . 18, 123-133}, suggesting a role for the lipid environment in the cation specificity . In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E . coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.

Ned Tijdschr Geneeskd, 1996 May 11, 140(19), 1022 - 5
{Fungi and yeasts isolated in mycological studies in skin and nail infections in The Netherlands, 1992-1993}; Kuijpers AF et al.; OBJECTIVE . To describe fungi and yeasts isolated from skin and nail infections in the Netherlands . DESIGN: Retrospective . SETTING . Centraalbureau voor Schimmelcultures (CBS), Baarn, the Netherlands . METHOD . Results of mycological investigation of skin and nail samples in the period 1992-1993 were analysed . After a clinical diagnosis of mycosis, performed by dermatologists and general practitioners, material was sent to the CBS for mycological research . RESULTS . The clinical diagnosis of onychomycosis was rather accurate, especially if made performed by dermatologists . Mycoses of the skin were sometimes confused with other skin diseases . When microscopical observation showed a positive result, 93% of the cultures were positive as well . The main agent of onychomycosis was Trichophyton rubrum; T . mentagrophytes was more frequently isolated from tinea manuum/pedis and T . tonsurans from tinea corporis/cruris . Epidermophyton floccosum was only isolated from skin lesions and Microsporum canis, T . soudanense and T . verrucosum only from tinea corporis/cruris . The most important yeasts isolated were Trichosporon mucoides, Candida guilliermondii, C . parapsilosis, C . famata and Malassezia furfur . Other fungi isolated were either pigmented (melanin, carotene), thermophilic or belonged to the order of the Onygenales . CONCLUSION . Mycological research to confirm the clinical diagnosis of a skin mycosis is advisable . Species isolated differed in their predilection for different parts of the human body . Yeasts were mainly isolated as double infections . Apart from the dermatophytes there is a special group of fungi which can cause mycoses.

Immunol Invest, 1996 May, 25(3), 177 - 83
Immunological detection of lipopolysaccharide antigens of thermophilic campylobacters captured on polymyxin-coated polyester cloth; Gomi K et al.; Cholate-extracted lipopolysaccharide (LPS) antigens from thermophilic campylobacters were captured on polymyxin-coated polyester cloth . The captured antigens were detected by sequential reactions with rabbit anti-Campylobacter antibody, anti-rabbit IgG peroxidase conjugate and chromogenic peroxidase substrate . A polyclonal rabbit antibody elicited against a single Campylobacter-jejuni strain detected the reference strains of the twenty most frequently isolated thermophilic campylobacters in the Lior serotyping scheme . Moreover, LPS antigens of six C . Jejuni Penner serotypes fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting were recognized by four antisera prepared against homologous and heterologous Penner serotypes . The results suggest the potential application of polymyxin-cloth enzyme immunoassay for rapid detection of thermophilic campylobacters where monoclonal antibodies can be raised to possible common LPS epitopes.

J Dairy Res, 1996 May, 63(2), 245 - 56
Detection and localization of peptidases in Lactococcus lactis with monoclonal antibodies; Laan H et al.; Monoclonal antibodies against peptidases of Lactococcus lactis were isolated and characterized: PEPN1-4 against a lysyl aminopeptidase PepN, PEPT1-5 against a tripeptidase PepT and PEPD1-3 against a dipeptidase PepD . These monoclonal antibodies reacted specifically with their respective antigens in crude cell extracts of Lc . lactis subspp . cremoris and lactis . A number of monoclonal antibodies cross reacted with proteins of other (lactic acid) bacteria . PEPT1, 2, 4 and 5 cross reacted weakly with a 35 kDa protein in Lactobacillus delbrueckii, while PEPT1 and PEPT2 reacted with proteins in the cell-free extract of Streptococcus thermophilus and Clostridium fervidus . Of the four isolated monoclonal antibodies against PEPN, only PEPN3 cross reacted weakly with a 90 kDa protein in Escherichia coli cell-free extract, and the other three antibody species against PEPN3 cross reacted with 80 kDa proteins of Lb . casei, Lb . delbrueckii, and Str . bovis, but not of Esch . coli . Of the three monoclonal antibodies against PepD, only PEPD1 and PEPD2 cross reacted with 40 kDa proteins of Lb . casei, Lb . delbrueckii and Str . bovis . All PEPN, PEPD and PEPT antibodies reacted with components in cell-free extracts of eleven different Lc . lactis strains, indicating that the peptidases of these strains were very similar to those of Lc . lactis subsp . cremoris WG2 . However, Lc . lactis subsp . hordniae appeared to differ from the other Lc . lactis subspecies since only PEPT1, 2 and 5 reacted with a protein in the cell-free extract . Immunogold labelling of Lc . lactis WG2 with the isolated monoclonal antibodies revealed that PepN, PepD and PepT were located intracellularly . The intracellular location of these peptidases is discussed in relation to the supply of essential amino acids and peptides.

J Biochem (Tokyo), 1996 May, 119(5), 1014 - 8
Conversion of the coenzyme specificity of isocitrate dehydrogenase by module replacement; Yaoi T et al.; The coenzyme specificity of isocitrate dehydrogenase from an extreme thermophilic bacterium was converted from NADP-dependent to NAD-dependent by replacing a "module" involved in the coenzyme binding site . The conversion was not possible with the replacement of a few residues that interact with the coenzyme . In addition, the module-replaced mutant dehydrogenase was as stable as the original, wild type enzyme . The results support a previous hypothesis that a module is a structural and functional unit of a protein.

Protein Eng, 1996 May, 9(5), 439 - 45
Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis; Frigerio F et al.; Using genetically engineered mutants of the neutral protease from Bacillus stearothermophilus (BsteNP), it had been shown that the surface-exposed structural motif constituted by Phe63 embedded in a four amino acid hydrophobic pocket is critical for the thermal stability of the thermophilic neutral proteases from Bacilli . To measure the stabilizing contribution of each hydrophobic interaction taking place between Phe63 and the hydrophobic pocket, we grafted this structural motif in the neutral protease from the mesophile Bacillus subtilis (BsubNP) . This was accomplished by first creating the Thr63-->Phe mutant of BsubNP and then generating a series of mutants in which the four amino acids which in thermolysin surround Phe63 and form the hydrophobic pocket were added one after the other . By analysing the thermal stability of each mutant it was found that the 2 degrees C destabilizing effect of the Thr63-->Phe substitution was completely suppressed by the addition of the four amino acid hydrophobic pocket, each replacement providing a stabilizing contribution of approximately 0.8-1 degrees C . These results are discussed in the light of the peculiar mechanism of thermal inactivation of proteolytic enzymes.

J Clin Microbiol, 1996 May, 34(5), 1216 - 9
Restriction fragment length polymorphism of flagellin genes of Campylobacter jejuni and/or C . coli isolates from Egypt; Mohran ZS et al.; The conservation of flagellin genes from thermophilic Campylobacter spp . strains isolated in Egypt was evaluated by a restriction fragment length polymorphism (RFLP) assay . The flaA and flaB genes were amplified from 59 independent clinical isolates and digested with EcoRI and PstI, and the resulting patterns were compared with each other and with previously described RFLP groups . The results indicate that the isolates fell into 14 groups for flaA and 11 groups for flaB, 9 of which have been described, and that considerable genetic variability exists among isolates belonging to the same LIO serogroup . In most cases, the flaB gene displayed the same RFLP pattern as that of the flaA gene of the same strain, although some variability was observed . The data suggest that more variability of flagellin genes exists within the LIO serogroups common to Campylobacter field isolates from Egypt than has previously been reported for North American isolates.

Biosci Biotechnol Biochem, 1996 May, 60(5), 840 - 6
Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp . E79 and the DNA sequence of the encoding gene; Lee JK et al.; A thermophilic Thermoactinomyces sp . E79 producing a highly thermostable alkaline protease was isolated from soil . The protease, produced extracellularly by Thermoactinomyces sp . E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography . The relative molecular mass was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis . Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease . The optimum temperature for the enzyme activity was 85 degrees C, and about 50% of the original activity remained after incubation at 90 degrees C for 10 min in the presence of Ca2+ . The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0 . The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment . Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a polypeptide- of 384 amino acids . The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids . The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.

Z Naturforsch {C}, 1996 May-Jun, 51(5-6), 319 - 28
Comparative immunological detection of lipids and carotenoids on peptides of photosystem I from higher plants and cyanobacteria; Makewicz A et al.; Photosystem I preparations were obtained from wild-type tobacco Nicotiana tabacum var . JWB, three chlorophyll-deficient tobacco mutants: Su/su, Su/su var . Aurea and yellow-green leaf patches of the variegated mutant NC 95, Spinacia oleracea and furthermore from the mesophilic cyanobacterium Synechococcus PCC 7942 and the thermophilic cyanobacterium Synechococcus sp. . Peptides from these preparations were analyzed by SDS polyacrylamide gel electrophoresis and transferred for detection of bound lipids and carotenoids according to the Western blot procedure to nitrocellulose membranes . The PS I preparations from the Nicotiana tabacum species and spinach consist of the core complex and the LHCP I complex, the latter containing, however, traces of the LHCP II polypeptides . The core complex consists of the two core peptides with the apparent molecular mass of 66 kDa each and peptides with molecular masses of 22, 20, 19, 17, 16, 10 and 9 kDa . The LHCP I complex contains 4 subunits with molecular masses of 28, 26, 25 and 24 kDa . The PS I preparations of the two mutants Su/su and Su/su var . Aurea contain as impurities traces of the core peptides (D1/D2) and the two chlorophyll-binding peptides (CP43/CP47) of photosystem II . The PS I preparation from the mesophilic and thermophilic cyanobacterium consists of the two core peptides with the apparent molecular mass of 66 kDa and peptides with molecular masses of 16, 14 and 10 kDa . The peptides of the PS I preparations were characterized by specific PS I, CP I and LHCP I antisera . The antiserum to the PS I complex reacts in the Western blot with the homologous peptides of PS I from higher plants, but only with the CP I complex from the two cyanobacteria . In comparative studies with PS II from higher plants the PS I antiserum reacts with the LHCP II complex as expected . The antiserum to the CP I complex reacts only with the 66 kDa peptides of PS I from all objects . There is no cross reaction with the 66 kDa peptides (heterodimer of the D1/D2 peptides) of PS II . The antiserum to the LHCP I complex reacts only with the four LHCP I peptides of PS I from higher plants and as expected with the LHCP II of PS II: Because cyanobacteria do not have LHCP complexes, there is no reaction with the LHCP I antiserum . By means of polyclonal monospecific antisera to lipids it was shown by Western blot procedure that only two lipid species are bound to PS I peptides . The galactolipid monogalactosyldiglyceride is bound to the CP I complex of the Nicotiana tabacum species, spinach and the two cyanobacteria as well as to the LHCP I complex of the higher plants . The phospholipid phosphatidylglycerol is only associated with the CP I complex of the analyzed higher plants and cyanobacteria . With polyclonal monospecific antisera to carotenoids it was demonstrated that beta-carotene, lutein, neoxanthin and zeaxanthin are associated with the CP I complex of the higher plants and the cyanobacteria analyzed . Violaxanthin is also bound to the CP I complex of the two cyanobacteria, whereas it is bound together with neoxanthin to the LHCP I complex of the higher plants.

J Eukaryot Microbiol, 1996 May-Jun, 43(3), 198 - 202
An improved method to obtain high molecular weight DNA from purified micro- and macronuclei of Tetrahymena thermophila; Chau MF et al.; An improved method to obtain high molecular weight DNA from purified macro- and micronuclei of Tetrahymena thermophila is described . Micro- and macronuclear DNA obtained using previously described protocols was degraded and not suitable for the cloning of large (>100 kb) DNA fragments . Based on the data reported here, we propose that DNA degradation is mainly due to nuclease activity; some micronuclear DNA degradation is due to mechanical shearing as a result of extended periods of blending . We have made modifications to reduce nuclease degradation by minimizing cell lysis, by the early addition of EDTA and by increasing the EDTA concentration (23 mM) . To reduce mechanical shearing, cell and nuclear suspensions were blended for shorter periods . High molecular weight micro- and macronuclear DNA was obtained using the new protocol.

FEMS Microbiol Rev, 1996 May, 18(2-3), 225 - 36
Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea; Zillig W et al.; We review and update the work on genetic elements, e.g., viruses and plasmids (exluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory . The viruses of Crenarchaeota represent four novel virus families . The Fuselloviridae represented by SSVI of S . shibatae and relatives in other Sulfolobus strains have the form of a tailed spindle . The envelope is highly hydrophobic . The DNA is double-stranded and circular . Members of this group have also been found in Methanococcus and Haloarcula . The Lipothrivciridae (e.g., T TV1 to 3) have the form of flexible filaments . They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane . The "Bacilloviridae" (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins . Both virus types carry on both ends structures involved in the attachment to receptors . Both types are represented in Thermoproteus and Sulfolobus . The droplet-formed novel Sulfolobus virus SNDV represents the "Guttaviridae" containing circular double-stranded DNA . Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated . SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states . The hosts of all but TTV1 survive virus production . We discuss the implications of the nature of these viruses for understanding virus evolution . The plasmids found so far range in size from 4.5 kb to about 40 kb . Most of them occur in high copy number, probably due to the way of their detection . Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction . The plasmid pTIK4 appears to encode a killer function . pNOB8 has been used as a vector for the transfer of the lac S (beta-galactosidase) gene into a mutant of S . solfataricus.

FEMS Microbiol Rev, 1996 May, 18(2-3), 215 - 24
Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: strategies of protein stabilization; Jaenicke R; The molecular origin of protein stability has been the subject of active research for more than a generation (R . Jaenicke (1991) Eur . J . Biochem . 202, 715-728) . Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential . In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima, it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds (delta delta Gstab approximately equal to 50 kJ/mol) . As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology . In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents.

Appl Environ Microbiol, 1996 May, 62(5), 1723 - 7
Isolation of Thermus strains from hot composts (60 to 80 degrees C); Beffa T et al.; High numbers (10(7) to 10(10) cells per g {dry weight}) of heterotrophic, gram-negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82 degrees C . These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windows, boxes with automated turning and aeration, and closed bioreactors with aeration) . Isolates grew fast on a rich complex medium at temperatures between 40 and 80 degrees C, with optimum growth between 65 and 75 degrees C . Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8 . These newly isolated T . thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem . Heterotrophic, ovalspore-forming, thermophilic bacilli were also isolated from hot composts, but none of the isolates was able to grow at temperatures above 70 degrees C . This is the first report of hot composts as habitats for a high number of thermophilic bacteria related to the genus Thermus . Our study suggests that Thermus strains play an important role in organic-matter degradation during the thermogenic phase (65 to 80 degrees C) of the composting process.

Appl Environ Microbiol, 1996 May, 62(5), 1574 - 82
The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators; Vaughan EE et al.; The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants . Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%) . This similarity was present both in the amino-terminal hydrophobic carrier domain, which is homologous to the E . coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain . The flanking regions of DNA surrounding lacS were also sequenced . Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95-amino-acid protein with a sequence similar to the amino-terminal portion of beta-galactosidase I from Bacillus stearothermophilus . The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria . The lacS gene was cloned in an E . coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S . thermophilus and in a pNZ63-cured strain, L . lactis NZ6091 . The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L . lactis or S . thermophilus plasmids harboring an intact lacS gene . Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L . lactis.

J Bacteriol, 1996 May, 178(9), 2695 - 700
Cloning, sequencing, and expression of ruvB and characterization of RuvB proteins from two distantly related thermophilic eubacteria; Tong J et al.; The ruvB genes of the highly divergent thermophilic eubacteria Thermus thermophilus and Thermotoga maritima were cloned, sequenced, and expressed in Escherichia coli . Both thermostable RuvB proteins were purified to homogeneity . Like E . coli RuvB protein, both purified thermostable RuvB proteins showed strong double-stranded DNA-dependent ATPase activity at their temperature optima (> or = 70 degrees C) . In the absence of ATP, T . thermophilus RuvB protein bound to linear double-stranded DNA with a preference for the ends . Addition of ATP or gamma-S-ATP destabilized the T . thermophilus RuvB-DNA complexes . Both thermostable RuvB proteins displayed helicase activity on supercoiled DNA . Expression of thermostable T . thermophilus RuvB protein in the E . coli ruvB recG mutant strain N3395 partially complemented the UV-sensitive phenotype, suggesting that T . thermophilus RuvB protein has a function similar to that of E . coli RuvB in vivo.

Virology, 1996 May 1, 219(1), 96 - 104
Site-specific spontaneous deletions in three genome regions of a temperate Streptococcus thermophilus phage; Bruttin A et al.; Site-specific spontaneous deletions were observed with high frequency in three regions of the genome of the temperate Streptococcus thermophilus phage phi SFi21 . Deletion sizes were 750 bp (type 1), 2.7 kb (type 2), and 1 kb (type 3) . Combinations of types 1 and 3 and 2 and 3 were observed . The mutants grew lytically although with reduced burst sizes . Type 2 mutants lost the capacity to lysogenize host cells . Upon serial passage, the deletion mutants overgrew the wild-type phage . No direct or inverted DNA repeats were associated with type 1 or 2 deletion sites . Several independent phage isolates showed deletions at identical nucleotide positions, suggesting a site-specific recombination system . Sequencing of an Xbal restriction fragment covering the type 1 deletion predicted a single long open reading frame (ORF) showing a high degree of amino acid similarity with two proteins from bacteriophage P1 implicated in its immunity control (KiIA, Ant) . Type 1 deletion leads to a loss of the conserved C-terminal part of this ORF.

DNA Res, 1996 Apr 30, 3(2), 87 - 92
Application of N-terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) to DNA sequencing and polymerase chain reactions: comparative study of delta Tth and wild-type Tth polymerases; Arakawa T et al.; N-Terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) lacking 5'-3' exonuclease activity was used for DNA sequencing and polymerase chain reaction (PCR) . In contrast to the high background of the sequencing ladder observed with the wild-type Tth polymerase, delta Tth polymerase gave readable sequencing patterns which extend up to more than 500 bases from the primer site on cycle sequencing and automated sequencing . The delta Tth polymerase was used for the standard and mutagenic PCR, and net amplification of the DNA and the mutations accumulated during PCR were analyzed . Under mutagenic PCR, the mutation rates were 7.0 x 10(-4) (Tth) and 8.3 x 10(-4) (delta Tth) per nucleotide per cycle of amplification, which were 4-9 times higher than the rates under standard PCR.

Biochim Biophys Acta, 1996 Apr 26, 1280(2), 207 - 16
A voltage-dependent chloride channel from Tetrahymena ciliary membrane incorporated into planar lipid bilayers; Fujiwara-Hirashima C et al.; Membrane vesicles from cilia of Tetrahymena thermophila were incorporated into a planar phospholipid bilayer membrane, and single-channel currents across the planar membrane were recorded under voltage-clamp conditions . A novel and reproducible chloride channel was observed when a mixture of phosphatidylethanolamine and phosphatidylcholine was used to form the planar lipid membrane but not when acidic phospholipid mixtures such as asolectin or a mixture containing phosphatidylserine . Using symmetrical 100 mM KCl solutions, the single-channel conductance of the fully open state (O1) was 73.1 pS, with sub-level (O2) conductance of 9.0 pS . The permeability ratio Pc1/Pk was calculated as 3.7, according to the Goldman-Hodgkin-Katz current equation . This channel exhibited characteristic voltage-dependent burst activities . With an increase in membrane potential, the lifetimes of both the burst and interburst states decreased . In the burst state, the frequency of transition between the O1 and O2 states was also voltage-dependent, mainly due to the decrease in the lifetime of the O1 state, with an increase in membrane potential . In addition, channel activity was inhibited by indanyloxyacetic acid-94 (IAA-94), an inhibitor of epithelial chloride channels.

J Biol Chem, 1996 Apr 19, 271(16), 9612 - 8
ATPase activity of UvrB protein form Thermus thermophilus HB8 and its interaction with DNA; Kato R et al.; Many living organisms remove wide range of DNA lesions from their genomes by the nucleotide excision repair system . The uvrB gene, which plays an essential role in the prokaryotic excision repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8 . Its nucleotide sequence was determined, and the deduced amino acid sequence showed it possessed a helicase motif, including a nucleotide-binding consensus sequence (Walker's A-type motif), which was also conserved in other UvrB proteins . The prokaryotic UvrB proteins and eukaryotic DNA repair helicases (Rad3 and XP-D) were classified into different groups by molecular phylogenetic analysis . The T . thermophilus uvrB gene product was overproduced in Escherichia coli and purified to apparent homogeneity . The purified T . thermophilus UvrB protein was stable up to 80 degrees C at neutral pH . T . thermophilus UvrB protein showed ATPase activity at its physiological temperature, whereas the E . coli UvrB protein alone has not been shown to exhibit detectable ATPase activity . The values of K(m) and k(cat) for the ATPase activity were 4.2 mM and 0.32 s-1 without DNA, and 4.0 mM and 0.46 s-1 with single-stranded DNA, respectively . This suggests that T . thermophilus UvrB protein could interact with single-stranded DNA in the absence of UvrA protein.

Biochemistry, 1996 Apr 16, 35(15), 4678 - 88
A synthetic model for triple-helical domains in self-splicing group I introns studied by ultraviolet and circular dichroism spectroscopy; Sarkar M et al.; Structural studies were performed on synthetic oligonucleotides with sequences corresponding to the P4/P6 and J3/4, J6/7 regions of the self-splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena thermophila intron . A 23-mer RNA was synthesized as a mixed ribo-deoxyribo oligonucleotide, modeling an expected base-paired region of P4 along with the J3/4 and P6 (5'-end bases of P6) regions . strand modeling the 3'-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA) . The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy . The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region . Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg2+ (>5mM) but not in presence of a monovalent cation like Na+ (up to 500 mM) . Studies on selected variants of both 7-mer and 23-mer RNAs were carried out . The results show that for the association of the two partner strands not only the formation of P6 helix but also triplet interactions between two strands are required . The association of the two strands in general follow a pattern predicted by comparative sequence analysis . Parallel studies on pure oligoribonucleotides having base sequence corresponding to those of the oligoribonucleotides showed no evidence of association under similar conditions, which could indicate that the 2'-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples . Molecular modeling studies on the proposed "plaited triple helix" formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2724 - 8
Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops; Tombline G et al.; In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension . Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products . The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase . Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 123 - 8
Cell survival and multiplication . The overriding need for signals: from unicellular to multicellular systems; Rasmussen L et al.; There are clear similarities in the control mechanisms for cell survival and multiplication in the two eukaryotes, the ciliate Tetrahymena thermophila and the yeast, Saccharomyces cerevisiae . Cell multiplication in both organisms is activated by the same compounds (phorbol esters, diacylglycerol, tetrapyrroles, etc.) . These compounds also affect cell multiplication and other activities in mammalian cell systems . This homology in control mechanisms in two distinct groups of unicellular eukaryotes on the one hand, and in cells from multicellular animals on the other, leads us to propose that these cytoplasmic control mechanisms for cell survival and multiplication originated in the unicellular eukaryotes.

Microbiology, 1996 Apr, 142 ( Pt 4), 829 - 36
The {Ni-Fe} hydrogenase from the thermophilic bacterium Acetomicrobium flavidum; Mura GM et al.; Biochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an alpha 2 beta 2 tetramer, with the alpha and beta subunits having a molecular mass of 50 kDa and 25 kDa, respectively . The most important biochemical properties of the enzyme are a specific activity of 77 mumol min-1 (mg protein)-1, a Km for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T50 of about 70 degrees C . In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air . By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced . When compared to the other hydrogenases so far characterized, the A . flavidum hydrogenase appears to be a typical {Ni-Fe} enzyme . The hydrogenase was expressed in Escherichia coli at high levels in a soluble form but it was not active . The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.

Microbiology, 1996 Apr, 142 ( Pt 4), 775 - 83
Characteristics of Sulfobacillus acidophilus sp . nov . and other moderately thermophilic mineral-sulphide-oxidizing bacteria; Norris PR et al.; Several isolates of Gram-positive, acidophilic, moderately thermophilic, ferrous-iron- and mineral-sulphide-oxidizing bacteria were examined to establish unequivocally the characteristics of Sulfobacillus-like bacteria . Two species were evident: Sulfobacillus thermosulfidooxidans with 48-50 mol% G+C and Sulfobacillus acidophilus sp . nov . with 55-57 mol% G+C . Both species grew autotrophically and mixotrophically on ferrous iron, on elemental sulphur in the presence of yeast extract, and heterotrophically on yeast extract . Autotrophic growth on sulphur was consistently obtained only with S . acidophilus.

Int J Syst Bacteriol, 1996 Apr, 46(2), 422 - 8
Thermothrix azorensis sp . nov., an obligately chemolithoautotrophic, sulfur-oxidizing, thermophilic bacterium; Odintsova EV et al.; A new aerobic, obligately chemolithoautotrophic, thermophilic, sulfur-oxidizing bacterium, Thermothrix azorensis, was isolated from a hot spring on Sao Miguel Island in the Azores . The cells of this organism are gram negative, nonsporulating, and rod shaped . Filament formation appears to occur as a response to nonoptimal growth conditions . Growth occurs at 63 to 86 degrees C, and the optimum temperature is 76 to 78 degrees C . The optimum pH range for growth is 7.0 to 7.5 . The G+C content of the DNA of our isolate is 39.7 mol% . This isolate uses thiosulfate, tetrathionate, hydrogen sulfide, and elemental sulfur as energy sources . Of particular interest are the absence of Calvin cycle enzymes and the initial appearance of sulfide during the lag phase of growth of aerobic cultures grown on elemental sulfur . The subsequent formation of thiosulfate is followed by oxidation of the thiosulfate to sulfate . T . azorensis differs from the only other Thermothrix species that has been described, Thermothrix thiopara, by having higher optimum and maximum growth temperatures, by being an obligate chemolithoautotroph, and by its close but separate position on a 16S rRNA sequence-based phylogenetic tree . Our T . azorensis isolate has been deposited in the American Type Culture Collection as strain ATCC 51754T (T = type strain).

Int J Syst Bacteriol, 1996 Apr, 46(2), 403 - 8
Thermus oshimai sp . nov., isolated from hot springs in Portugal, Iceland, and the Azores, and comment on the concept of a limited geographical distribution of Thermus species; Williams RA et al.; We examined aerobic, thermophilic, gram-negative bacteria that were isolated from hot springs in Portugal and were identified as Thermus strains and placed in phenetic groups on the basis of their phenotypic characteristics . We determined the composition of the peptidoglycan, identified the respiratory quinones, and determined the mean base composition of the DNA, and the levels of DNA-DNA homology were determined by both the filter hybridization and reassociation rate methods . Thermus aquaticus, Thermus brockianus, and Thermus filiformis were not detected in this collection of organisms, although three Thermus thermophilus strains were identified . We propose that the isolates that belonged to phenetic clusters E and F are members of a new species, Thermus oshimai; the type strain of T . oshimai is strain SPS17.

FEBS Lett, 1996 Apr 1, 383(3), 277 - 83
The Tetrahymena chaperonin subunit CCT eta gene is coexpressed with CCT gamma gene during cilia biogenesis and cell sexual reproduction; Cyrne L et al.; We report here the cloning and the characterization of the T . pyriformis CCT eta gene (TpCCT eta) and also a partial sequence of the corresponding T . thermophila gene (TtCCT eta) . The TpCCt eta gene encodes a protein sharing a 60.3% identity with the mouse CCT eta . We have studied the expression of these genes in Tetrahymena exponentially growing cells, cells regenerating their cilia for different periods and during different stages of the cell sexual reproduction . These genes have similar patterns of expression to those of the previously identified TpCCt gamma gene . Indeed, the Tetrahymena CCT eta and CCT gamma genes are up-regulated at 60-120 min of cilia recovery, and in conjugation when vegetative growth was resumed and cell division took place . Our results seem to indicate that both CCT subunits play an important role in the biogenesis of the newly synthesized cilia of Tetrahymena and during its cell division.

J Biochem Biophys Methods, 1996 Apr, 32(1), 1 - 6
Rapid purification of two thermophilic proteinases using dye-ligand chromatography; Cowan DA et al.; Dye-ligand chromatography has been used successfully for the purification of extracellular thermostable proteinases from thermophilic Bacillus and Thermus cultures . Single-step purification factors of up to 115-fold (for Thermus protease) and 2195-fold (for Bacillus protease) were obtained . Elution studies suggested that the mode of binding involved the enzyme active sites . The method was readily scalable to 600 1 volume.

Antonie Van Leeuwenhoek, 1996 Apr, 69(3), 235 - 41
Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetraspora flexuosa SIIX; Berens S et al.; Two endoxylanases were isolated from the xylanolytic enzyme system of the thermophilic actinomycete Microtetraspora flexuosa SIIX, and purified by ammonium sulfate fractionation, DEAE-Sepharose chromatography, gel filtration on Sephacryl S 200 and fast protein liquid chromatography on Q-Sepharose . The molecular masses of xylanase I and II were 26.3 and 16.8 kDa, and isoelectric points were 8.4 and 9.45, respectively . Optimal enzyme activities were obtained at 80 degrees C and pH 6.0 . The thermostability of both xylanases was greatly diminished during purification but could be restored by preincubation of the purified enzymes in the presence of xylan . The half-lives at 80 degrees C were approximately 25 min . The kinetic constants of xylanases I and II determined with Remazol-brilliant-blue xylan were Vmax of 1537 and 353 mumol.min-1.mg protein-1 and K(m) values of 2.44 and 1.07 mg.ml-1, respectively . Purified xylanases utilized xylan as well as small oligosaccharides such as xylotriose as substrate . They did not exhibit xylobiase or debranching activities . The predominant products of arabinoxylan hydrolysis were xylobiose and xylotriose, the latter being hydrolysed to xylobiose and xylose upon further incubation . In addition, fragments containing arabinose side chains accumulated . The xylanases did not act on crystalline or amorphous cellulose indicating a possible application in biobleaching processes.

Mol Microbiol, 1996 Apr, 20(2), 385 - 9
A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing; Kurosaka K et al.; The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence . In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65 degrees C . The 38 kDa precursor protein is separated as a soluble form from denatured E . coli proteins after heat treatment . Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography . The purified precursor protein gave a single band on SDS-polyacrylamide gels . The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme . The purified precursor protein was processed to the mature enzyme upon heat treatment . The processing was inhibited by diisopropyl fluorophosphate . The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.

Biochemistry, 1996 Mar 26, 35(12), 3810 - 5
Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process; Shao Y et al.; Protein splicing involves the self-catalyzed formation of a branched intermediate, which then resolves into the excised intervening sequence and the spliced protein . A possible mechanism for branched intermediate formation is an N-O rearrangement of the peptide bond involving the amino group of the conserved serine/cysteine residue at the upstream splice junction to yield a linear peptide ester intermediate . This possibility was examined in using an in vitro splicing system involving the intervening sequence from the DNA polymerase of the extremely thermophilic archeon, Pyrococcus sp . GB-D . Because thioesters react much more rapidly with nitrogen nucleophiles at neutral pH than do oxygen esters, protein-splicing precursors in which the serine residue of interest was replaced by cysteine were constructed and purified . In the presence of 0.25 M hydroxylamine or 0.1 M ethylene diamine at pH 6 or higher, these constructs underwent rapid cleavage at the upstream splice junction, consistent with the aminolysis of a thioester . The site of hydroxylaminolysis was identified by analysis of the C-terminus of the polypeptide cleavage products . Comparison of the C-terminal peptide hydroxamate with the synthetic peptide hydroxamates with respect to chromatographic mobility, colorimetric assay, amino acid composition, and high-resolution mass spectrometry showed that the hydroxylamine-sensitive site in the splicing precursor was the peptide bond adjacent to the serine residue at the upstream splice junction . These results provide evidence that the peptide bond at the upstream splice junction can undergo a self-catalyzed N-O or N-S acyl rearrangement to yield a linear polypeptide ester intermediate and suggest that this kind of rearrangement constitutes the first step in protein splicing.

JAMA, 1996 Mar 20, 275(11), 870 - 6
Biotherapeutic agents . A neglected modality for the treatment and prevention of selected intestinal and vaginal infections; Elmer GW et al.; OBJECTIVE: To evaluate the potential of biotherapeutic agents (microorganisms with therapeutic properties) for the prevention and/or treatment of selected intestinal and vaginal infections . DATA SOURCES: The MEDLINE database was searched for all relevant articles published between 1966 and September 1995 . Search terms used were biotherapeutic agent, probiotic, Lactobacillus, Saccharomyces, Bifidobacterium, Candida, gastrointestinal- system, vaginitis, vaginosis-bacterial, and related terms . The bibliographies of obtained articles were also reviewed . STUDY SELECTION AND DATA EXTRACTION: All placebo-controlled human studies on biotherapeutic agents were reviewed . English-language open trials, case series and reports, and animal studies were reviewed only if they were especially relevant to providing information on the potential efficacy, adverse effects, or mechanisms of action of these agents . DATA SYNTHESIS: Placebo-controlled studies have shown that biotherapeutic agents have been used successfully to prevent antibiotic-associated diarrhea (Lactobacillus caseiGG, bifidobacterium longum, B longum with L acidophilus, and Saccharomyces boulardii), to prevent acute infantile diarrhea (Bifidobacterium bifidum with Streptococcus thermophilus), to treat recurrent Clostridium difficile disease (S boulardii), and to treat various other diarrheal illnesses (Enterococcus faecium SF68, L caseiGG, and S boulardii) . There is also evidence for Lactobacillus acidophilus in the prevention of candidal vaginitis . Few adverse effects have been reported . However, many of the studies tested only small numbers of patients or volunteers . CONCLUSIONS: There is now evidence that administration of selected microorganisms is beneficial in the prevention and treatment of certain intestinal and, possibly, treatment of vaginal infections . In an effort to decrease the reliance on antimicrobials, the time has come to carefully explore the therapeutic applications of biotherapeutic agents.

Biochemistry, 1996 Mar 19, 35(11), 3447 - 56
Biosynthesis of methanopterin; White RH; This paper establishes the pathway for the biosynthesis of methanopterin in Methanosarcina thermophila to proceed by the following series of reactions . First, 5-phospho-alpha-D-ribosyl diphosphate (PRPP) and 4-aminobenzoic acid condense together to produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate, which then reacts with 6-hydroxymethyl-7,8-H2pterin pyrophosphate to produce 7,8-H2pterin-6-ylmethyl-4-(beta-D-ribofuranosyl)aminobenz ene 5'-phosphate (3') . Compound 3' is then reduced to 7,8-H2pterin-6-ylmethyl-l-(4-aminophenyl)-1-deoxy-D-ribitol 5'-phosphate (4') in a reaction stimulated by the addition of FMN or factor F(420) . Dephosphorylation of compound 4' leads to 7,8-H2pterin-6-ylmethyl-1-(4-aminophenyl)-1-deoxy-D-ribitol (5') . Compound 5' then condenses with another molecule of PRPP to form 7,8-H2pterin-6-ylmethyl-1-(4-aminophenyl)-1-deoxy-5-{1-alpha -D- ribofuranosyl 5-phosphate}-D-ribitol (9') . Compound 9', in the presence of ATP, then condenses with (S)-2-hydroxyglutaric acid to form demethylated H2methanopterin, a known precursor to methanopterin . The occurrence of this pathway was confirmed by (a) the chemical and/or biochemical synthesis of most of the proposed intermediates, (b) the detection of these intermediates in cell-free extracts, and (c) by the measurement of their conversion to demethylated methanopterin and/or other intermediates in the pathway.

FEMS Microbiol Lett, 1996 Mar 15, 137(1), 31 - 5
Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector; Elferink MG et al.; We describe a transformation system for extremely thermophilic archaea of the genus Sulfolobus in the kingdom Crenarchaeota . We have constructed in vitro a recombinant derivative of the recently described conjugative plasmid pNOB8, containing a beta-galactosidase gene downstream of a strong promotor . Transformation of a beta-galactosidase negative mutant of Sulfolobus solfataricus with this construct resulted in its spreading through the culture containing the primary transformants and in efficient restoration of beta-galactosidase activity.

Eur J Biochem, 1996 Mar 15, 236(3), 1010 - 24
Isolation, characterization and electron microscopy analysis of a hemidiscoidal phycobilisome type from the cyanobacterium Anabaena sp . PCC 7120; Ducret A et al.; In this work we present the characterization of a hemidiscoidal phycobilisome type of the heterocyst-forming cyanobacterium Anabaena sp . PCC 7120 . The phycobilisome of this organism contains allophycocyanin, phycocyanin and phycoerythrocyanin, similar to the closely related thermophilic cyanobacterium Mastigocladus laminosus . Intact phycobilisomes exhibit an absorption maximum at 619 nm and two fluorescence maxima at 664 nm and 680 nm, corroborating the presence of a complete energy pathyway along the antenna . Upon dissociation, the phycobiliproteins were released from the phycobilisome . One phycoerythrocyanin, one phycocyanin and three allophycocyanin complexes were isolated by ion-exchange chromatography and characterized by absorption and fluorescence spectroscopy and by SDS/PAGE . The amino-terminal sequences of the polypeptides belonging to the phycoerythrocyanin and phycocyanin families were identical with the derived sequences of their corresponding genes . Partial amino-terminal sequences of the polypeptides belonging to the allophycocyanin family are presented here . Our results show that the phycobiliproteins and linker polypeptides from Anabaena sp . PCC 7120 are similar to the phycobilisome components characterized in other cyanobacteria . The phycobilisome of Anabaena sp . PCC 7120 was extensively analyzed by electron microscopy . It differs from the common hemidiscoidal tricylindrical, six-rod phycobilisome type by a core domain consisting of five core cylinders surrounded by up to eight rods radiating in a hemidiscoidal manner . One rod is linked to each basal core cylinder, whereas the remaining core cylinders bind two rods each . On the basis of the data presented in this work, a revised model for the hemidiscoidal pentacylindrical phycobilisome of Anabaena sp . PCC 7120, M . laminosus and Anabaena variabilis is proposed . This model accounts more accurately for the 'grape' pattern typically exhibited by these phycobilisomes in electron micrographs.

EMBO J, 1996 Mar 15, 15(6), 1350 - 9
Crystal structure of the RNA binding ribosomal protein L1 from Thermus thermophilus; Nikonov S et al.; L1 has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding mRNA . The crystal structure of L1 from Thermus thermophilus has been determined at 1.85 angstroms resolution . The protein is composed of two domains with the N- and C-termini in domain I . The eight N-terminal residues are very flexible, as the quality of electron density map shows . Proteolysis experiments have shown that the N-terminal tail is accessible and important for 23S rRNA binding . Most of the conserved amino acids are situated at the interface between the two domains . They probably form the specific RNA binding site of L1 . Limited non-covalent contacts between the domains indicate an unstable domain interaction in the present conformation . Domain flexibility and RNA binding by induced fit seems plausible.

Biochem J, 1996 Mar 15, 314 ( Pt 3), 833 - 8
Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A; Lopez-Camacho C et al.; Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme . Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively . The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change . CD spectra indicated that the E96K replacement caused an increase in alpha-helix content . The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.

Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 86 - 93
Cloning, sequencing and overexpression in Escherichia coli of a xylanase gene, xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor; Dwivedi PP et al.; A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in lambda ZapII and screened for the expression of xylanase activity . One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity . Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism . The xylanase gene, xynA was shown to code for a multi-domain protein . Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains . Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host . The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70 degrees C for at least 12 h at pH 7 . The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.

Biosci Biotechnol Biochem, 1996 Mar, 60(3), 543 - 5
Isolation and identification of Alicyclobacillus acidoterrestris from acidic beverages; Yamazaki K et al.; Thermophilic bacteria isolated from spoiled acidic beverages were identified on the basis of DNA-DNA homology and 16S ribosomal DNA sequence similarity to Alicyclobacillus acidoterrestris . This result suggested that the isolates were a new type of spoilage bacterium in acidic beverages in Japan.

Protein Sci, 1996 Mar, 5(3), 511 - 6
A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes; Hayashi-Iwasaki Y et al.; The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC) . The enzyme is a homodimer with a subunit containing two structural domains . The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T . thermophilus wild-type enzyme had one peak . The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition . Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent . When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating . These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11 . Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains . The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.

Mol Microbiol, 1996 Mar, 19(5), 911 - 22
Cation and sugar selectivity determinants in a novel family of transport proteins; Poolman B et al.; A new family of homologous membrane proteins that transport galactosides-pentoses-hexuronides (GPH) is described . By analysing the aligned amino acid sequences of the GPH family, and by exploiting their different specificities for cations and sugars, we have designed mutations that yield novel insights into the nature of ligand binding sites in membrane proteins . Mutants have been isolated/constructed in the melibiose transport proteins of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium, and the lactose transport protein of Streptococcus thermophilus which facilitate uncoupled transport or have an altered cation and/or substrate specificity . Most of the mutations map in the amino-terminal region, in or near amphipathic alpha-helices II and IV, or in interhelix-loop 10-11 of the transport proteins . On the basis of the kinetic properties of these mutants, and the primary and secondary structure analyses presented here, we speculate on the cation binding pocket of this family of transporters . The regulation of the transporters through interaction with, or phosphorylation by, components of the phosphoenolpyruvate:sugar phosphotransferase system is also discussed.

J Biochem (Tokyo), 1996 Mar, 119(3), 482 - 6
Intra- and inter-complex cross-linking of subunits in the quinol oxidase super-complex from thermophilic Bacillus PS3; Tanaka T et al.; Gram-positive thermophilic Bacilli contain quinol-cytochrome c reductase and cytochrome c oxidase as two major respiratory complexes of the electron transfer chain, and these enzymes can be extracted with mild detergents as an associated quinol oxidase super-complex . The reductase is composed of three subunits; cytochrome b6, cytochrome c1, and FeS protein, whereas cytochrome c oxidase consists of four subunits numbered 1 through 4 . In order to clarify the interactions between the subunits, the super-complex isolated from Bacillus PS3 was cross-linked with three bifunctional cross-linkers; disuccinimidyl tartrate, 3,3'-dithiobis(succinimidylpropionate), and ethylene glycolbis(sulfosuccinimidylsuccinate) . The most prominent cross-linking was observed for the combination of subunit 1 plus 2 in cytochrome c oxidase, and for that of cytochrome b6 plus cytochrome c1 in the reductase . In addition to these intra-complex cross-linkings, inter-complex linking was observed for the combination of cytochrome b6 plus subunit 1 with ethylene glycolbis(sulfosuccinimidylsuccinate), and for the combinations of cytochrome b6 plus subunit 1 and cytochrome b6 plus subunit 2 with 3,3'-dithiobis(succinimidylpropionate) . Incubation in the presence of Triton X-100, which was confirmed to cleave the two enzyme complexes, selectively reduced the inter-complex cross-linking, suggesting that the chemical cross-linking reflect the spatial arrangement of subunits in the super-complex.

C R Acad Sci III, 1996 Mar, 319(3), 161 - 9
Pathways for conformational change in seryl-tRNA synthetase from Thermus thermophilus; El-Kettani MA et al.; Seryl t-RNA synthetase of the bacterium Thermus thermophilus contains a long arm, consisting of an antiparallel coiled coil, that is involved in binding of tRNA . Two crystallographic structures exist for this protein, in which the arm is in different conformations . Here, we use computational methods employing an empirical potential energy function to investigate the flexibility of the long arm . A conformational pathway is calculated between the 2 crystallographic structures using a method based on molecular dynamics simulation . The pathway is analyzed in terms of sequential phi and psi backbone angle changes . Several transient phi and psi displacements are present along the pathway that are not visible in the end states and may be required for transition between them . Energy maps are constructed by rotating the arm around its principal axes of inertia and energy minimizing . The map identifies 2 regions of relatively low energy which might be accessible to the arm.

J Bacteriol, 1996 Mar, 178(6), 1680 - 90
Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6; Stingele F et al.; We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis . This report is the first genetic work concerning EPS production in a food microorganism . The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-{alpha-D-Galp-(1-->6)}-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1--> . The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants . Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames . EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host . Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae . Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis . Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.

J Bacteriol, 1996 Mar, 178(6), 1539 - 47
Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp . strain JW/SL-YS 485 in Escherichia coli; Liu SY et al.; The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp . strain JW/SL-YS 485 was cloned and expressed in Escherichia coli . A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria . This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene . This fragment was cloned into E . coli, and recombinant clones expressed significant levels of xylanase activity . The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485 . The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E . coli . The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases . A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria . These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.

Mol Cell Biol, 1996 Mar, 16(3), 1267 - 74
New telomere formation coupled with site-specific chromosome breakage in Tetrahymena thermophila; Fan Q et al.; Programmed chromosome breakage occurs in many ciliated protozoa and is accompanied by efficient new telomere formation . In this study, we have investigated the relationship between programmed chromosome breakage and telomere formation in Tetrahymena thermophila . Using specially constructed DNA clones containing the breakage signal Cbs in transformation studies, we have determined the locations of telomere addition around the breakage sites . They occur at variable positions, over 90% of which are within a small region (less than 30 bp) starting 4 bp from Cbs . This distribution is independent of the nucleotide sequence in the region or of the orientation of Cbs . In five of six cases determined, these sites occur at or before a T, and in the remaining case, the site occurs at or before a G . When sequences devoid of G or T are placed in this region, telomere addition still occurs within the region to maintain a similar distance relationship with Cbs . This efficient and healing process appears to be associated specifically with Cbs-directed breakage, since it does not occur when DNA ends are generated by restriction enzyme digestion . These results suggest a strong mechanistic link between chromosome breakage and telomere formation.

Arch Biochem Biophys, 1996 Mar 1, 327(1), 98 - 106
A novel invertase from a thermophilic fungus Thermomyces lanuginosus: its requirement of thiol and protein for activation; Chaudhuri A et al.; Unlike the invertases from the mesophilic fungi and yeasts, invertase from a thermophilic fungi, Thermomyces lanuginosus, was unusually unstable both in vivo and in vitro . The following observations suggested that the unstable nature of the enzyme activity in the cell-free extracts was due to the oxidation of the cysteine residue(s) in the enzyme molecule: (a) the addition of dithiothreitol or reduced glutathione stabilized invertase activity during storage of the extracts and also revived enzyme activity in the extracts which had become inactive with time; (b) N-ethylmaleimide, iodoacetamide, oxidized glutathione, cystine, or oxidized coenzyme A-inactivated invertase; (c) invertase activity was low when the ratio reduced/oxidized glutathione was lower and high when this ratio was higher, suggesting regulation of the enzyme by thiol/disulfide exchange reaction . In contrast to the activation of invertase by the thiol compounds and its inactivation by the disulfides in the cell-free extracts, the purified enzyme did not respond to these compounds . Following its inactivation, the purified enzyme required a helper protein in addition to dithiothreitol for maximal activation . A cellular protein was identified that promoted activation of invertase by dithiothreitol and it was called "PRIA" for the protein which helps in restoring invertase activity . The revival of enzyme activity was due to the conversion of the inactive invertase molecules into an active form . A model is presented to explain the modulation of invertase activity by the thiol compounds and the disulfides, both in the crude cell-free extracts and in the purified preparations . The requirement of free sulfhydryl group(s) for the enzyme activity and, furthermore, the reciprocal effects of the thiols and the disulfides on invertase activity have not been reported for invertase from any other source . The finding of a novel invertase which shows a distinct mode of regulation demonstrates the diversity in an enzyme that has figured prominently in the development of biochemistry.

J Mol Biol, 1996 Mar 1, 256(3), 611 - 22
Crystal structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1; Knegtel RM et al.; The crystal structure of the cyclodextrin glycosyltransferase (CGTase) from the thermophilic microorganism Thermoanaerobacterium thermosulfurigenes EM1 has been elucidated at 2.3 A resolution . The final model consists of all 683 amino acid residues, two calcium ions and 343 water molecules, and has a crystallographic R-factor of 17.9% (Rfree 24.9%) with excellent stereochemistry . The overall fold of the enzyme is highly similar to that reported for mesophilic CGTases and differences are observed only at surface loop regions . Closer inspection of these loop regions and comparison with other CGTase structures reveals that especially loops 88-95, 335-339 and 534-539 possibly contribute with novel hydrogen bonds and apolar contacts to the stabilization of the enzyme . Other structural features that might confer thermostability to the T . thermosulfurigenes EM1 CGTase are the introduction of five new salt-bridges and three Gly to Ala/Pro substitutions . The abundance of Ser, Thr and Tyr residues near the active site and oligosaccharide binding sites might explain the increased thermostability of CGTase in the presence of starch, by allowing amylose chains to bind non-specifically to the protein . Additional stabilization of the A/E domain interface through apolar contacts involves residues Phe273 and Tyr187 . No additional or improved calcium binding is observed in the structure, suggesting that the observed stabilization in the presence of calcium ions is caused by the reduced exchange of calcium from the protein to the solvent, rendering it less susceptible to unfolding . The 50% decrease in cyclization activity of the T . thermosulfurigenes EM1 CGTase compared with that of B . circulans strain 251 appears to be caused by the changes in the conformation and amino acid composition of the 88-95 loop . In the T . thermosulfurigenes EM1 CGTase there is no residue homologous to Tyr89, which was observed to take part in stacking interactions with bound substrate in the case of the B . circulans strain 251 CGTase . The lack of this interaction in the enzyme-substrate complex is expected to destabilize bound substrates prior to cyclization . Apparently, some catalytic functionality of CGTase has been sacrificed for the sake of structural stability by modifying loop regions near the active site.

Arch Microbiol, 1996 Mar, 165(3), 179 - 86
Pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase from Streptomyces arenae is closely related to GAPDH from thermostable eubacteria and plant chloroplasts; Frohlich KU et al.; Streptomyces arenae produces the antibiotic pentalenolactone, a highly specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . During the phase of pentalenolactone production, S . arenae expresses a pentalenolactone-insensitive GAPDH isoform; otherwise, a pentalenolactone-sensitive form is expressed . The gene of the pentalenolactone-insensitive GAPDH was cloned and sequenced . Regulatory elements typical for genes encoding antibiotic resistance and production are localized upstream and downstream of the open reading frame . No expression of pentalenolactone-insensitive GAPDH was detected in Streptomyces lividans transformed with the gene . In Escherichia coli, the gene was expressed from an induced lac promoter . Amino-terminal sequencing of the heterologously expressed GAPDH proved its identity with pentalenolactone-insensitive GAPDH from S . arenae . Sequence comparisons with GAPDH from other organisms showed a close relationship to GAPDH of plant chloroplasts, of other gram-positive bacteria, and of thermophilic gram-negative bacteria . Pentalenolactone-insensitive GAPDH differs from all closely related GAPDHs only in a few residues, none of which are directly involved in catalysis or substrate binding . The total amino acid composition is more similar to GAPDH of thermophilic species than to that of mesophilic species . The purified enzyme was moderately thermotolerant, which could be a side effect of the structural changes causing pentalenolactone-resistance.

Biochemistry, 1996 Feb 27, 35(8), 2597 - 609
Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 25 Angstroms Resolution; Tanner JJ et al.; The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution . To study the determinants of thermostability, we compare our structure to four other GAPDHs . Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed . We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners . These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links . The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds . Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner . Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles . There is, however, an association between thermostability and decreasing enzyme surface to volume ratio . Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs . These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues.

Gene, 1996 Feb 22, 169(1), 135 - 6
Sequence of the Bacillus stearothermophilus gene encoding aspartokinase II; Cantoni R et al.; The complete nucleotide sequence of the gene encoding aspartokinase (Ask) II from thermophilic Bacillus stearothermophilus has been determined . Degenerate oligodeoxyribonucleotides primed the amplification of a 932-bp gene . This sequence was successively used for constructing new primers applied in inverse polymerase chain reaction using, as template, self-ligating DNA fragments . The deduced amino-acid sequence is 68.7% identical with the sequence of the Bacillus sp . strain MGA3 Ask II.

Mutat Res, 1996 Feb 19, 350(1), 199 - 200
Antimutagenicity of yogurt; Bakalinsky AT et al.; Yogurt is milk fermented by a mixture of two bacteria: Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus salivarius ssp . thermophilus . Epidemiological studies have correlated a reduced risk of colon cancer with yogurt consumption . Independent studies have established that yogurt and extracts thereof are antimutagenic . Although multiple explanations can account for yogurt's putative anticarcinogenicity, we are interested in testing the hypothesis that antimutagenic compounds produced during fermentation are responsible . We recently reported on the antimutagenicity of an acetone extract of yogurt against the experimental carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 3.2'dimethyl-4-aminobiphenyl (DMAB) (Mutation Res . (1995) 334, 213-224) . We are now aware that palmitic acid is an active ingredient against MNNG.

Eur J Biochem, 1996 Feb 15, 236(1), 222 - 7
Elongation factor Ts from Thermus thermophilus-- overproduction in Escherichia coli, quaternary structure and interaction with elongation factor Tu; Blank J et al.; The gene encoding the elongation factor Ts from Thermus thermophilus was sequenced, cloned and the protein overproduced in Escherichia coli . In comparison to the EF-Ts from E . coli with 282 amino acid residues, EF-Ts from T . thermophilus is considerably shorter, differing by 86 amino acids . EF-Ts from the thermophile is stable at high temperatures, which facilitates its separation from E . coli proteins . Purified T . thermophilus EF-Ts forms a homodimer with a disulfide bridge between the two cysteine residues at position 190 . The modification of Cys19O by iodoacetamide affects neither the dimerization nor the ability of EF-Ts to facilitate the nucleotide exchange of elongation factor Tu . The disulfide bridge was detected only in purified EF-TS, but not in protein extracts immediately after cell disruption . The physiological role of this disulfide bridge remains, therefore, unclear . Besides the quaternary (EF-TU . EF-Ts)2 complex, a ternary EF-TU . EF-Ts2 complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis . Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF-Ts, that does not bind to EF-Tu but is still capable of forming homodimers.

Biochim Biophys Acta, 1996 Feb 15, 1273(2), 129 - 38
Nucleotide and amino acid sequences for cytochrome caa3-type oxidase of Bacillus stearothermophilus K1041 and non-Michaelis-type kinetics with cytochrome c; Kusano T et al.; A pseudo-sigmoidal cytochrome c-dependence curve of oxidase activity was observed with cytochrome oxidase from the Bacillus stearothermophilus strain K1041, while the other thermophilic Bacillus PS3 which has been extensively studied possessed normal Michaelis-Menten type kinetics . The genes coding for four subunits of cytochrome caa3-type oxidase and for heme O synthase were isolated from a genomic DNA library of K1041 by using a PS3 DNA fragment containing the highly-conserved region of the largest subunit as a probe, and sequenced . Most residues in subunits I (COI/caaB product), III (COIII/caaC product), and IV (COIV/caaD product) of K1041 were highly conserved when compared with those of PS3 . However, the sequence of K1041 subunit II (COII/caaA product) was distinctly different from that of the PS3 subunit II . These Bacillus COIIs have an additional sequence for cytochrome c after the CuA binding protein portion with two transmembrane segments which is homologous to the mitochondrial counterpart, and represents the site of electron ingress . Several charged residues in the vicinity of cytochrome c moiety are replaced by oppositely charged residues . It is likely that these amino acid replacements in subunit II are the cause of the abnormal sigmoidal saturation curve for extrinsic cytochromes c of the K1041 enzyme.

Nucleic Acids Res, 1996 Feb 15, 24(4), 640 - 7
Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8; Takamatsu S et al.; The mutS gene, implicated in DNA mismatch repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8 . Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa . Its predicted amino acid sequence showed 56 and 39% homology with Escherichia coli MutS and human hMsh2 proteins, respectively . The T.thermophilus mutS gene complemented the hypermutability of the E.coli mutS mutant, suggesting that T.thermophilus MutS protein was active in E.coli and could interact with E.coli MutL and/or MutH proteins . The T.thermophilus mutS gene product was overproduced in E.coli and then purified to homogeneity . Its molecular mass was estimated to be 91 kDa by SDS-PAGE but approx . 330 kDa by size-exclusion chromatography, suggesting that T.thermophilus MutS protein was a tetramer in its native state . Circular dichroic measurements indicated that this protein had an alpha-helical content of approx . 50%, and that it was stable between pH 1.5 and 12 at 25 degree C and was stable up to 80 degree C at neutral pH . Thermus thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80 degrees C . The kinetic parameters of the ATPase activity at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(-1) . Thermus thermophilus MutS protein bound specifically with G-T mismatched DNA even at 60 degrees C.

Biochim Biophys Acta, 1996 Feb 8, 1292(2), 303 - 11
A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability; Xu F et al.; A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability . Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs . NHE), while other oxidases have potentials near 0.5 V . It is observed that higher redox potential correlates with higher activity . By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series . At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively . The enzymes are more stable in the neutral-alkaline pH range . The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase . Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.

Biochemistry, 1996 Feb 6, 35(5), 1560 - 70
pH dependencies of the Tetrahymena ribozyme reveal an unconventional origin of an apparent pKa; Knitt DS et al.; The L-21 ScaI ribozyme derived from the Tetrahymena thermophila pre-rRNA group I intron catalyzes a site-specific endonucleolytic cleavage of RNA, DNA, and chimeric RNA/DNA oligonucleotides: CCCUCUA5 + G-->CCCUCU + GA5 . The pH-rate dependence was determined for the reaction of the E.G complex with the oligonucleotide substrate d(CCCUC)r(U)d(A5) {(kcat/Km)S conditions} . Although it was shown that the pH dependence is not affected by specific buffers, there is inhibition by specific monovalent cations . The intrinsic pH-rate dependence is log-linear with slope 1 below pH 7, displays an apparent pKa of 7.6, remains nearly level until pH 8.5, and then begins to fall . Two models to explain the apparent pKa were ruled out: (1) the pKa represents loss of a proton from the nucleophilic 3' OH of G, and (2) the pKa arises from a change in rate-limiting step from a pH-dependent to a pH-independent step . In addition, these models, or others involving a single titration, cannot account for the decrease in activity at high pH . A third, unconventional, model is consistent with all of the data . It involves inactivation of the ribozyme by any of several independent titrations of groups with pKa values considerably higher than the apparent pKa of 7.6 . The data are consistent with loss of catalytic function upon release of a proton from any one of 19 independent sites with pKa = 9.4 (the unperturbed pKa of N1 of G and N3 of U in solution) . Independent experiments investigating the effect of pH on different reaction steps supported this model and suggested the identity of some of the required protons . This mechanism of inactivation is expected to generally affect the behavior of RNAs at pH values removed from the pKa of the titrating bases.

J Biol Chem, 1996 Feb 2, 271(5), 2433 - 8
Structural asymmetry of F1-ATPase caused by the gamma subunit generates a high affinity nucleotide binding site; Kaibara C et al.; The alpha 3 beta 3 gamma and alpha 3 beta 3 complexes of F1-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the alpha 3 beta 3 complex . The results of equilibrium dialysis show that the alpha 3 beta 3 gamma complex has a high affinity nucleotide binding site and several low affinity sites, whereas the alpha 3 beta 2 complex has only low affinity sites . This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the beta subunit . Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the alpha 3 beta 3 gamma complex is accelerated by the chase addition of excess ATP, whereas that by the alpha 3 beta 3 complex is not . We further examined the complexes containing mutant beta subunits (Y341L, Y341A, and Y341C) . Surprisingly, in spite of very weak affinity of the isolated mutant beta subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K, Yutani, K., and Yoshida, M . (1994) J . Biochem . (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the beta subunit in the mutant alpha 3 beta 3 gamma complexes where single-site TNP-ATP hydrolysis can occur . ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex . The mutant alpha 3 beta 3 complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration . Thus, the gamma subunit is responsible for the generation of a high affinity nucleotide binding site on the beta subunit in F1-ATPase where cooperative catalysis can proceed.

Bioseparation, 1996 Feb, 6(1), 41 - 5
Affinity purification of cellulose-binding enzymes of Clostridium stercorarium; Bronnenmeier K et al.; Cellulose-affinity chromatography proved to be a fast and efficient purification procedure for exoenzymes of the thermophilic anaerobe Clostridium stercorarium, suitable for the preparation of large amounts of enzymes for technical applications . The cellulose-binding enzymes could be identified as the C . stercorarium; cellulolytic enzymes Avicelase I and Avicelase II characterized previously as endo-1,4-beta-glucanase and exo-1,4-beta-glucanase . A third protein was identified as xylanase A, the major endo-1,4-beta-xylanase of this organism.

Microbiology, 1996 Feb, 142 ( Pt 2), 401 - 10
Mapping of 61 genes on the refined physical map of the chromosome of Thermus thermophilus HB27 and comparison of genome organization with that of T . thermophilus HB8; Tabata K et al.; We have constructed refined physical maps of the chromosome (1 center dot 82 Mb) and the large plasmid pTT27 (250 kb) of Thermus thermophilus HB27 . A total of 49 cleavage sites with five restriction enzymes, EcoRI, SspI, MunI, EcoRV and ClaI, were determined on the maps . The location of 61 genes was determined by using as probes 64 genes cloned from T . thermophilus or other Thermus strains . Comparison of the genomic organization of the chromosomes of T . thermophilus HB27 and HB8 revealed that they were basically identical, but some genes were located in different regions . Among 32 genes whose locations were determined on both the HB27 and the HB8 chromosomes, the copy number of rpsL-rpsG-fus-tufA, the locations of glyS, pol, and one copy of nusG-rplK-rplA were different . The IS1000 sequence was located only in one region on the HB27 chromosome . In contrast, IS1000 sequences were scattered over four regions on the chromosome of HB8 . As each region in which glyS, pol, or one copy of nusG-rplK-rplA are present also contained IS1000 in HB8, it is suggested that IS1000 may play an important role in genomic rearrangements in Thermus strains.

Biochem Mol Biol Int, 1996 Feb, 38(1), 181 - 8
Identification of peptide fragments chemically cross-linked in cytochrome c oxidase from thermophilic Bacillus PS3; Kuge S et al.; In order to study steric arrangement of subunits in cytochrome c oxidase isolated from thermophilic Bacillus PS3, we developed a simple procedure including chemical cross-linking, two consecutive runs of electrophoresis, proteolytic digestion, and peptide sequencing for simultaneous identification of two cross-linked fragments . By this procedure, the cytochrome c domain of subunit 2 was found cross-linked to C-terminal region of subunit 1 including two hydrophobic transmembrane segments, suggesting that these two regions were located close each other . The present simple procedure might be applicable to proteins whose crystal structures are not revealed.

Biotechnol Appl Biochem, 1996 Feb, 23 ( Pt 1), 47 - 54
Purification and characterization of NADH oxidase from the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus; Masullo M et al.; The enzyme NADH oxidase (EC 1.6.99.3) has been isolated from the two thermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus and characterized . In both organisms the enzyme oxidizes specifically beta-NADH in the presence of molecular oxygen and requires the presence of a flavin cofactor, showing a high specificity for FAD . A stoicheiometric amount of hydrogen peroxide to NADH is formed as the end product of the reaction, indicating that both enzymes are two-electron donors . The purified enzymes exhibit quite different molecular properties . S . acidocaldarius NADH oxidase is a monomeric protein with an estimated molecular mass of about 27 kDa, whereas S . solfataricus NADH oxidase is a dimeric protein with a molecular mass of 35 kDa per subunit; S . solfataricus NADH oxidase is purified as an FAD-containing protein, whereas S . acidocaldarius NADH oxidase does not contain a flavin molecule . Furthermore, a comparison of the N-terminal amino acid sequence shows no similarities either between the two proteins or to any other NADH oxidases . Both enzymes are essentially thermophilic . In the temperature range 20-80 degrees C, the energy of activation is almost the same for both activities, suggesting that similar energetic parameters are required . Also both oxidases display a great stability to heat . The half-life of heat inactivation is about 180 min at 90 degrees C for S . acidocaldarius NADH oxidase and 77 min at 98 degrees C for the S . solfataricus enzyme . The activity of the two enzymes is inhibited by urea and guanidine and are regulated very differently by several organic solvents.

Biotechnol Appl Biochem, 1996 Feb, 23 ( Pt 1), 41 - 5
Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus; Ianniciello G et al.; The elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was expressed in Escherichia coli and purified . The SsEF-1 alpha gene was amplified by PCR and cloned in the Ndel site of the pT7-7 expression vector, under the control of the promoter of T7 RNA polymerase . Upon induction with isopropyl beta-D-thiogalactopyranoside, the recombinant SsEF-1 alpha (recSsEF-1 alpha) was purified from the E . coli S-100 extract by a two-step procedure . From 1 litre of cell culture, about 2 mg of purified recSsEF-1 alpha was obtained . The N-terminal sequence of the first 30 amino acid residues of recSsEF-1 alpha was identical with that translated from the nucleotide sequence of the corresponding gene, except for the initial residue, which in recSsEF-1 alpha was Ser instead of Met . The M(r) of recSsEF-1 alpha (determined by electrospray MS) was almost coincident with that of the naturally occurring SsEF-1 alpha (SsEF-1 alpha) . The thermal-inactivation and thermophilicity profiles of SsEF-1 alpha and recSsEF-1 alpha were identical . Concerning the functional properties, recSsEF-1 alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to elicit an NaCl-dependent GTPase activity {Masullo, De Vendittis and Bocchini (1994) J . Biol . Chem . 269, 20376-20379} with the same efficiency as that displayed by SsEF-1 alpha.

Biotechniques, 1996 Feb, 20(2), 240 - 8
Cycling probe technology with RNase H attached to an oligonucleotide; Bekkaoui F et al.; A streptavidin-RNase H gene fusion was constructed by cloning the Thermus thermophilus RNase H coding sequence in the streptavidin expression vector pTSA18F . The gene was expressed in Escherichia coli, and the resulting fusion protein was purified to apparent homogeneity . The fusion protein was shown to have a molecular weight of 128 kDa and to consist of four subunits . Furthermore, heat treatment of the fusion enzyme showed that it was stable as a tetramer at 65 degrees C . The fusion enzyme was shown to have both biotin binding and RNase H catalytic properties . Using cycling probe technology (CPT), the fusion enzyme was compared to the native RNase H with a biotinylated probe at different ratios of probe:enzyme and varying amounts of synthetic target DNA . At a ratio of 1:1, the fusion enzyme was active in CPT, but the native enzyme was not; both enzymes were active at a 1:5000 ratio of probe:enzyme . The fusion enzyme was further tested using biotinylated and non-biotinylated probes and was shown to be active at a 1:1 ratio with the biotinylated probe but not with the non-biotinylated probe . These experiments show that through binding of the streptavidin-RNase H fusion enzyme to the biotinylated probe, the efficiency of the cycling probe reaction is enhanced.

Proteins, 1996 Feb, 24(2), 269 - 71
Crystallization and preliminary X-ray characterization of the Methanothermus fervidus histones HMfA and HMfB; Decanniere K et al.; HMfA and HMfB are histone proteins from the thermophilic archaeon Methanothermus fervidus . They wrap DNA into nucleosome-like structures and appear to represent the basic core histone fold . HMfA was crystallized in space groups P4(2)2(1)2 and P2(1)2(1)2(1) . HMfB crystallized in space group P2(1)2(1)2, while a selenomethionine-substituted variant, SeMet-HMfB, yielded crystals in C222(1) . In all crystal forms HMfA, HMfB, or SeMet-HMfB may be present as homodimers.

Int J Biochem Cell Biol, 1996 Feb, 28(2), 239 - 46
Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70 degrees C; Guagliardi A et al.; The biocatalysts isolated from thermophilic microorganisms are the object of ever-growing scientific interest for (i) the comprehension of the molecular basis of their thermal tolerance, and (ii) their use in different bio-industrial fields . Here we report the purification and characterization of an alcohol dehydrogenase (designated ADH-hT) from the novel strain LLD-R of Bacillus stearothermophilus which grows at 70 degrees C . ADH-hT was obtained in pure form by anion exchange chromatography and two affinity chromatographies, with a final yield of about 30% . ADH-hT was found to be a tetramer of 37 kDa-subunits, and to have a pI of 4.9 . ADH-hT displayed a broad substrate specificity; its activity was highest for aldehydes, and decreased progressively for alcohols and ketones . ADH-hT was endowed with catalytic activity and resistance in the presence of several denaturing agents (organic solvents, detergents, chaotropic agents) . ADH-hT shared with ADH 1503 (the alcohol dehydrogenase from B . stearothermophilus strain NCA 1503 which grows at 55 degrees C) the optimal temperature of 65 degrees C, but it was more resistant than ADH 1503 towards heating . In conclusion, due to its stability and broad substrate specificity ADH-hT could be utilized in bio-industrial processes . Furthermore, we believe that ADH-hT could represent a good model system for studying the mechanism(s) which proteins exploit to gain heat resistance.

Cell Struct Funct, 1996 Feb, 21(1), 73 - 80
Involvement of active cellular mechanisms on the disorganization of oral apparatus in amicronucleate cells in Tetrahymena thermophila; Haremaki T et al.; Ciliated protozoa, a group of unicellular eukaryotes, have two kinds of nuclei, a macronucleus (somatic nucleus) and a micronucleus (germinal nucleus) in a single cell . We previously reported that amicronucleate cells of Tetrahymena thermophila induced by nocodazole gradually lost their oral apparatus (OA) and ciliary rows but that amacronucleate cells did not . Since the macronucleus is responsible for the gene expression in the vegetative phase, the effects of actinomycin D and cycloheximide on the disorganization of the OA in amicronucleate cells induced by nocodazole were investigated . These inhibitors prevented the disorganization of the OA in amicronucleate cells . Amicronucleate cells did not grow even in the medium supplemented with high concentration of Fe, Cu and folinic acid which allow cells to grow without formation of food vacuoles . The results suggest that the macronucleus in the amicronucleate cells plays an active role in the induction of disorganization of the OA and malfunctions of nutrient uptake from the cell surface and/or in the fundamental cell division mechanisms, resulting in the death of amicronucleate cells.

Genetika, 1996 Feb, 32(2), 197 - 203
{Construction and analysis of transgenic plants of Nicotiana tabacum L . expressing a bacterial gene for beta-1,3-glucanase . I . Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts}; Darbinian NS et al.; We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells . The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum . To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained . Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme . The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively . Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia . Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase . We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

Zoolog Sci, 1996 Feb, 13(1), 89 - 96
A substance secreted from Tetrahymena and mammalian sera act as mitogens on Paramecium tetraurelia; Tokusumi Y et al.; We previously isolated and purified Paramecium growth factor (ParGF) from a cell-free fluid of an early stationary mass culture of Paramecium tetraurelia (Tanabe et al., 1990) . The mitogenic activity of the purified ParGF and of the crude sample (ca . a 100-fold concentrate obtained by ultrafiltration of cell-free fluid) has been assessed based on restoration of the fission rate of the jumyo mutant of P . tetraurelia in daily reisolation cultures . With this assay system, we found that crude samples of Tetrahymena pyriformis and T . thermophila showed mitogenic activity . This suggests that Tetrahymena cells secrete a mitogenic factor(s) like ParGF . To some extent, fetal bovine serum (FBS) and calf serum (CS) also acted as mitogens on the jumyo mutant . Of nine mammalian growth factors assayed for their mitogenic effects on the jumyo mutant, epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) were slightly and occasionally effective . These results support the idea of actual use of similar kind of growth factors to control cell divisions from protozoa to mammals.

Eur J Biochem, 1996 Feb 1, 235(3), 779 - 88
ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin . 2 . Relationships between proton motive force and ATP synthesis; Pitard B et al.; The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3 . By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient . Maximal rates of ATP synthesis (up to to 200 nmol ATP x min(-1) x mg protein (-1) were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25 . It was demonstrated that this was the maximal deltapH attainable at 40 degrees C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates . In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40 degrees C, contributing to an increase in the rate of ATP synthesis . The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient . Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of delta pH remained the same or, at very high bacteriorhodopsin levels, decreased . Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content . Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96-->Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase . Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient . Similar results were obtained with chloroplast ATP synthase . Furthermore, ATP synthase activities induced by delta pH/delta psi transitions were independent of bacteriorhodopsin or anionic lipid levels . These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.

Eur J Biochem, 1996 Feb 1, 235(3), 769 - 78
ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin . 1 . Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin; Pitard B et al.; Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined . Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process . After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40 degrees C . The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions . The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes . The conditions for reconstitutions were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1 . It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TFO-F1 . Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity . Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used . In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids . Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1 . Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP x min(-1) x mg TF0F1(-1) in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis TF0F1 measured under similar conditions . This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.

J Appl Bacteriol, 1996 Feb, 80(2), 157 - 64
Ribotypes and AP-PCR fingerprints of thermophilic campylobacters from marine recreational waters; Hernandez J et al.; Thirty-two strains of thermophilic campylobacters isolated from marine recreational water and seven reference strains were biotyped and analysed by chromosomal DNA HaeIII ribopatterns and AP-PCR profiles based on a random 10-mer primer (5'-CAA TCG CCG T-3') . The majority of seawater isolates (90%) were Campylobacter coli, and three strains were Camp . jejuni . Southern blot hybridization analysis showed differences between the strains, and in a numerical analysis three main clusters were formed at the 45% similarity level, that corresponded to Camp . jejuni subsp . jejuni, Camp . coli, and a combination of Camp . coli and Camp . jejuni subsp . doylei . AP-PCR profiles also differentiated between the species but were less discriminatory than ribotyping because six strains (17%) could not be typed by this method . Numerical analysis gave four main clusters at the 45% similarity level, corresponding to Camp . jejuni subsp . jejuni, Camp . coli (two clusters) and Camp . lari . The study shows that strains within each species are diverse genomically . Both molecular methods were highly discriminatory, although some strains with identical ribotypes could be distinguished by AP-PCR, and they are valuable new alternatives to traditional typing in epidemiological studies of environmental campylobacters.

EMBO J, 1996 Feb 1, 15(3), 650 - 7
Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu; Rudinger J et al.; During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor . tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g . the SELB factor in prokaryotes . An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp . However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP . A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP . This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem . The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP . The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.

J Bacteriol, 1996 Feb, 178(3), 914 - 6
dTMP biosynthesis in Archaea; Nyce GW et al.; The biosynthesis of dTMP has been studied in cell extracts of two different members of the domain Archaea, Methanosarcina thermophila and Sulfolobus solfataricus . In M . thermophila, the dTMP was formed from dUMP and {methylene-2H2}-5,10-methylenetetrahydrosarcinapterin generated in situ from added {methylene-2H2} formaldehyde and the tetrahydrosarcinapterin present in the cell extract . In S . solfataricus, the 5,10-methyl-enetetrahydro derivative of a synthetic fragment of sulfopterin, the modified folate present in these cells, served as the C1 donor . These data indicate that the Archaea thymidylate synthases carry out the same basic reaction which occurs in other organisms but use the 5,10-methylenetetrahydro derivatives of modified folates as C1 donors.

J Bacteriol, 1996 Feb, 178(3), 723 - 7
Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method; Kotsuka T et al.; We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, by a suppressor mutation method . We previously constructed a chimeric IPMDH consisting of portions of thermophile and mesophile enzymes . The chimeric enzyme is less thermostable than the thermophile enzyme . The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T . thermophilus . The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained . The leuB genes from these three mutants were cloned and analyzed . The sequence analyses revealed Ala-172-->Val substitution in all of the mutants . The thermal stability of the thermophile IPMDH was improved by introducing the amino acid substitution.

Biochemistry, 1996 Jan 30, 35(4), 1195 - 200
Glycine-15 in the bend between two alpha-helices can explain the thermostability of DNA binding protein HU from Bacillus stearothermophilus; Kawamura S et al.; On the basis of sequence comparison of thermophilic and mesophilic DNA binding protein HUs, Bacillus stearothermophilus DNA binding protein HU (BstHU) seems to gain thermostability with a change in amino acid residues present on the molecular surface . To evaluate the contribution of exchange of each amino acid to the thermostability of BstHU, we constructed three mutants, BstHU-T13A (Thr13 to Ala), BstHU-G15E (Gly15 to Glu), and BstHU-T33L (Thr33 to Leu), in which the amino acids in BstHU were changed to the corresponding ones in Bacillus subtilis DNA binding protein HU (BsuHU) . Stability of the mutant proteins was determined from thermal-denaturation curves . Replacement of Gly15 located in the turn region between alpha 1 and alpha 2 helices (HTH motif), with Glu (BstHU-G15E), resulted in a decrease in thermostability, and the Tm value was 54.0 degrees C compared to the Tm value of 63.9 degrees C for BstHU . The mutants, BstHU-T13A and BstHU-T33L, were, by contrast, slightly more stable (Tm values of 67.0 and 65.6 degrees C for BstHU-T13A and BstHU-T33L, respectively) than the wild type . We then generated the BsuHU mutant protein BsuHU-E15G, where Glu15 in BsuHU was in turn replaced by Gly, and we analyzed the thermostability . This substitution clearly enhanced the melting temperature by 11.8 degrees C (Tm value: 60.4 degrees C for BsuHU-E15G) compared to the value for BsuHU (Tm: 48.6 degrees C) . Thus, Gly15 in the HTH motif of BstHU has an important role in the thermostability of BstHU . Characterization of the structure of the BstHU-G15E by 1H-NMR analysis showed that solvent accessibility of amide proton of Ala21 in the mutant was significantly increased compared with that of wild type, which means that the structure of the HTH motif in the N-terminal region in the mutant was changed to a more open conformation, thereby avoiding the interaction of Ala21 with either Ser17 by hydrogen bond or Ala11 by hydrophobic interaction.

FEBS Lett, 1996 Jan 29, 379(2), 127 - 31
Purification and structural characterization of the thermosome from the hyperthermophilic archaeum Methanopyrus kandleri; Andra S et al.; From Methanopyrus kandleri, the most thermophilic methanogen known so far, we have purified to homogeneity a protein complex of high molecular mass . Image analysis of transmission electron micrographs revealed a barrel-shaped particle composed of two rings with 8-fold symmetry . Only one type of subunit could be detected . The corresponding gene has been cloned and sequenced . The deduced amino acid sequence shows high homology with the members of group II chaperonins . The structure of the projection and the sequence homology suggest that this particle is the first thermosome isolated from a methanogen.

Biochim Biophys Acta, 1996 Jan 19, 1299(2), 167 - 74
Tetrahymena thermophila: analysis of phospholipids and phosphonolipids by high-field 1H-NMR; Adosraku RK et al.; The phospholipids of control and lipid-modified Tetrahymena thermophila were identified and quantified, using 1-D and 2-D COSY proton NMR spectroscopy on intact lipids, before and after HPLC separation . The results are comparable to those obtained using classical lipid analytical techniques . The results indicate that the study of enzyme pathways and other metabolic processes involving phospholipids in Tetrahymena and related protozoa can be carried out using proton NMR spectroscopy as the investigating technique.

Biochemistry, 1996 Jan 16, 35(2), 648 - 58
A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme; Bevilacqua PC et al.; A simple model system is described which mimics the second step of splicing and reverse cyclization reactions of the self-splicing intron from Tetrahymena thermophila . This model is based on the L-21 Sca I catalyzed ligation reaction between exogenously added oligomers: cucu + UCGa L-21 Sca I cucua + UCG . Steady-state kinetics for the forward and reverse direction were measured at 15 degrees C to find oligonucleotides that exhibit Michaelis-Menten behavior with acceptable KMS . CUCU and UCGA fit both criteria and were chosen for further studies . Steady-state kinetics reveal a lag that appears to be an RNA folding step that is eliminated by preincubation of the ribozyme with 2 mM and higher {Mg2+} and by UCGA . At constant ionic strength, the Mg2+ dependence of steady-state rates exhibits a sharp maximum near 5 mM Mg2+ . Pre-steady-state and steady-state kinetics, along with active-site titrations, explain the Mg2+ profile: the rate of reaction up to and including chemistry increases with Mg2+ concentration, while the fraction of active ribozyme and the rate of postchemistry steps decrease with Mg2+ concentration . The rate-limiting step at 5 mM Mg2+ for the reaction mimicking the second step of splicing is either chemistry or a conformational change before chemistry involving ribozyme bound with substrates . The rate-limiting step at 50 mM Mg2+ appears to be a postchemistry conformational change of the ribozyme or product release . At 50 mM Mg2+, single-turnover experiments support ordered binding of substrates with 5'-exon mimic binding before 3'-splice site mimic . Moreover, the 3'-splice site mimic binds and reacts in the presence of 5'-exon mimics predocked into the catalytic core . Results also indicate that Mg2+ ions associate with the ribozyme upon docking.

Biochemistry, 1996 Jan 16, 35(2), 593 - 600
Reactivity of a paramagnetic enzyme--CO adduct in acetyl-CoA synthesis and cleavage; Grahame DA et al.; Partial reactions of acetyl-CoA cleavage by the Methanosarcina barkeri acetyl-CoA decarbonylase synthase enzyme complex were investigated by UV--visible and electron paramagnetic resonance (EPR) spectroscopy . Reaction of the enzyme complex with carbon monoxide generated an EPR-detectable adduct with principal g values of 2.089, 2.076, and 2.028, and line widths of 13.76, 16.65, and 5.41 G, respectively . The EPR signal intensity was dependent upon both enzyme and carbon monoxide concentration . A second signal with gav = 2.050 was generated by storage of the CO-exposed enzyme for 17 months at -70 degrees C . Reaction of the enzyme complex with low levels of CO caused reduction of the enzyme complex, but did not result in immediate formation of the NiFeC signal (designated NiFeC based on isotopic substitution studies carried out by others in analogous systems from Clostridium thermoaceticum and Methanosarcina thermophila) . Further addition of CO generated the NiFeC signal, and the signal amplitude then increased progressively with increasing CO concentration . UV-visible spectra showed that enzyme Fe-S and corrinoid centers were already fully reduced at levels of CO significantly lower than needed for maximal EPR signal intensity . This result indicated that the EPR signal is formed by reaction of the reduced enzyme with CO (or a reduced one-carbon species), rather than with a one-carbon unit at the oxidation level of CO2 . Addition of coenzyme A, acetyl-CoA, or tetrahydrosarcinapterin had no effect on the EPR signal . In contrast, addition of N5-methyltetrahydrosarcinapterin (CH3-H4SPt) abolished the EPR signal . EPR spectra recorded at 20-21 K revealed that reaction with CH3-H4SPt affects only the enzyme NiFeC signal, and does not influence other EPR-detectable Fe-S center(s) . The results suggest that the enzyme--CO adduct reacts with CH3-H4SPt to form an EPR-silent enzyme-acetyl species . Preincubation of the enzyme complex with CO and CH3-H4SPt, both of which were required, produced an approximately 44-fold increase in the turnover rate of acetyl-CoA synthesis . The relevance of these findings to mechanisms involving possible reductive methylation of the enzyme and/or acetyl-enzyme formation is discussed.

J Biochem Biophys Methods, 1996 Jan 11, 31(1-2), 31 - 7
Rapid purification of two thermophilic proteinases using dye-ligand chromatography; Cowan DA et al.; Dye-ligand chromatography has been used successfully for the purification of extracellular thermostable proteinases from thermophilic Bacillus and Thermus cultures . Single step purification factors of up to 115-fold (for Thermus protease) and 2195-fold (for Bacillus protease) were obtained . Elution studies suggested that the mode of binding involved the enzyme active sites . The method was readily scaleable to 600 1 volume.

Dev Biol, 1996 Jan 10, 173(1), 174 - 84
Effects of the transcription inhibitor actinomycin D on postzygotic development of Tetrahymena thermophila conjugants; Ward JG et al.; During Tetrahymena thermophila conjugation, new somatic macronuclei develop from a common zygotic nucleus derived from meiotic products of the germline, and the old parental somatic nucleus is destroyed . The transcription inhibitor actinomycin D disrupts many events of postzygotic conjugation (cycloheximide causes indistinguishable effects) . Early treatment causes a block of all postzygotic development, suggesting a transcription requirement for conjugants to pass a checkpoint, allowing entry into postzygotic development . Thereafter, pair separation, resorption of the old macronucleus, and elimination of one of the new micronuclei are blocked if actinomycin D is added at least 1.5 hr before each of these events normally occurs . Treatment just before DNA rearrangements in the developing macronuclei (anlagen) causes aberrant anlage DNA loss, suggesting that this DNA loss may be caused by inhibition of gene expression involved in genome rearrangements . DNA loss, and correlated lethality, appear to require previous gene expression, since actinomycin D added earlier causes cells to arrest in development without anlage DNA loss, and these conjugants can (at some frequency) complete conjugation and make viable progeny once actinomycin D is removed . The old macronucleus already had been inactivated before most actinomycin D treatments were initiated, indicating that the various induced defects we observed are the result of inhibition of postzygotic gene expression, presumably in anlagen . The defects induced by actinomycin D are similar to defects previously observed in conjugants harboring nullisomic germline deficiencies but proficient old macronuclei.

FEBS Lett, 1996 Jan 8, 378(2), 199 - 201
Immunochemical detection of ADP-ribosylating enzymes in the archaeon Sulfolobus solfataricus; Faraone-Mennella MR et al.; Polyclonal antibodies raised against eukaryotic mono-(ADPribose)transferase and poly(ADPribose)polymerase were used to test the presence of antigenic determinants in a crude extract of Sulfolobus solfataricus, a thermophilic archaeon . Samples from eukaryotic (bull testis) and bacterial (E . coli) sources were analysed for comparison . All tested antibodies reacted with the sulfolobal sample with a specificity comparable to that of the eukaryotic preparation, as revealed by ELISA test, activity assays in the presence of antibodies and immunoblot experiments . After electrophoresis and western blot of sulfolobal proteins, a band at a mass around 50 kDa was detected by immunostaining.

Biochim Biophys Acta, 1996 Jan 4, 1292(1), 197 - 205
Properties and stabilization of an extracellular alpha-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN1: enzyme activity at 130 degrees C; Piller K et al.; An extracellular alpha-glucosidase from the thermophilic archaebacterium Thermococcus strain AN1 was purified 875-fold in five steps (Hiload Q-Sepharose, phenyl Sepharose, HPHT-hydroxyapatite, gel filtration and Mono Q chromatography) with a yield of 4% . It is a monomer with a molecular mass of about 60 kDa and a pI around 5 . At 98 degrees C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of 1% (w/v) dithiothreitol and 1% (w/v) BSA . Dithiothreitol (1%, w/v) and BSA (0.4%, w/v) also substantially increase the enzyme activity . The Km at 75 degrees C is 0.41 mM with pNP-alpha-D-glucopyranoside as substrate . The substrate preference of the enzyme is: pNP-alpha-D-glucoside > nigerose > panose > palatinose > isomaltose > maltose and turanose . No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and beta-gentiobiose . A variety of techniques including immobolization (e.g., on epoxy and glass beads), chemical modification (cross- and cocross-linking) and the use of additives (including polyhydroxylic molecules, BSA, salts, etc.) were applied to enhance stability at temperatures above 100 degrees C . The half-life could be increased from about 4 min at 100 degrees C to 30-60 min at 130 degrees C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and 1% (w/v) BSA, and by cross-linking with BSA in the presence of 90% (w/v) sorbitol . The stabilized enzyme showed good activity at 130 degrees C.

Ciba Found Symp, 1996, 202, 150 - 70; discussion 170-3
Fossilization processes in siliceous thermal springs: trends in preservation along thermal gradients; Cady SL et al.; To enhance our ability to extract palaeobiological and palaeoenvironmental information from ancient thermal spring deposits, we have studied the processes responsible for the development and preservation of stromatolites in modern subaerial thermal spring systems in Yellowstone National Park (USA) . We investigated specimens collected from silica-depositing thermal springs along the thermal gradient using petrographic techniques and scanning electron microscopy . Although it is known that thermophilic cyanobacteria control the morphogenesis of thermal spring stromatolites below 73 degrees C, we have found that biofilms which contain filamentous thermophiles contribute to the microstructural development of subaerial geyserites that occur along the inner rims of thermal spring pools and geyser effluents . Biofilms intermittently colonize the surfaces of subaerial geyserites and provide a favoured substrate for opaline silica precipitation . We have also found that the preservation of biotically produced microfabrics of thermal spring sinters reflects dynamic balances between rates of population growth, decomposition of organic matter, silica deposition and early diagenesis . Major trends in preservation of thermophilic organisms along the thermal gradient are defined by differences in the mode of fossilization, including replacement, encrustation and permineralization.

Ciba Found Symp, 1996, 202, 131 - 45; discussion 145-9
The Rhynie cherts: an early Devonian ecosystem preserved by hydrothermal activity; Trewin NH; The Rhynie cherts contain a remarkable early Devonian terrestrial to freshwater biota preserved in siliceous sinter by the action of a precious-metal-bearing hot spring system . Arthropods, vascular and non-vascular plants, algae, fungi and cyanobacteria are present . Preservation ranges from perfect 3D cellular permineralization to compacted coalified films, and can be related to both silicification processes and stages of biological and physical degradation of the plants at the time of silicification . Plants occasionally have original subaerial vertical axes preserved in growth position, and rhizomes bearing rhizoids . The plant litter of the substrate is also partly silicified . Silicification of organic material took place in hot spring pools, by surface flooding of areas with growing plants, and by permeation of the substrate . Sinters recognized include botryoidal geyserite typical of vent margins, and laminated sinter comparable with that of modern sinter terraces . Massive, vuggy, brecciated and nodular sinter textures are also present . At the microscopic level, textures associated with filamentous elements of the biota, and with the preservation of plants, closely match those present in modern sinters . Oxygen isotope and organic geochemical data from the Rhynie cherts indicate a temperature of 90-120 degrees C . This is apparently greater than the temperature at which elements of the biota were preserved and represents subsequent shallow burial in the hot spring system . The range of temperature and chemistry present at the surface provided high local environmental gradients . Current work attempts to identify thermophilic elements of the biota and document environmental zonation of biota relative to hot spring vents.

Ciba Found Symp, 1996, 202, 112 - 27; discussion 127-30
Ancient hydrothermal ecosystems on earth: a new palaeobiological frontier; Walter MR; Thermal springs are common in the oceans and on land . Early in the history of the Earth they would have been even more abundant, because of a higher heat flow . A thermophilic lifestyle has been proposed for the common ancestor of extant life, and hydrothermal ecosystems can be expected to have existed on Earth since life arose . Though there has been a great deal of recent research on this topic by biologists, palaeobiologists have done little to explore ancient high temperature environments . Exploration geologists and miners have long known the importance of hydrothermal systems, as they are sources for much of our gold, silver, copper, lead and zinc . Such systems are particularly abundant in Archaean and Proterozoic successions . Despite the rarity of systematic searches of these by palaeobiologists, already 12 fossiliferous Phanerozoic deposits are known . Five are 'black smoker' type submarine deposits that formed in the deep ocean and preserve a vent fauna like that in the modern oceans; the oldest is Devonian . Three are from shallow marine deposits of Carboniferous age . As well as 'worm tubes', several of these contain morphological or isotopic evidence of microbial life . The oldest well established fossiliferous submarine thermal spring deposit is Cambro-Ordovician; microorganisms of at least three or four types are preserved in this . One example each of Carboniferous and Jurassic sub-lacustrine fossiliferous thermal springs are known . There are two convincing examples of fossiliferous subaerial hydrothermal deposits . Both are Devonian . Several known Proterozoic and Archaean deposits are likely to preserve a substantial palaeobiological record, and all the indications are that there must be numerous deposits suitable for study . Already it is demonstrable that in ancient thermal spring deposits there is a record of microbial communities preserved as stromatolites, microfossils, isotope distribution patterns and hydrocarbon biomarkers.

Ciba Found Symp, 1996, 202, 24 - 32; discussion 32-9
Phylogenetic perspective on microbial life in hydrothermal ecosystems, past and present; Barns SM et al.; Understanding hydrothermal ecosystems, both past and present, requires basic information on the types of organisms present . Traditional methods, which require cultivation of microorganisms, fail to detect many taxa . We have used phylogenetic analyses of small subunit rRNA sequences obtained from microorganisms of a hot spring in Yellowstone National Park to explore the archael (archaebacterial) diversity present . Analysis of these sequences reveals several novel groups of archaea, greatly expanding our conception of the diversity of high temperature microorganisms, and demonstrating that hydrothermal systems harbour a rich variety of life . Many of these groups diverged from the archael line of descent early during evolution, and an understanding of their common properties may assist in inference of the nature of the last common ancestor of all life . The data also show a specific relationship between low-temperature marine archaea and some hot spring archaea, consistent with a thermophilic origin of life . Future use of rRNA-sequence-based techniques in exploration of hydrothermal systems should greatly facilitate study of modern thermophiles and give us insight into the activities of extinct communities as well.

Pneumonol Alergol Pol, 1996, 64 Suppl 1, 78 - 89
{The influence of organic dust on the chemotaxis of lung cells . Experimental studies}; Milanowski J; Mechanisms of chemotaxis of alveolar macrophages (AMs) and neutrophils (PMNs) in response to microbial products derived from organic dust were studied using blindwell chemotaxis chamber technique . Seven different agents (extract and endotoxin of Erwinia herbicola, extracts from Thermoactinomyces vulgaris and Aspergillus fumigatus, thermophilic protease and two preparations of glucans), were used for experiments . These agents were evaluated for their ability to direct attraction of AMs and PMNs and stimulation of alveolar macrophages to release chemotactic factors for other AMs and PMNs . The microbial products were able to attract both AMs and PMNs directly in a dose-dependent manner and the exposure of cultured AMs to most agents were stimulatory for production of chemotactic activity for AMs and PMNs . The generation and release of this activity by AMs may provide a mechanisms for the initiation and amplification of inflammatory reactions in the lung after inhalation of organic dust . Results of these in vitro studies may be relevant to the pathogenesis of alveolitis in organic dust-induced lung diseases.

Biochimie, 1996, 78(11-12), 915 - 9
A ribosomal protein from Thermus thermophilus is homologous to a general shock protein; Gryaznova OI et al.; The gene encoding the ribosomal protein from Thermus thermophilus, TL5, which binds to the 5S rRNA, has been cloned and sequenced . The codon usage shows a clear preference for G/C rich codons that is characteristic for many genes in thermophilic bacteria . The deduced amino acid sequence consists of 206 residues . The sequence of TL5 shows a strong similarity to a general shock protein from Bacillus subtilis, named CTC . The protein CTC is homologous in its N-terminal part to the 5S rRNA binding protein, L25, from E coli . An alignment of the TL5, CTC and L25 sequences displays a number of residues that are totally conserved . No clear sequence similarity was found between TL5 and other proteins which are known to bind to 5S rRNA . The evolutionary relationship of a heat shock protein in mesophiles and a ribosomal protein in thermophilic bacteria as well as a possible role of TL5 in the ribosome are discussed.

Folia Microbiol (Praha), 1996, 41(2), 165 - 74
Purification and properties of two forms of glucoamylase from Aspergillus niger; Amirul AA et al.; A . niger produced alpha-glucosidase, alpha-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source . The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography . The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively . The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5-9.0 . Both enzymes were thermophilic in nature with temperature optimum of 60 and 65 degrees C, respectively, and were stable for 1 h at temperatures of up to 60 degrees C . The kinetic parameters Km and V showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose . GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.

Folia Microbiol (Praha), 1996, 41(2), 146 - 8
Influence of culture conditions on thermostable lipase production by a thermophilic alkalitolerant strain of Bacillus sp; Kambourova M et al.; The effect of different culture conditions on thermostable lipase production by Bacillus sp . was studied in shake flasks . A maximum enzyme activity of 67-75 nkat/mL was observed in a medium consisting of 0.5% soybean flour and 0.1% stearyl glycerol esters or natural fats . A lipase activity of about 117 nkat/mL was established when the cultivation was carried out in a laboratory fermentor at 20% minimal dissolved oxygen level, the enzyme production being increased 1.5 fold compared to that in a flask culture.

Folia Microbiol (Praha), 1996, 41(1), 26 - 8
Lowering of ochratoxin A level in milk by yoghurt bacteria and bifidobacteria; Skrinjar M et al.; A decrease in the content of ochratoxin A (OA) was observed in milk samples fermented by yoghurt bacteria and bifidobacteria . OA was added to the milk before fermentation at a rate of 0.05-1.5 mg/L . No residues of OA were found in samples containing 0.05 and 0.1 mg/L of OA, fermented by S . Salivarius subsp . thermophilus, L . delbrueckii subsp . bulgaricus and B . bifidum . Yoghurt bacteria (S . salivarius subsp . thermophilus and L . delbrueckii subsp . bulgaricus) were the most effective since no residues were detected even in fermented samples containing originally 0.5 mg/L OA.

Annu Rev Genet, 1996, 30, 557 - 78
Genome downsizing during ciliate development: nuclear division of labor through chromosome restructuring; Coyne RS et al.; The ciliated protozoa divide the labor of germline and somatic genetic functions between two distinct nuclei . The development of the somatic (macro-) nucleus from the germinal (micro-) nucleus occurs during sexual reproduction and involves large-scale, genetic reorganization including site-specific chromosome breakage and DNA deletion . This intriguing process has been extensively studied in Tetrahymena thermophila . Characterization of cis-acting sequences, putative protein factors, and possible reaction intermediates has begun to shed light on the underlying mechanisms of genome rearrangement . This article summarizes the current understanding of this phenomenon and discusses its origin and biological function . We postulate that ciliate nuclear restructuring serves to segregate the two essential functions of chromosomes: the transmission and expression of genetic information.

Biochimie, 1996, 78(7), 605 - 23
Aspartate identity of transfer RNAs; Giege R et al.; Structure/function relationships accounting for specific tRNA charging by class II aspartyl-tRNA synthetases from Saccharomyces cerevisiae, Escherichia coli and Thermus thermophilus are reviewed . Effects directly linked to tRNA features are emphasized and aspects about synthetase contribution in expression of tRNA(Asp) identity are also covered . Major identity nucleotides conferring aspartate specificity to yeast, E coli and T thermophilus tRNAs comprise G34, U35, C36, C38 and G73, a set of nucleotides conserved in tRNA(Asp) molecules of other biological origin . Aspartate specificity can be enhanced by negative discrimination preventing, eg mischarging of native yeast tRNA(Asp by yeast arginyl-tRNA synthetase . In the yeast system crystallography shows that identity nucleotides are in contact with identity amino acids located in the catalytic and anticodon binding domains of the synthetase . Specificity of RNA/protein interaction involves a conformational change of the tRNA that optimizes the H-bonding potential of the identity signals on both partners of the complex . Mutation of identity nucleotides leads to decreased aspartylation efficiencies accompanied by a loss of specific H-bonds and an altered adaptation of tRNA on the synthetase . Species-specific characteristics of aspartate systems are the number, location and nature of minor identity signals . These features and the structural variations in aspartate tRNAs and synthetases are correlated with mechanistic differences in the aminoacylation reactions catalyzed by the various aspartyl-tRNA synthetases . The reality of the aspartate identity set is verified by its functional expression in a variety of RNA frameworks . Inversely a number of identities can be expressed within a tRNA(Asp) framework . From this emerged the concept of the RNA structural frameworks underlying expression of identities which is illustrated with data obtained with engineered tRNAs . Efficient aspartylation of minihelices is explained by the primordial role of G73 . From this and other considerations it is suggested that aspartate identity appeared early in the history of tRNA aminoacylation systems.

Biomed Pharmacother, 1996, 50(6-7), 306 - 8
Physical and biochemical changes of thermal mud after maturation; Galzigna L et al.; Thermal mud is a therapeutic agent whose antirheumatic effectiveness is optimized by a process of maturation . The maturation of thermal mud was followed at 48 degrees C under controlled conditions by measuring physical and biochemical changes due to the growth of colonizing thermophilic microorganisms . Thermogravimetric measurements allowed us to identify the building up of an organic component including phospholipids and in particular a previously recognized sulfoglycolipid, which was further purified . The compound may be responsible for the antirheumatic effect of the mud and is produced by the colonizing species which develop in a period of maturation subsequent to that of production of photosynthetic pigments.

Comp Biochem Physiol B Biochem Mol Biol, 1996 Jan, 113(1), 157 - 62
Control of ornithine decarboxylase activity by polyamines and absence of antizyme in Tetrahymena; Koguchi K et al.; 1 . In cells of Tetrahymena pyriformis and thermophila, ODC activity was significantly suppressed but ODC decay was not stimulated by putrescine . 2 . Free antizyme and ODC-antizyme complex were both not detected in extracts of cells of T . pyriformis treated with putrescine . 3 . It was concluded that in Tetrahymena, unlike vertebrate cells, ODC is not subject to polyamine-induced destabilization mediated by antizyme.

Nutr Cancer, 1996, 26(3), 365 - 80
Lactobacillus- and bifidobacterium-mediated antigenotoxicity in the colon of rats; Pool-Zobel BL et al.; Lactic acid bacteria (LAB) are proposed to have several beneficial effects, including the inactivation of carcinogens . We have studied the potential of Lactobacillus acidophilus (from a commercially available yogurt), Lactobacillus gasseri (P79), Lactobacillus confusus (DSM20196), Streptococcus thermophilus (NCIM 50083), Bifidobacterium breve and Bifidobacterium longum (from human infant stool) to prevent the induction of DNA damage by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 7.5 mg/kg body wt) in colon cells of the rat . Using the new technique of single cell microgel electrophoresis, all investigated strains were antigenotoxic toward MNNG after a single dose of 10(10) viable cells/kg body wt p.o . eight hours before the carcinogen . One-half and one-tenth of this initial dose resulted in a loss of protective activity . High doses of heat-treated L . acidophilus strains were also not antigenotoxic . One mechanism of the preventive effect could be that bacterial metabolites or components are responsible . Accordingly, selected examples were investigated in vitro in colon cells of the rat . Metabolically active L . acidophilus cells, as well as an acetone extract of the culture, prevented MNNG-induced DNA damage . Different cell fractions from L . acidophilus (cytoplasm, cell wall skeleton, cell wall) were devoid of antigenotoxic activity, whereas the peptidoglycan fraction and whole freeze-dried cells were antigenotoxic . As a second carcinogen, 1,2-dimethylhydrazine (DMH) was used . A dose- and time-response study was first performed to assess the effects of DMH in several segments of the gastrointestinal (GI) tract . Exposure for 16 hours to 15 or 25 mg DMH/kg body wt p.o . induced DNA damage in cells of the distal colon of rats, whereas no cytotoxicity was seen . Pretreatment orally with LAB on four consecutive mornings before DMH gavage (8 hours after the last LAB application) revealed that L . acidophilus, L . confusus, L . gasseri, B . longum, and B . breve inhibited the genotoxic effect of DMH . One of four S . thermophilus and one of three Lactobacillus delbrueckeii ssp . bulgaricus strains were also protective . Heat-treated L . acidophilus did not inhibit DMH-induced genotoxicity . A few aliquots of the colon cells were processed immunohistochemically for the presence of the "proliferation cell nuclear antigen" (PCNA) . DMH treatment did not increase PCNA, nor was there any modulation by LAB . The effect of L . acidophilus on foreign compound-metabolizing enzymes (Phase I and Phase II) in liver and colon cells of rats revealed only one parameter to be modulated, namely, a two- to three-fold increase in the levels of NADPH-cytochrome P-450 reductase . The meaning of this finding, in terms of possible chemoprevention by LAB, remains unclear . In conclusion, our studies show that most, but not all, LAB tested could strongly inhibit genotoxicity in the GI tract of the rat and that viable LAB organisms are required for the protective effect in vivo . The comet assay technique is a powerful tool to elucidate such in vivo antigenotoxic activities in tumor target tissues.

J Biochem (Tokyo), 1996 Jan, 119(1), 135 - 44
An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8; Okamoto A et al.; The aspartate aminotransferase gene (AspAT, EC 2.6.1.1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced . The purified T . thermophilus AspAT was stable up to about 80 degrees C at neutral pH . T . thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids . The gene coding for T . thermophilus AspAT showed that it comprised 1,155 bp with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42,050 . The amino acid sequence of T . thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp . YM-2, and Sulfolobus solfataricus, respectively . When the amino acid sequence of T . thermophilus AspAT was compared with that of E . coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures . Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37 . As shown by the E . coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule . This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule . Different strategies of different proteins as to prolyl contribution to thermostability have been suggested . Despite the high degree of conservation of active-site residues, Arg292 in E . coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T . thermophilus AspAT . Arg89 may complement the function of Arg292.

J Biochem (Tokyo), 1996 Jan, 119(1), 80 - 4
Nucleotide sequence of the uricase gene from Bacillus sp . TB-90; Yamamoto K et al.; The nucleotide sequence of the uricase gene from the thermophilic bacterium Bacillus sp . TB-90 was determined . The primary structure of the uricase deduced from the nucleotide sequence comprised 332 amino acids, with a total molecular mass of 37,994 Da . The molecular mass of the subunit of the uricase produced by the transformant of Escherichia coli agreed well with this value . However, the molecular mass of a subunit of the uricase produced by Bacillus sp . TB-90 was found to be 34,000 Da by SDS-PAGE . The difference between these molecular masses was attributed to processing of the C-terminal 13 amino acid residue in Bacillus sp . TB-90 . Comparison of the enzymatic properties of both uricases showed that the thermostability of the uricase produced by the transformant was enhanced by about 10 degrees C in comparison to that produced by Bacillus sp . TB-90.

Int J Vitam Nutr Res, 1996, 66(3), 244 - 9
The effect of 6'-galactooligosaccharides on bone mineralization of rats adapted to different levels of dietary calcium; Chonan O et al.; 6'-galactooligosaccharides (6'-GOS), a mixture of galactosyl oligosaccharides formed from lactose by the transgalactosyl reaction with beta-D-galactosidase derived from Aspergillus oryzae and Streptococcus thermophillus, are unhydrolyzed in the small intestine and are fermented by the intestinal bacteria . The effects of 6'-GOS on calcium (Ca) absorption and bone mineralization were examined in male Wistar rats adapted to different levels of dietary Ca for 30 days . Dietary 6'-GOS (5 g/100 g of diet) were more potent than control in stimulating Ca absorption in rats fed the Normal-Ca diet (0.5 g of Ca/100 g of diet) after 8-10 days and 18-20 days, and the bone (femur and tibia) Ca content of rats fed the Normal-Ca diet with 6'-GOS were significantly higher than those of the control animals . However, in rats fed the Low-Ca diet (0.05 g of Ca/100 g of diet), 6'-GOS feeding did not affect both the absorption of Ca and the bone mineralization . Ca content in the liquid phase of the cecal digesta was significantly elevated by 6'-GOS feeding in the rats fed the Normal-Ca diet, however, this was unchanged in the rats fed the Low-Ca diet . We conclude that the effect of 6'-GOS on the bone mineralization is affected by dietary Ca concentration used in the experiment, and the stimulatory effect of 6'-GOS on Ca absorption may be partly associated with increased solubility of Ca in the intestinal digesta.

Biol Cell, 1996, 86(2-3), 111 - 20
Developmentally programmed DNA rearrangement in Tetrahymena thermophila: isolation and sequence characterization of three new alternative deletion systems; Chau MF et al.; Extensive developmentally programmed DNA rearrangements, including thousands of internal deletions, occur in the differentiating somatic macronucleus in Tetrahymena thermophila . Some deletion systems involve the use of multiple alternative deletion sites . We report here the cloning and the sequences of three new alternative deletion systems (RR, RP and B) obtained using genomic subtraction . The RP and RR deletion systems are 2 kb apart on chromosome 1R, and both involve the removal of < 2 kb of micronuclear sequences . The B deletion system is on chromosome 5 and involves a deletion of > 5 kb . All three deleted regions are very AT rich (approximately 80%) and do not appear to encode any protein . Sequences of the regions flanking the deletion junctions of all three systems revealed no sequence similarity among them nor with any previously reported deletion systems, suggesting that different cis-acting elements are involved for rearrangement . Unlike other deletion systems in ciliates, the B deletion system lacks short terminal direct repeats . Our results suggest an average of at least one alternative deletion system per 134 kb of micronuclear DNA and lead to an estimate that at least 25% of all deletion systems in Tetrahymena utilize alternative ends . The genomic subtraction method employed in this study could prove useful for the isolation of alternatively deleted DNA in special-purpose cases in Tetrahymena and other ciliates . The hybridization parameters for genomic subtraction worked out here for highly AT-rich DNA may have wider usefulness.

Microbiol Immunol, 1996, 40(8), 553 - 60
Role of intestinal bacteria in ileal ulcer formation in rats treated with a nonsteroidal antiinflammatory drug; Uejima M et al.; The role of intestinal bacteria in induction and repression of ulcer formation in the ileum of rats treated with one of the nonsteroidal antiinflammatory drugs (NSAIDs), 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl) thiophene (BFMeT), was examined in this study . BFMeT was administered by intragastric gavage once at doses of 500-1,500 mg/kg of body weight to Wistar rats treated with and without antibiotics (bacitracin, neomycin, streptomycin), germ-free rats and gnotobiotic rats, and 72 hr later their gastrointestinal tracts were examined for ulcer formation . A single oral administration of BFMeT induced ileal ulcers in specific pathogen-free rats . However, the rats given antibiotics to reduce the intestinal bacteria had no ulcers . BFMeT-treated germ-free rats and gnotobiotic rats mono-associated with Bifidobacterium adolescentis or Lactobacillus acidophilus also had no intestinal ulcers . However, the drug induced ileal ulcers in gnotobiotic rats mono-associated with Eubacterium limosum or Escherichia coli . An overnight culture of B . adolescentis or L . acidophilus or yogurt containing Bifidobacterium breve and Streptococcus thermophilus, when given as drinking water, inhibited ulcer formation in the ileum of rats treated with BFMeT . Gram staining of the ileal contents of normal rats revealed that 97.4% of the stained microorganisms were Gram-positive rods and only 1.2% were Gram-negative rods . In the group of rats with ulcers induced by BFMeT, the Gram-positive rods decreased by 56.4% and the Gram-negative rods including Escherichia coli, Klebsiella, Proteus and Bacteroides increased by 37.3% . However, in the group of rats administered the Bifidobacterium culture, the Lactobacillus culture or yogurt, the percentages of the Gram-negative rods were decreased . Although Lactobacillus was a major bacterium in the ileum of normal rats, the Gram-negative facultatively anaerobic rods E.coli, Klebsiella and Proteus were increased in the ulcerated ileum of rats treated with BFMeT, suggesting that these bacteria are associated with ulcer formation in rats treated with NSAIDs, and that Lactobacillus and Bifidobacterium inhibit it by repressing the growth of ulcer-inducing bacteria.

Microbiol Immunol, 1996, 40(1), 55 - 8
Tumor-specific transplantation resistance in mice after treatment of initial tumors with Streptococcus thermophilus; Kaklij GS et al.; Antitumor activity observed by treatment with Streptococcus thermophilus was further investigated . The mice cured from fibrosarcoma by treatment with heat-killed preparation of S . thermophilus, when challenged with fibrosarcoma failed to take up the tumor . However, these cured mice when challenged with sarcoma-180 or Ehrlich ascites carcinoma, did not show significant changes in tumor take and/or survival compared to their respective controls . Similarly, mice cured from sarcoma-180 were challenged with fibrosarcoma, sarcoma-180 or Ehrlich ascites carcinoma . Though there was no change in the mean survival time (MST) of the dying mice regarding sarcoma-180 or Ehrlich ascites carcinoma, there was 50 and 30% increase in the number of mice that showed total regression respectively over controls . However, there was no difference in the growth rate of fibrosarcoma . Similar observations were made with mice cured from Ehrlich ascites carcinoma, challenged with these tumors . These findings thus suggest that the antitumor response was tumor-specific and that tumor-associated antigens may have a role in imparting this specificity . Bacterial treatment non-specifically augmented this primary response.

PDA J Pharm Sci Technol, 1996 Jan-Feb, 50(1), 51 - 4
Discussion of the Z-value to use in calculating the F0-value for high-temperature sterilization processes; Pflug IJ; The appropriate z-value to use in integrating heat process time-temperature data in the temperature range of 120.0-140.0 degrees C (248.0-284.0 degrees F) is discussed . We conclude that for control of Clostridium botulinum there is little risk in extrapolating a public health F0-value for C . botulinum to temperatures in the 132.0-138.0 degrees C (270.0-280.0 degrees F) range using a z-value of 10.0 degrees C (18.0 degrees F) . It would seem prudent, at this time, when extrapolating data to conditions in the 132.0-138.0 degrees C (270.0-280.0 degrees F) range, that as a starting point an F0-value of 3.0 minutes be used as the minimum public health process . A design z-value of 10.0 degrees C (18.0 degrees F) is appropriate for Clostridium sporogenes to temperatures of 140.0 degrees C (284.0 degrees F) . To control thermophilic microorganisms such as Bacillus stearothermophilus and Bacillus coagulans with processes at temperatures from 120.0-140.0 degrees C (248.0-284.0 degrees F), the effective z-value will be in the range of 7.0-8.0 degrees C (12.6-14.4 degrees F) instead of 10.0 degrees C (18.0 degrees F) . This means that when we design and calculate processes at temperatures from 120.0-140.0 degrees C (248.0-284.0 degrees F), using a z-value of 10.0 degrees C (18.0 degrees F), the lethal effect against these organisms will be much larger than indicated by the F0-value of the process.

Braz J Med Biol Res, 1996 Jan, 29(1), 95 - 9
Recent progress in understanding the temporal behavior of unicellular organisms; Balzer I; This survey summarizes the findings concerning endogenous oscillations of three unicellular organisms: the dinophyte Gonyaulax polyedra, the ciliate Tetrahymena thermophila and the euglenophyte Euglena gracilis . All of them behave rhythmically and show the common features of zeitgeber action, differential sensitivity and temperature compensation; however, they exhibit some species-specific peculiarities that make each of them suitable for addressing particular chronobiological questions . Although ultradian rhythms have been described for Tetrahymena thermophila and Euglena gracilis, they appear under different conditions: in the first case, a modulation of the period in relation to the concentration of nutrients is observed, whereas Euglena oscillates in an ultradian and circadian fashion simultaneously . Transitions between periodic and aperiodic states can be induced in Euglena gracilis and Gonyaulax polyedra: Euglena gracilis can enter an aperiodic state after repeated exposure to short light pulses (up to 10 sec) given at intervals of 40 min or less, whereas in Gonyaulax polyedra the circadian oscillator is arrested at temperatures below 12 degrees C . In the arrhythmic state, the oscillator might be driven into singularity within the phase space of a limit cycle attractor; re-initiation from the holding point occurs by transition to a relatively precisely defined new phase . Photoperiodism as another important chronobiological phenomenon can be studied in Gonyaulax polyedra: cells enter the dormant stage of an asexual cyst under short days and a temperature below 16 degrees C . This response can be mimicked by 5-methoxylated indoleamines such as melatonin and 5-methoxytryptamine, which are synthesized by this organism . Melatonin concentration exhibit an endogenous circadian rhythm characterized by a rapid increase shortly after the onset of darkness . Encystment, as induced by indoleamines, is associated with stimulations of bioluminescence . The coupling of the two processes involves, as a common element, the release of protons from an acidic vacuole.

Antonie Van Leeuwenhoek, 1996 Jan, 69(1), 1 - 14
Limitations of thermophilic anaerobic wastewater treatment and the consequences for process design; van Lier JB; Thermophilic anaerobic digestion offers an attractive alternative for the treatment of medium- and high-strength wastewaters . However, literature reports reveal that thermophilic wastewater treatment systems are often more sensitive to environmental changes than the well-defined high-rate reactors at the mesophilic temperature range . Also, in many cases a poorer effluent quality is experienced while the carry over of suspended solids in the effluent is relatively high . In this paper recent achievements are discussed regarding the process stability of thermophilic anaerobic wastewater treatment systems . Laboratory experiments reveal a relatively low sensitivity to temperature changes if high-rate reactors with immobilized biomass are used . Other results show that if a staged process is applied, thermophilic reactors can be operated for prolonged periods of time under extreme loading conditions (80-100 kg chemical oxygen demand.m-3.day-1), while the concentrations of volatile fatty acids in the effluent remain at a low level.

J Dairy Sci, 1996 Jan, 79(1), 33 - 43
Modulation of proliferation, second messenger levels, and morphotype expression of the rat intestinal epithelial cell line IEC-6 by fermented milk; Thoreux K et al.; Trophic effects of milk fermented with Lactobacillus helveticus, Lactobacillus paracasei ssp . paracasei, Bifidobacterium sp., or the combination of Lactobacillus bulgaricus and Streptococcus thermophilus (yogurt) were studied on the IEC-6 intestinal epithelial cell line . Incorporation of {methyl-3H}thymidine, mitochondrial dehydrogenase activities, cyclic AMP production, and differentiation of levels of the IEC-6 strain were evaluated between the 15th and 30th passage in culture . All fermented and unfermented milks enhanced trophic responses of IEC-6 cells in a dose-dependent manner . Compared with the corresponding milks, supernatant fractions were more effective in stimulating mitochondrial dehydrogenase response . Fermented milk supernatants were also more effective than the corresponding unfermented fractions . Increases in DNA synthesis and cyclic AMP confirmed the activation observed with mitochondrial dehydrogenase . Yogurt induced the more trophic response with an increased number of the more differentiated cell morphotype . Fermentation with L . casei also demonstrated an important trophic adaptation of IEC-6 cells . Milk processing by lactic acid bacteria enhanced trophic and proliferation responses of intestinal epithelial cell line IEC-6 . These results suggested that IEC-6 cells could represent an accurate and easy in vitro model for testing the trophic quality of various nutrients and for an optimization of physiological digestive functions.

Appl Biochem Biotechnol, 1996 Spring, 57-58, 599 - 604
Cellulose degradation and ethanol production by thermophilic bacteria using mineral growth medium; Ahn HJ et al.; Growth of thermophilic cellulase-utilizing bacteria in a vitamin-free growth medium is reported for both a previously described strain, Clostridium thermoclelum 31549, and now isolates HJA1 and HJA2 . Formation of fermentation products with and without vitamins was similar for strains HJA1 and HJA2 as well as for the enrichment cultures from which these stains were derived . Strain HJA2 was maintained in continuous culture on a vitamin-free mineral medium with Avicel as the carbon source for over a week . At a 38 h residence time, Avicel conversion was higher (81%) at pH 6.42 than at pH 6.97 (73%) or at 6.01 (58%) . Ethanol and acetate were produced in significant amounts by strain JHA2 at all pH values tested (6.97, 6.42, 6.01) . Lactic acid was the primary fermentation product at pH 6.97, but was not a significant product at both the lower values . Efforts to grow thermophilic, cellulose-utilizing bacteria at pH < 6.0 were unsuccessful for described strains, new isolates, and enrichment cultures.

J Vet Med Sci, 1996 Jan, 58(1), 85 - 6
Phagocytosis of splenetic neutrophils of mice enhanced by orally administered peptidoglycan from Bifidobacterium thermophilum; Sasaki T et al.; A preparation of peptidoglycan (PG) of swine Bifidobacterium thermophilum was orally administered to SPF-BALB/C and ICR mice and its effect on phagocytosis splenetic neutrophils from PG administered mice was measured by chemiluminescent response (CL) and fluorometric analysis and the result was compared with that of non-treated mice . PG stimulated phagocytosis of neutrophils in a dose-dependent manner, whereas dosage exceeding the optimum concentration (500 microgram) inhibited phagocytosis . The maximum effect on phagocytosis of neutrophils was observed at 3 days after administration of PG 500 microgram . The result of fluorometric analysis was almost similar to that of CL . These results indicate that orally administered PG enhances the activity of the phagocytosis of splenetic neutrophils from mice.

Vet Res, 1996, 27(1), 3 - 12
Antagonism of lactic acid bacteria towards Staphylococcus aureus and Escherichia coli on agar plates and in milk; Fang W et al.; The antagonistic effect of lactic acid bacteria (LAB, including Lactobacillus acidophilus, L . bulgaricus, L . casei and Streptococcus thermophilus) on Staphylococcus aureus and Escherichia coli was evaluated on MRS agar with the deferred and cross-streaking techniques, and in milk with the plate counting method . All LAB were repressive to S aureus and E coli on the agar medium . However, their suppressive activity was significantly reduced when the agar medium was buffered to pH 7.2 . In normal milk, L acidophilus strains A and B, S thermophilus and its combinations with L acidophilus A and L bulgaricus 6032 were inhibitory to S aureus, while in mastitic milk, only S thermophilus and its combinations showed inhibition . L acidophilus A and L bulgaricus 34104 were repressive to E coli growth in normal milk . S thermophilus and its combinations were inhibitory to E coli in both the normal and mastitic milk samples . These results indicate that the antagonistic activity of LAB on pathogenic bacteria varied with the type of media in which the tests were done, and that testing of in vitro antagonism in milk would be more informative than that in artificial media for in vivo tests concerning the possible roles of competitive microbiological ecology in mastitis control.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 137 - 42
A leader open reading frame is essential for the expression in Escherichia coli of GC-rich leuB gene of an extreme thermophile, Thermus thermophilus; Ishida M et al.; To improve expression efficiency of the leuB gene of an extreme thermophile . Thermus thermophilus, in Escherichia coli, the gene was placed under a potent promoter, tac . However, the expression was hardly improved, despite increased transcription . The expression under tac promoter was significantly improved by introducing a leader open reading frame in front of the gene . Similar improvement under a weak promoter, tet, with a leader open reading frame had been described previously . The present results provide evidence that the major limiting step in the expression of a GC-rich thermophile gene in E . coli is translation, and that the addition of a leader open reading frame is more crucial for high level expression of the gene than the use of a potent promoter.

Can J Microbiol, 1996 Jan, 42(1), 1 - 5
Purification and characterization of a beta-glucosidase from solid-state cultures of Humicola grisea var . thermoidea; Ferreira Filho EX; The thermophilic fungus Humicola grisea var . thermoidea produced beta-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source . A beta-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) on a 12.5% (w/v) slab gel . The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS-PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits . The purified enzyme was thermostable at 60 degrees C for 1 h with a half-life of 15 min at 65 degrees C, and displayed optimum activity at 60 degrees C and a pH range . of 4.0-4.5 . The Km and Vmax values for p-nitrophenyl beta-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU.mL-1, respectively . D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+ inhibited beta-glucosidase activity . The enzyme activity was competitively inhibited by D-glucose (ki = 0.6 mM) . The purified enzyme was very active against cellobiose and p-nitrophenyl beta-D-glucopyranoside.

Int J Syst Bacteriol, 1996 Jan, 46(1), 265 - 9
Fervidobacterium gondwanense sp . nov., a new thermophilic anaerobic bacterium isolated from nonvolcanically heated geothermal waters of the Great Artesian Basin of Australia; Andrews KT et al.; A new thermophilic, carbohydrate-fermenting, obligately anaerobic bacterial species was isolated from a runoff channel formed from flowing bore water from the geothermally heated aquifer of the Great Artesian Basin of Australia . The cells of this organism were nonsporulating, motile, gram negative, and rod shaped and generally occurred singly or in pairs . The optimum temperature for growth was 65 to 68 degrees C, and no growth occurred at temperatures below 44 degrees C or above 80 degrees C . Growth was inhibited by 10 micrograms of lysozyme per ml, 10 micrograms of penicillin per ml, 10 micrograms of tetracycline per ml, 10 micrograms of phosphomycin per ml, 10 micrograms of vancomycin per ml, 10 micrograms of vancomycin per ml, and NaCl concentrations greater than 0.2% . The optimum pH for growth was 7.0, and no growth occurred at pH 5.5 or 8.5 . The DNA base composition was 35 mol% guanine plus cytosine, as determined by thermal denaturation . The end products of glucose fermentation were lactate, acetate, ethanol, CO2, and H2 . Sulfur, but not thiosulfate, sulfite, or sulfate, was reduced to sulfide . Phase-contrast microscopy of whole cells and an electron microscopic examination of thin sections of cells revealed the presence of single terminal spheroids, a trait common in members of the genus Fervidobacterium . However, a phylogenetic analysis of the 16S rRNA sequence revealed that the new organism could not be assigned to either of the two previously described Fervidobacterium species . On the basis of these observations, we propose that the new organism should be designated a new Fervido-bacterium species, Fervidobacterium gondwanense . The type strain of this species is strain AB39 (= Australian Collection of Microorganisms strain ACM 5017.

Int J Syst Bacteriol, 1996 Jan, 46(1), 123 - 7
Characterization of a new obligately anaerobic thermophile, Thermoanaerobacter wiegelii sp . nov; Cook GM et al.; An obligately anaerobic, extremely thermophilic Thermoanaerobacter species was isolated from a freshwater pool formed from a geothermally heated (56 to 69 degrees C) water outlet in Government Gardens, Rotorua, New Zealand . This organism was a spore-forming, gram-negative, rod-shaped bacterium . Strain Rt8.B1T (= DSM 10319T) (T = type strain) fermented a wide variety of mono-, di-, and polysaccharides and produced ethanol, acetate, lactate, propionate, and hydrogen . Sugar alcohols were also fermented, but organic acids and amino acids were not utilized . On the basis of its morphological characteristics, DNA G + C content, obligately anaerobic, thermophilic, polysaccharolytic nature, and levels of 16S rRNA sequence homology, we propose that strain Rt8.B1T should be classified in the genus Thermoanaerobacter as a new species, Thermoanaerobacter wiegelii.

FASEB J, 1996 Jan, 10(1), 84 - 92
Stability and folding of ultrastable proteins: eye lens crystallins and enzymes from thermophiles; Jaenicke R; Soluble globular proteins exhibit marginal stabilities, equivalent to only a few weak intermolecular interactions . Extreme conditions in the biosphere, as well as acute physiological stress, require either mutative adaptation or stabilization by accessory proteins or extrinsic factors such as metabolites, cofactors, or compatible solvent components . No general strategies of stabilization have yet been established . However, certain contributions to stability have been elucidated by analyzing extremely stable proteins, such as crystallins from the eye lens, or proteins from hyperthermophilic microorganisms . Relating the structure and stability of homologous proteins from mesophiles and extremophiles, it becomes clear that stability increments may accumulate from 1) local interactions, 2) secondary or supersecondary structure, 3) packing and docking of domains, 4) association of subunits, and 5) conjugation with prosthetic groups, carbohydrate moieties, or nucleic acids, etc . Single and multiple point mutations, nicking and swapping of folding units in domain proteins, grafting of linker peptides between domains, and dissociation-reassociation of oligomeric proteins give insight into the cumulative nature of protein stability and its relation to the hierarchy of protein structure and folding . In this review, beta gamma-crystallins and enzymes from hyperthermophilic microorganisms are used as models to discuss mechanisms of protein stabilization.

Ann Allergy Asthma Immunol, 1996 Jan, 76(1), 41 - 50
Microflora and acarofauna of bed dust from homes in Upper Silesia, Poland; Horak B et al.; BACKGROUND: Pyroglyphid mites are considered a major cause of house dust allergy . The occurrence and possible pathogenic role of other biologic components of house dust, in particular bacteria, has received less attention . OBJECTIVE: The aim of this study was to examine bacteria present in the samples of house dust from beds, in comparison to fungi and mites recovery . METHODS: Samples of bed dust were collected from 40 homes in Upper Silesia (Poland) . Of these, 19 came from the homes of people with asthma caused by house dust and 21 from the homes of people without allergy . The concentrations of bacteria, fungi, mites, and endotoxin and species composition of microflora and acarofauna were determined . RESULTS: The overall mean concentrations of mesophilic bacteria, thermophilic bacteria and fungi, including yeasts, were, respectively, 1.6 x 10(6), 1.7 x 10(3), and 1.6 x 10(4) CFU/g . Samples contained an average of 8.4 mites/g and the ten samples assayed for bacterial endotoxin averaged 80.4 ng/mg . A total of 55 species of bacteria, 40 of fungi and 13 of mites were found . Gram-positive cocci (mostly Staphylococcus spp.) were the predominant mesophilic bacteria, followed by corynebacteria and Bacillus spp . Thermophilic bacteria were represented only by actinomycetes, with Thermoactinomyces vulgaris predominant . The most numerous fungi were Penicillium spp . and Aspergillus spp . followed by yeasts . The most abundant mites were Dermatophagoides spp . which formed > 85% of the total count . There were no significant differences between the homes of allergic and nonallergic people in the concentrations of total bacteria, fungi, and mites . Bacillus, Aspergillus and total filamentous fungi (molds), but not yeasts, were significantly more numerous in the homes of people with asthma caused by house dust . CONCLUSION: The results suggest that some species of bacteria and filamentous fungi should be considered potential causes of house dust allergy.

J Eukaryot Microbiol, 1996 Jan-Feb, 43(1), 5 - 11
Analyses of 22S dynein binding to Tetrahymena axonemes lacking outer dynein arms; Sullivan J et al.; Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature of 39 degrees C . Axonemes isolated from nonmotile oad mutants (oad 39 degrees C axonemes) lack approximately 90% of their outer dynein arms and are deficient in 22S dynein . Here we report that oad 39 degrees C axonemes contain 40% of the 22S dynein heavy chains that wild-type axonemes contain and that oad axonemes do not undergo ATP-induced microtubule sliding in vitro . Wild-type 22S dynein will bind to the outer arm position in oad axonemes and restore ATP-induced microtubule sliding in those axonemes . Unlike wild-type 22S dynein, oad 22S dynein does not bind to the outer arm position in oad axonemes . These data indicate that the oad mutation affects some component of the outer arm dynein itself rather than the outer arm dynein binding site . These data also indicate that oad axonemes can be used to assay outer dynein arm function.

J Bacteriol, 1996 Jan, 178(2), 357 - 65
slpM, a gene coding for an "S-layer-like array" overexpressed in S-layer mutants of Thermus thermophilus HB8; Olabarria G et al.; S-layer deletion mutants of Thermus thermophilus HB8 overproduce a regular array which surrounds groups of several cells . Averages of two-dimensional projections revealed a detailed architecture similar in general morphology and unit cell dimensions to that of the S-layer but having a different mass distribution . The structural components of these "S-layer-like arrays" are a group of three proteins of 52 (P52), 50 (P50), and 36 (P36) kDa, which are overexpressed in S-layer mutants . These three proteins specifically bind antibodies against P52, suggesting that the smaller proteins correspond to fragments derived from P52 . This hypothesis was demonstrated by the identity of the trypsin digestion products of P52 and P50 . The gene slpM, responsible for the synthesis of P52, was cloned by using synthetic oligonucleotides designed from partial amino acid sequences of P52 and P50 . When slpM was expressed in Escherichia coli, proteins specifically recognized by anti-P52 antiserum whose electrophoretic mobilities were similar to those of P52 and P36 were detected . The sequence of slpM revealed the existence of an open reading frame in which the amino termini of P52, P50, and P36 were identified . The unprocessed product of slpM is a 469-amino-acid-long polypeptide whose theoretical M(r) (52,131) was in good agreement with the electrophoretic mobility of P52 . The properties deduced for the product of slpM are very different from those of any S-layer protein so far sequenced . The possible roles of SlpM in wild-type cells are discussed.

J Bacteriol, 1996 Jan, 178(2), 340 - 6
Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid/iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila; Maupin-Furlow J et al.; The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma) . The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced . The cdhD gene is upstream of and separated by 3 bp from cdhE . Both genes are preceded by apparent ribosome-binding sites . Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD . The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum . The CdhE sequence had a four-cysteine motif with the potential to bind a 4Fe-4S cluster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy . A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli . The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration . The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom.

J Bacteriol, 1996 Jan, 178(1), 6 - 11
Reduced sulfur compound oxidation by Thiobacillus caldus; Hallberg KB et al.; The oxidation of reduced inorganic sulfur compounds was studied by using resting cells of the moderate thermophile Thiobacillus caldus strain KU . The oxygen consumption rate and total oxygen consumed were determined for the reduced sulfur compounds thiosulfate, tetrathionate, sulfur, sulfide, and sulfite in the absence and in the presence of inhibitors and uncouplers . The uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl-hydrazone had no affect on the oxidation of thiosulfate, suggesting that thiosulfate is metabolized periplasmically . In contrast, the uncouplers completely inhibited the oxidation of tetrathionate, sulfide, sulfur, and sulfite, indicating that these compounds are metabolized in the cytoplasm of T . caldus KU . N-Ethylmaleimide inhibited the oxidation of tetrathionate and thiosulfate at the stage of elemental sulfur, while 2-heptyl-4-hydroxyquinoline-N-oxide stopped the oxidation of thiosulfate, tetrathionate, and elemental sulfur at the stage of sulfite . The following intermediates in the oxidation of the sulfur compounds were found by using uncouplers and inhibitors: thiosulfate was oxidized to tetrathionate, elemental sulfur was formed during the oxidation of tetrathionate and sulfide, and sulfite was found as an intermediate of tetrathionate and sulfur metabolism . On the basis of these data we propose a model for the metabolism of the reduced inorganic sulfur compounds by T . caldus KU.

J Bacteriol, 1996 Jan, 178(1), 248 - 57
Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima; Kletzin A et al.; Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin oxidoreductase (POR) . These enzymes catalyze the synthesis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dependent oxidative decarboxylation reaction with reduction of ferredoxin . We report here on the molecular analysis of the POR (por) and 2-ketoisovalerate ferredoxin oxidoreductase (vor) genes from P . furiosus and of the POR gene from T . maritima, all of which comprise four different subunits . The operon organization for P . furiosus POR and VOR was porG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzymes . The operon organization for T . maritima POR was porGDAB . The three enzymes were 46 to 53% identical at the amino acid level . Their delta subunits each contained two ferredoxin-type {4Fe-4S} cluster binding motifs (CXXCXXCXXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain . Amino-terminal sequence comparisons show that POR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P . furiosus all belong to a phylogenetically homologous OR family . Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochondriate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequence identity) . Three of these subunits are also homologous to the dimeric POR from a mesophilic archaeon, Halobacterium halobium (21% identity) . A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits.

Mol Cell Biol, 1996 Jan, 16(1), 66 - 75
Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase; Gilley D et al.; The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA . Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization . Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues . We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues . Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation . In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension . This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.

Gene, 1995 Dec 29, 167(1-2), 141 - 5
Direct linkage of str-, S10- and spc-related gene clusters in Thermus thermophilus HB8, and sequences of ribosomal proteins L4 and S10; Pfeiffer T et al.; We have analyzed the genomic region harboring str-S10-spc-related gene clusters in the thermophilic bacterium, Thermus thermophilus (Tt) HB8 . This study was initiated for the purpose of isolating the gene encoding ribosomal (r-) protein S10 which is assumed to be involved in the antitermination of transcription at the rRNA-encoding genes in Bacteria . The S10-related gene cluster encodes the same set of r-proteins as in Escherichia coli . However, the gene coding for elongation factor Tu (the last gene of the str operon in E . coli) is separated by only eight nucleotides (nt) from the gene encoding r-protein S10 (the first gene of the S10 operon in E . coli), and the genes encoding r-protein S17 (the last gene of the S10 operon in E . coli) and L14 (the first gene of the spc operon in E . coli) overlap . This suggests that all three gene clusters are cotranscribed from a single promoter preceding the str-related operon . In addition, we determined the complete nt sequences of the Tt genes encoding r-proteins L4 and S10 . Tt L4 shows the lowest degree of conservation among the known L4 r-proteins from Bacteria . Tt S10 has the highest proportion of basic amino acids (aa) and the lowest number of acidic aa, as compared with its homologues from Bacteria and Archaea, which might be related to its possible role in binding to the boxA RNA of nascent rRNA transcripts at high temperatures.

FEBS Lett, 1995 Dec 27, 377(3), 408 - 12
2,8-Diazido-ATP--a short-length bifunctional photoaffinity label for photoaffinity cross-linking of a stable F1 in ATP synthase (from thermophilic bacteria PS3); Schafer HJ et al.; To demonstrate the direct interfacial position of nucleotide binding sites between subunits of proteins we have synthesized the bifunctional photoaffinity label 2,8-diazidoadenosine 5'-triphosphate (2,8-DiN3ATP) . UV irradiation of the F1-ATPase (TF1) from the thermophilic bacterium PS3 in the presence of 2,8-DiN3ATP results in a nucleotide-dependent inactivation of the enzyme and in a nucleotide-dependent formation of alpha-beta crosslinks . The results confirm an interfacial localization of all the nucleotide binding sites on TF1.

Biochim Biophys Acta, 1995 Dec 27, 1264(3), 279 - 83
Cloning and sequencing of the gene coding for topoisomerase I from the extremely thermophilic eubacterium, Thermotoga maritima; Bouthier de la Tour C et al.; A 2767 bp fragment containing a gene coding for a topoisomerase I from the extremely thermophilic eubacterium Thermotoga maritima (Tm TopA) has been cloned and sequenced . The protein is composed of 633 amino acids with a calculated molecular mass of 72,695 Da . It shares significant similarity with the topoisomerases I of mesophilic eubacteria . The highest score is obtained with Bacillus subtilis (44% identity); in particular, T . maritima and B . subtilis possess an insertion of 7-8 amino acids in the vicinity of the active site, that is absent in topoisomerases of other organisms . A specific feature of T . maritima topoisomerase I is its low cysteine content compared to its mesophilic homologs . It contains 5 cysteine residues, of which 4 could constitute a zinc finger motif . Finally, analysis of the regions flanking the gene reveals that Tm TopA is surrounded by two other ORFs, suggesting the occurrence of a polycistronic transcriptional unit.

Biochemistry, 1995 Dec 19, 34(50), 16412 - 8
The alpha 3 beta 3 gamma complex of the F1-ATPase from thermophilic Bacillus PS3 containing the alpha D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites; Jault JM et al.; ATP hydrolyses by the wild-type alpha 3 beta 3 gamma and mutant (alpha D261N)3 beta 3 gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3 have been compared . The wild-type complex hydrolyzes 50 microM ATP in three kinetic phases: a burst decelerates to an intermediate phase, which then gradually accelerates to a final rate . In contrast, the mutant complex hydrolyzes 50 microM or 2 mM ATP in two kinetic phases . The mutation abolishes acceleration from the intermediate phase to a faster final rate . Both the wild-type and mutant complexes hydrolyze ATP with a lag after loading a catalytic site with MgADP . The rate of the MgADP-loaded wild-type complex rapidly accelerates and approaches that observed for the wild-type apo-complex . The MgADP-loaded mutant complex hydrolyzes ATP with a more pronounced lag, and the gradually accelerating rate approaches the slow, final rate observed with the mutant apo-complex . Lauryl dimethylamide oxide (LDAO) stimulates hydrolysis of 2 mM ATP catalyzed by wild-type and mutant complexes 4- and 7.5-fold, respectively . The rate of release of {3H}ADP from the Mg{3H}ADP-loaded mutant complex during hydrolysis of 40 microM ATP is slower than observed with the wild-type complex . LDAO increases the rate of release of {3H}ADP from the preloaded wild-type and mutant complexes during hydrolysis of 40 microM ATP . Again, release is slower with the mutant complex . When the wild-type and mutant complexes are irradiated in the presence of 2-N3-{3H}ADP plus Mg2+ or 2-N3-{3H}ATP plus Mg2+ and azide, the same extent of labeling of noncatalytic sites is observed . Whereas ADP and ATP protect noncatalytic sites of the wild-type and mutant complexes about equally from labeling by 2-N3-{3H}ADP or 2-N3-{3H{ATP, respectively, AMP-PNP provides little protection of noncatalytic sites of the mutant complex . The results suggest that the substitution does not prevent binding of ADP or ATP to noncatalytic sites, but rather that it affects cross-talk between liganded noncatalytic sites and catalytic sites which is necessary to promote dissociation of inhibitory MgADP.

FEBS Lett, 1995 Dec 18, 377(2), 253 - 7
Site-directed mutagenesis of Thermus thermophilus EF-Tu: the substitution of threonine-62 by serine or alanine; Ahmadian MR et al.; The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus . The altered proteins were overproduced in Escherichia coli, purified and characterized . The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu . In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity . The affinity of aminoacyl-tRNA to the EF-Tu T62A.GTP complex is almost 40 times lower as compared to the native EF-Tu.GTP . These observations are in agreement with the tertiary structure of EF-Tu.GTP, in which threonine-62 is interacting with the Mg2+ ion, gamma-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 183 - 8
Overproduction, purification and characterization of GroES and GroEL from thermophilic Bacillus stearothermophilus; Schon U et al.; To facilitate purification of the two chaperonins GroES and GroEL encoded by the thermophilic Bacillus stearothermophilus, an Escherichia coli strain was constructed in which the geoESL operon was replaced by that of B . stearothermophilus . This strain is perfectly viable, demonstrating that the B . stearothermophilus operon is functionally interchangeable with that of E . coli . To increase the amount of GroES, the groES gene was fused to an IPTG-inducible promoter . Both proteins GroES and GroEL, were purified from E . coli using the standard protocol with some modifications . This method should be applicable in all cases where a foreign groE operon can substitute that of E . coli . A preliminary characterization of GroEL, revealed that it has the same secondary structural elements as the E . coli homologue, but its thermodynamic stability is significantly increased.

Eur J Biochem, 1995 Dec 15, 234(3), 897 - 902
Recognition of tRNAPhe by phenylalanyl-tRNA synthetase of Thermus thermophilus; Moor NA et al.; The tRNA(Phe) nucleotides required for recognition by phenylalanyl-tRNA synthetase of Thermus thermophilus have been determined using Escherichia coli tRNA(Phe) transcripts with various mutations . The anticodon nucleotides are shown to be the most important recognition elements . The discriminator nucleotide, A73, involved in the recognition set of yeast, E . coli and human phenylalanyl-tRNA synthetases contributes only slightly to tRNA(Phe) recognition by Th . thermophilus phenylalanyl-tRNA synthetase . Nucleotide 20 and some tertiary nucleotides, including the conserved G19.C56 base pair, are proposed to participate in stabilization of the precise tRNA conformation required for efficient aminoacylation . The role of the 3'-CCA terminus, common to all tRNAs, in the specific interaction of tRNA with phenylalanyl-tRNA synthetase is discussed.

Biochim Biophys Acta, 1995 Dec 12, 1232(3), 208 - 16
Isolation and characterization of a Photosystem II complex from the red alga Cyanidium caldarium: association of cytochrome c-550 and a 12 kDa protein with the complex; Enami I et al.; A Photosystem II (PS II) complex was purified from an acidophilic as well as a thermophilic red alga, Cyanidium caldarium . The purified PS II complex was essentially devoid of phycobiliproteins and other contaminating components, and showed a high oxygen-evolving activity of 2375 mumol O2/mg Chl per h using phenyl-p-benzoquinone as the electron acceptor . The expression of this high activity did not require addition of exogenous Ca2+, although EDTA reduced the activity by 40% . This effect of EDTA can be reversed not only by Ca2+ but also by Mg2+; a similar Mg2+ effect has been observed in purified cyanobacterial PS II but not in higher plant PS II . Immunoblotting analysis indicated the presence of major intrinsic polypeptides commonly found in PS II from cyanobacteria and higher plants as well as the extrinsic 33 kDa protein . Antibodies against the extrinsic 23 and 17 kDa proteins of higher plant PS II, however, did not crossreact with any polypeptides in the purified PS II, indicating the absence of these proteins in the red alga . In contrast, two other extrinsic proteins of 17 and 12 kDa were present in the red algal PS II; they were released by 1 M Tris or Urea/NaCl treatment but not by 1 M NaCl . The 17 kDa polypeptide was identified to be cytochrome c-550 from heme-staining, immunoblot analysis and N-terminal amino acid sequencing, and the 12 kDa protein was found to be homologous to the 12 kDa extrinsic protein of cyanobacterial PS II from its N-terminal sequence . These results indicate that PS II from the red alga is closely related to PS II from cyanobacteria rather than to that from higher plants, and that the replacement of PS II extrinsic cytochrome c-550 and the 12 kDa protein by the extrinsic 23 and 17 kDa proteins occurred during evolution from red algae to green algae and higher plants.

FEBS Lett, 1995 Dec 11, 377(1), 77 - 81
A three-dimensional structure model of the complex of glutamyl-tRNA synthetase and its cognate tRNA; Tateno M et al.; A docking model of glutamyl-tRNA synthetase (GluRS) and tRNAGlu was constructed, on the basis of the distinguished similarity between the X-ray crystallographic three-dimensional structures of the N-terminal halves of the Thermus thermophilus GluRS in the free state and the Escherichia coli glutaminyl-tRNA synthetase in a complex with tRNAGln . The modeled structure is energetically favorable and is also well consistent with the results of site-directed mutagenesis studies . The model indicates that the GluRS-specific insertions 2 and 3 fit and bind to the acceptor stem and the D arm, respectively, of the cognate tRNA without affecting other contacts . In particular, insertion 3 strongly interacts with the two D-stem base pairs that are essential for the tRNA-GluRS recognition.

FEBS Lett, 1995 Dec 11, 377(1), 6 - 8
Close evolutionary relatedness among functionally distantly related members of the (alpha/beta)8-barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region; Janecek S; A short conserved sequence equivalent to the fifth conserved sequence region of alpha-amylases (173_LPDLD, Aspergillus oryzae alpha-amylase) comprising the calcium-ligand aspartate, Asp-175, was identified in the amino acid sequences of several members of the family of (alpha/beta)8-barrel glycosyl hydrolases . Despite the fact that the aspartate is not invariantly conserved, the stretch can be easily recognised in all sequences to be positioned 26-28 amino acid residues in front of the well-known catalytic aspartate (Asp-206, A . oryzae alpha-amylase) located in the beta 4-strand of the barrel . The identification of this region revealed remarkable similarities between some alpha-amylases (those from Bacillus megaterium, Bacillus subtilis and Dictyoglomus thermophilum) on the one hand and several different enzyme specificities (such as oligo-1,6-glucosidase, amylomaltase and neopullulanase, respectively) on the other hand . The most interesting example was offered by B . subtilis alpha-amylase and potato amylomaltase with the regions LYDWN and LYDWK, respectively . These observations support the idea that all members of the family of glycosyl hydrolases adopting the structure of the alpha-amylase-type (alpha/beta)8-barrel are mutually closely related and the strict evolutionary borders separating the individual enzyme specificities can be hardly defined.

FEBS Lett, 1995 Dec 4, 376(3), 190 - 4
Conformational dynamics monitored by His-179 and His-200 of isolated thermophilic F1-ATPase beta subunit which reside at the entrance of the 'conical tunnel' in holoenzyme; Tozawa K et al.; When monitored by 1H NMR at various pH values, most of the C-2 proton signals from 12 His residues of the isolated beta subunit of thermophilic F1-ATPase (TF1) could be separately observed . Two of them were assigned to His-179 and His-200 which reside at the entrance of a 'conical tunnel' to reach catalytic site in the crystal structure of F1-ATPase . His-200 gave doublet, suggesting that this region is not a rigid alpha-helix in the isolated beta subunit . The binding of Mg.AMP-PNP changed the chemical shifts of His-179 and His-200 significantly . Although His-119 located at the opposite side of the conical tunnel was not affected by the nucleotide-binding, it contributed to the stability of beta subunit and the efficiency of the catalysis of the holoenzyme.

Biol Trace Elem Res, 1995 Dec, 50(3), 181 - 91
Doublet cells in Tetrahymena as indicators of culture media composition; Christopher GK et al.; Stomatogenesis in ciliates is a complex and carefully orchestrated event . The exo- mutant SB255 of Tetrahymena thermophila has defects in mucocyst formation and docking and can also have one or two mouths . Three common culture media (proteose peptone, Medium 357, and yeast extract) were analyzed for total C, N, and inorganic elements and then tested for their effect on the number of mouths present in SB255 . Cultures of SB255 grown in Medium 357 consisted of a mixed population of cells with either two mouths (doublet) or one mouth . Cultures from the same original stock grown in Medium 357 (SBm) and in 1% proteose peptone (SBpp) had different percentages of doublet cells in 1-, 2-, 3-, and 4-d-old cultures . When transferred to and grown in 1% yeast medium, both SBpp and SBm cultures had increased percentages of doublets over a 4-d culture period . When grown in 0.1, 0.5, or 1% yeast medium for 2 d, both SBpp and SBm cultures had more doublets in 1% than in either 0.1 or 0.5% yeast medium . Cultures of SBm grown in Medium 357 or 1% yeast medium for 2 d had a 10-fold increase in doublet cells compared to the inoculum . After 2 d in 1% proteose peptone, SBm cultures had percentages of doublet cells almost equal to that of the inoculum . Immunofluorescence and scanning electron microscopy (SEM) were used to examine cellular morphology of the doublet cells . These findings suggest that enriched media promote the growth of doublet cells . Furthermore, these doublets could prove to be a useful model system for the study of biological roles of trace elements.

Nat Struct Biol, 1995 Dec, 2(12), 1109 - 14
Crystal structure of NADH oxidase from Thermus thermophilus; Hecht HJ et al.; The crystal structures of the flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) containing isoforms of NADH oxidase from Thermus thermophilus have been determined by isomorphous and molecular replacement and refined to 2.3 A and 1.6 A resolution with R-values of 18.5% and 18.6% respectively . The structure of the homodimeric enzyme consists of a central 4-stranded antiparallel beta-sheet covered by helices, a more flexible domain formed by two helices, and a C-terminal excursion connecting the subunits . The active sites are located in a deep cleft between the subunits . The binding site of the flavin cofactor lacks the common nucleotide binding fold and is different from the FMN binding site found in flavodoxins.

Mol Microbiol, 1995 Dec, 18(5), 925 - 32
Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea; van de Vossenberg JL et al.; Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea . At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions . In some thermophiles, sodium is the sole energy-coupling ion . To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature . The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures . Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature . In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature . The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.

J Biochem (Tokyo), 1995 Dec, 118(6), 1261 - 7
Orotate phosphoribosyltransferase from Thermus thermophilus: overexpression in Escherichia coli, purification and characterization; Bunnak J et al.; Orotate phosphoribosyltransferase (OPRTase, EC2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP) . To obtain heat-stable OPRTase and to elucidate the mechanism of heat stability, this enzyme from Thermus thermophilus was expressed in Escherichia coli and purified . The pyrE gene of T . thermophilus which encodes OPRTase, contains an open reading frame of 549 base pairs with 69% G+C content . Since this gene expressed itself inefficiently in E . coli, the 5' and 3' ends of the coding regions were replaced with synonymous codons which contain more A+T and corresponds to major codons for E . coli . Introduction of the modified gene fragments into a plasmid having a tac promoter resulted in production of a polypeptide of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) in E . coli . This protein represented as much as 16% of the bacterial total protein and showed the OPRTase activity . Three purification steps, consisting of heat treatment at 65 degrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypeptide . The optimum activity of the purified OPRTase was observed at 150 mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 microM PRPP . The activation energy of this enzyme reaction was 20.3 kJ/mol . The Km of this enzyme for orotate as a substrate was 75 microM and the maximum specific activity was 300 units/mg protein under the optimum conditions . The purified OPRTase was stable for 20 min at 85 degrees C.

J Dairy Sci, 1995 Dec, 78(12), 2624 - 8
Observation of encapsulated lactic acid bacteria using confocal scanning laser microscopy; Hassan AN et al.; The confocal scanning laser microscope allows direct observation of milk and cultured milks in their natural state . The microscope was used to observe the capsules of lactic acid bacteria growing in milk . Capsule production was confirmed by microscopic observation of cells suspended in latex beads . Some strains of Streptococcus thermophilus were surrounded by a capsule 4 to 5 microns in diameter, and others by capsules 2 microns in diameter . Lactobacillus delbrueckii ssp . bulgaricus strains showed capsule sizes from 1.5 to 3 micron in diameter . Of four strains of lactococci tested, three were unencapsulated, and one had capsule sizes of 1.5 and 2 microns around some cells . Encapsulated strains produced less acid in milk than did unencapsulated strains . Growth in Elliker's broth produced smaller capsules than did growth in milk . Capsules acted as a barrier to acid diffusing from the cell.

J Med Virol, 1995 Dec, 47(4), 378 - 85
Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis; Casas I et al.; Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF) . Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed . The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide-stained gels . Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR . Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification . In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube . Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders . However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis.

Genetics, 1995 Dec, 141(4), 1315 - 25
Genetic map of randomly amplified DNA polymorphisms closely linked to the mating type locus of Tetrahymena thermophila; Lynch TJ et al.; We have used the PCR-based randomly amplified polymorphic DNA (RAPD) method to efficiently identify and map DNA polymorphisms in the ciliated protozoan Tetrahymena thermophila . The polymorphisms segregate as Mendelian genetic markers . A targeted screen, using DNA from pooled meiotic segregants, yielded the polymorphisms most closely linked to the mat locus . A total of 10 polymorphisms linked to the mat-Pmr segment of the left arm of micronuclear chromosome 2 have been identified . This constitutes the largest linkage group described in T . thermophila . We also provide here the first crude estimate of the frequency of meiotic recombination in the mat region, 20 kb/cM . This frequency is much higher than that observed in most other eukaryotes . Special features of Tetrahymena genetics enhanced the power of the RAPD method: the ability to obtain in a single step meiotic segregants that are whole-genome homozygotes and the availability of nullisomic strains permitting quick deletion mapping of polymorphisms to micronuclear chromosomes or chromosome segments . The RAPD method appears to provide a practical and relatively inexpensive approach to the construction of a high-resolution map of the Tetrahymena genome.

Biophys J, 1995 Dec, 69(6), 2728 - 38
Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus; Yu X et al.; The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo . Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E . coli protein . We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus . This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C . We show that the active filament formed by the T . aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E . coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament . This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well . We have also characterized bundles of filaments formed by the T . aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined . The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 405 - 12
Highly bioluminescent Streptococcus thermophilus strain for the detection of diary-relevant antibiotics in milk; Jacobs MF et al.; Inefficient translational initiation is often the cause of poor foreign gene expression in gram-positive organisms . The expression of bacterial luciferase (lux) genes in Streptococcus thermophilus (bioluminescence) was improved by addressing this problem in two ways; by ribosome-binding site (RBS) replacement, and by enhancing lux RBS access by polymerase chain reaction modification either alone or combined with translational coupling to a truncated upstream open- reading frame (orf') having its own RBS . Lactococcal expression signals were employed for plasmid-based lux expression . The same constructs were used to monitor bioluminescence in Lactococcus lactis, as well as two non-lactic bacterial strains, for comparison . High lux expression was achieved in all four organisms with a heterodimeric thermostable enzyme . Surprisingly, where ready access to the lux RBS was predicted, translational coupling to the lactococcal orf remained a prerequisite for detectable lux expression in L . lactis . In contrast, high bioluminescence in S . thermophilus was independent of coupling . Consistent with these observations, inspection of published gene sequences suggests that RBS "strength" may be a more important factor in translation in S . thermophilus than in L . lactis . Using reduced light production in highly bioluminescent S . thermophilus as an indicator of biocide presence in milk, test times could be significantly shortened compared with a commercial test utilizing the related non-bioluminescent strain . lux genes appear to be sensitive, exponential-phase reporters of gene activity in S . thermophilus, an organism with molecular biology and genetics that remain largely unstudied.

Comp Biochem Physiol B Biochem Mol Biol, 1995 Dec, 112(4), 727 - 32
Comparison of axonemal proteins from two kinds of Tetrahymena . I . Different characteristics of dyneins in heat stability; Takaya C et al.; Tetrahymena thermophila could still swim after incubation of the cell body at 40 degrees C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment . Turbidity measurements revealed that axonemes of T . pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T . thermophilia still had the activity under the same conditions . In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena . Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment . Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T . thermophilia is more stable in maintaining native configuration than that of T . pyriformis against the heat treatment, although both treated dyneins keep three (alpha, beta and gamma) subunits . Analysis by peptide mapping demonstrated that beta- and gamma-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species . These results imply that dynein of T . thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.

J Mol Evol, 1995 Dec, 41(6), 1096 - 104
Structure-based sequence alignment of elongation factors Tu and G with related GTPases involved in translation; Avarsson A; The G domain and domain II in the crystal structure of Thermus thermophilus elongation factor G (EF-G) were compared with the homologous domains in Thermus aquaticus elongation factor Tu (EF-Tu) . Sequence alignment derived from the structural superposition was used to define conserved sequence elements in domain II . These elements and previously known conserved sequence elements in the G domain were used to guide the alignment of the sequences of Sulfolobus acidocaldarius elongation factor 2, human elongation factor 2, and Escherichia coli initiation factor 2 and release factor 3 to the aligned sequences of EF-G and EF-Tu . This alignment, which deviates from previously published alignments, has evolutionary implications and leads to alternative interpretations of biochemical data concerning the interaction of elongation factors with the alpha-sarcin/ricin region of the ribosome . A single conserved sequence motif in domain II was identified and used to further characterize the GTPase subfamily of translation factors and related proteins . It was shown that the motif is found in most if not all the members of the family . Apparently, the common characteristic of these GTPases is an extensive consensus structural unit that possibly accounts for a similar interaction with the ribosome and is composed of two domains homologous to the G domain and domain II in EF-Tu and EF-G.

J Clin Microbiol, 1995 Dec, 33(12), 3345 - 6
Evaluation of API Campy in comparison with conventional methods for identification of thermophilic campylobacters; Huysmans MB et al.; API Campy was compared with conventional biochemical methods for its ability to identify 100 thermophilic campylobacter isolates . When the results were read according to the manufacturer's instructions, API Campy showed 92% agreement with conventional methods . Extended incubation of the assimilation strip resulted in the correct identification of an additional two isolates . Discrepant results occurred for six isolates . Overall, API Campy offered no advantages over conventional methods.

Protein Sci, 1995 Dec, 4(12), 2594 - 604
Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus . An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions; Delboni LF et al.; The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A . The structure was solved by molecular replacement using XPLOR . Twofold averaging and solvent flattening was applied to improve the quality of the map . Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the "closed" conformation in either subunit . The crystallographic R-factor is 17.6% with good geometry . The two subunits have an RMS deviation of 0.29 A for 248 C alpha atoms and have average temperature factors of 18.9 and 15.9 A2, respectively . In both subunits, the active site Lys 10 adopts an unusual phi, psi combination . A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B . stearothermophilus TIM . Although the ratio Arg/(Arg+Lys) is higher in B . stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B . stearothermophilus enzyme . A higher number of prolines contributes to the higher stability of B . stearothermophilus TIM . Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family . Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B . stearothermophilus enzyme . However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B . stearothermophilus TIM compared to all five other known TIM structures.

Appl Microbiol Biotechnol, 1995 Dec, 44(1-2), 81 - 7
Identification of new enzyme activities of several strains of Thermus species; Berger JL et al.; New thermostable enzyme activities of seven Thermus strains were compared using the API ZYM system . All the strains exhibited high levels of alpha- and beta-glycosidases, esterase (C4) and esterase-lipase (C8) activities intracellularly . Only T . thermophilus HB8 (ATCC 27634) showed alpha-glucosidase and esterase activities in the supernatant . According to the intensity of beta-galactosidase activity, Thermus strains were divided in three groups . Group 0, which showed a weak beta-galactosidase activity, included Thermus spp . ATCC 31674 (T351) and 27978 (X-1) as well as T . thermophilus ATCC 27634 (HB8) . Group I which consisted of T . aquaticus ATCC 25104 (YT-1), ATCC 25105 (Y-VII-51B) and Thermus sp . ATCC 27737 (T2), had a specific activity of approximately 40.0 U mg-1 and galactose as inducer . T . aquaticus ATCC 31558 (group 2) was particularly effective for beta-galactosidase production (2840 U) with a specific activity of 98 U mg-1 . For each strain, galactose (0.5%) was a better inducer of beta-galactosidase production than lactose (1%) . The detection of beta-galactosidase activity was dependent on the derivative chromogenic substrates used (naphthyl or nitrophenol coupled to sugar) . Oligosaccharides were synthesized from cellobiose, lactulose, maltose or lactose as substrates at high temperature in some strains of Thermus.

Biotechnol Appl Biochem, 1995 Dec, 22 ( Pt 3), 261 - 8
Industrial-scale production and rapid purification of an archaeal beta-glycosidase expressed in Saccharomyces cerevisiae; Morana A et al.; The application of enzymes isolated from extreme thermophiles in biotechnological processes is hampered by their unconventional fermentation conditions . The expression, in mesophilic hosts, of genes encoding for thermophilic proteins enables these difficulties to be overcome and permits the production of enzymes in high yield by using conventional fermentation plants and an efficient enzyme purification utilizing heat precipitation of host proteins . The beta-glycosidase gene from Sulfolobus solfataricus, a thermoacidophilic archaeon growing at 87 degrees C and pH 3.5, has been cloned and expressed in Saccharomyces cerevisiae (baker's yeast) . The fermentation of a S . cerevisiae strain on a 100-litre scale and the two-step purification of the expressed beta-glycosidase by cell autolysis and extracts thermal precipitation is described . This procedure, after 72 h of autolysis, gave a yield 56-fold higher with respect to that obtained with the beta-glycosidase from S . solfataricus.

Epidemiol Infect, 1995 Dec, 115(3), 495 - 500
Effect of changes in processing to improve hygiene control on contamination of poultry carcasses with campylobacter; Mead GC et al.; Examination of neck skin and caecal samples taken at a commercial processing plant from 15 randomly chosen poultry flocks showed that all flocks were contaminated initially with thermophilic Campylobacter spp., even in the apparent absence of caecal carriage . During processing, numbers of campylobacter on skin samples were reduced by between 10 and 1000-fold . To improve hygiene control generally, chlorinated-water sprays were used to limit microbial contamination on equipment and working surfaces . In addition, chlorine concentrations in process water were increased and any unnecessary carcass contact surfaces in the processing plant were removed . When comparing flocks before and after the changes, it was found that numbers of campylobacter on packaged carcasses were significantly lower after the changes had been made (P 0.001) . In practice, however, the reduction would be likely to have little impact on consumer exposure to campylobacter infection.

Can J Microbiol, 1995 Dec, 41(12), 1071 - 80
Microbial ecology of simultaneous thermophilic microbial leaching and digestion of sewage sludge; Shooner F et al.; The microbial population encountered during a simultaneous thermophilic microbial leaching and digestion process at 50 degrees C, based on microbial sulfur oxidation, was investigated . The cell count of the sulfuric acid producer Thiobacillus thermosulfatus increased, followed by a decrease . In the absence of sulfur (control: conventional thermophilic digestion), Thiobacillus thermosulfatus population decreased under the detection limit . Acidophilic and neutrophilic heterotrophic populations increased during the leaching process, and the final acidophilic population count was higher than the neutrophilic population . During the thermophilic digestion (control), the final neutrophilic population count was higher than the acidophilic . Six heterotrophic bacterial strains were isolated and partially characterized . Bacillus was the most predominant genus . The type of bacterial populations in thermophilic microbial leaching and digestion, as well as the thermophilic digestion process (control), were the same, while only the relative concentrations changed . In both processes, the bacterial indicators decreased under the detection limit after 12 h . Mesophilic heterotrophic population was more affected by the thermophilic microbial leaching process than by thermophilic digestion . Sludge mineralization was probably more influenced by the final cell concentration rather than the presence of an individual species or mixed population.

Appl Environ Microbiol, 1995 Dec, 61(12), 4403 - 8
Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate; Gibbs MD et al.; A genomic library of the Dictyoglomus sp . strain Rt46B.1 was constructed in the phage vector lambda ZapII and screened for xylanase activity . A plaque expressing xylanase activity, designated B6-77, was isolated and shown to contain a genomic insert of 5.3 kb . Subcloning revealed that the xylanase activity was restricted to a internal 1,507-bp PstI-HindIII fragment which was subsequently sequenced and shown to contain a single complete open reading frame coding for a single-domain xylanase, XynA, with a putative length of 352 amino acids . Homology comparisons show that XynA is related to the family F group of xylanases . The temperature and pH optima of the recombinant enzyme were determined to be 85 degrees C and pH 6.5, respectively . However, the enzyme was active across a broad pH range, with over 50% activity between pH 5.5 and 9.5 . XynA was shown to be a true endo-acting xylanase, being capable of hydrolyzing xylan to xylotriose and xylobiose, but it could not hydrolyze xylobiose to monomeric xylose . XynA was also shown to hydrolyze xylan present in Pinus radiata kraft pulp, indicating that it may be of use as an aid in pulp bleaching . The equivalent xylanase gene was also isolated from the related bacterium Dictyoglomus thermophilum, and DNA sequencing showed these genes to be identical, which, together with the 16S small-subunit rRNA gene sequencing data, indicates that Rt46B.1 and D . thermophilum are very closely related.

Mol Cell Biol, 1995 Dec, 15(12), 7117 - 26
An intramolecular recombination mechanism for the formation of the rRNA gene palindrome of Tetrahymena thermophila; Butler DK et al.; Large palindromic DNAs are found in a wide variety of eukaryotic cells . In Tetrahymena thermophila, a large palindrome is formed from a single rRNA gene (rDNA) during nuclear differentiation . We present evidence that a key step in the formation of the rDNA palindrome of T . thermophila involves homologous intramolecular recombination . Heteroduplex micronuclear rDNA molecules were constructed in vitro and microinjected into developing macronuclei, where they formed palindromes . Analysis of the resulting palindromes indicated that both strands of the microinjected rDNA are used to form the same palindrome . This study, together with a previous study (L . F . Yasuda and M.-C . Yao, Cell 67:505-516, 1991), is the first to define a molecular pathway of palindrome formation . The process is initiated by chromosome breakage at sites flanking the micronuclear rDNA . An intramolecular recombination reaction, guided by a pair of short inverted repeats located at the 5' end of the excised rDNA, covalently joins the two strands of micronuclear rDNA in a giant hairpin molecule . Bidirectional DNA replication converts the giant hairpin molecule to a palindrome . We suggest that the general features of this pathway are applicable to palindrome formation in other cell types.

Infect Immun, 1995 Dec, 63(12), 4682 - 5
Molecular characterization and T-cell-stimulatory capacity of Mycobacterium leprae antigen T5; Wieles B et al.; A Mycobacterium leprae lambda gt11 clone designated T5 has previously been selected with sera from tuberculoid leprosy patients . Sequence analysis of this clone revealed the presence of two overlapping open reading frames (ORFs) present on the two cDNA strands . The first ORF codes for the serologically recognized antigen, which was fused with the lacZ gene in the lambda gt11 clone . The second ORF, present on the complementary strand, displays strong sequence homology with the aspartyl-tRNA synthetase genes of Escherichia coli and Thermus thermophilus . Here we show that the purified T5-derived product, overexpressed in E . coli, is recognized by T cells of the majority of the leprosy patients tested, including lepromatous leprosy patients who do not respond to whole M . leprae bacilli.

J Biol Chem, 1995 Dec 1, 270(48), 28617 - 22
A proteasome from the methanogenic archaeon Methanosarcina thermophila; Maupin-Furlow JA et al.; A 645-kDa proteasome was purified from Methanosarcina thermophila which had chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities and contained alpha (24-kDa) and beta (22-kDa) subunits . Processing of both subunits was suggested by comparison of N-terminal sequences with the sequences deduced from the alpha- and beta-encoding genes (psmA and psmB) . Alignment of deduced sequences for the alpha and beta subunits revealed high similarity; however, the N-terminal sequence of the alpha subunit contained an additional 24 amino acids that were not present in the beta subunit . The alpha and beta subunits had high sequence identity with alpha- and beta-type subunits of proteasomes from eucaryotic organisms and the distantly related archaeon Thermoplasma acidophilum . The psmB gene was transcribed in vivo as a monocistronic message from a consensus archaeal promoter . The results suggest that proteasomes are more widespread in the Archaea than previously proposed . Southern blotting experiments suggested the presence of ubiquitin-like sequences in M . thermophila.

Nucleic Acids Res, 1995 Nov 25, 23(22), 4598 - 602
Footprinting of tRNA(Phe) transcripts from Thermus thermophilus HB8 with the homologous phenylalanyl-tRNA synthetase reveals a novel mode of interaction; Kreutzer R et al.; The phosphates of the tRNA(Phe) transcript from Thermus thermophilus interacting with the cognate synthetase were determined by footprinting . Backbone bond protection against cleavage by iodine of the phosphorothioate-containing transcripts was found in the anticodon stem-loop, the D stem-loop and the acceptor stem and weak protection was also seen in the variable loop . Most of the protected phosphates correspond to regions around known identity elements of tRNA(Phe) . Enhancement of cleavage at certain positions indicates bending of tRNAPhe upon binding to the enzyme . When applied to the three-dimensional model of tRNA(Phe) from yeast the majority of the protections occur on the D loop side of the molecule, revealing that phenylalanyl-tRNA synthetase has a rather complex and novel pattern of interaction with tRNAPhe, differing from that of other known class II aminoacyl-tRNA synthetases.

Biochim Biophys Acta, 1995 Nov 22, 1240(1), 83 - 8
Stability against temperature and external agents of vesicles composed of archael bolaform lipids and egg PC; Fan Q et al.; The bolaform lipid PLE extracted from the thermophilic archaeon Sulfolobus solfataricus and its mixtures with egg phosphatidyl-choline (egg PC) have been used to prepare sonicated vesicles . The leakage of entrapped calcein was continuously monitored by fluorescence dequenching . The half times of leakage have been used to compare vesicle stability under different conditions of temperature, lipid composition and presence of destabilizing agents like Ca2+ ions and poly(ethylene glycol) (PEG) . It has been found that leakage is primarily modulated by the monopolar/bipolar lipid ratio . In particular, the half time of leakage for vesicles formed from a mixture of the polar lipid extract (PLE) and egg PC is characterized by a maximum at about 1:2 molar ratio . The free energy of mixing has been evaluated from pressure-area isotherms on monolayers at the air/water interface . The results indicate a non monotonous behaviour of the excess free energy of mixing as a function of the molar ratio and the occurrence of a minimum at a fixed molar ratio . The possible formation of a complex is discussed and compared with previous calorimetric measurements on similar compounds.

Eur J Biochem, 1995 Nov 15, 234(1), 132 - 9
Properties of isolated domains of the elongation factor Tu from Thermus thermophilus HB8; Nock S et al.; The relative contributions of the three domains of elongation factor Tu (EF-Tu) to the factor's function and thermal stability were established by dissecting the domains apart with recombination techniques . Domain I (EF-TuI), domains I/II (EF-TuI/II) and domain III (EF-TuIII) of the EF-Tu from Thermus thermophilus HB8 comprising the amino acids 1-211, 1-312 and 317-405, respectively, were overproduced in Escherichia coli and purified . A polypeptide consisting of domain II and III (EF-TuII/III) was prepared by limited proteolysis of native EF-Tu with V8 protease from Staphylococcus aureus {Peter, M . E., Reiser, C . O . A., Schirmer, N . K., Kiefhaber, T., Ott, G., Grillenbeck, N . W . & Sprinzl, M . (1990) Nucleic Acids Res . 18, 6889-6893} . As determined by circular dichroism spectrometry, the isolated domains have the secondary structure elements found in the native EF-Tu . GTP and GDP binding as well as GTPase activity are maintained by the EF-TuI and EF-TuI/II; however, the rate of GDP dissociation from EF-TuI . GDP and EF-TuI/II . GDP complex is increased as compared to native EF-Tu . GDP, reflecting a constraint imposed by domain III on the ability to release the nucleotide from its binding pocket located in domain I . A weak interaction of Tyr-tRNATyr with the EF-TuI . GTP suggests that domain I provides a part of the structure interacting with aminoacyl-tRNA . The domain III is capable of regulating the rate of GTPase in EF-Tu, since the polypeptide consisting only of domains I/II has a 39-fold higher intrinsic GTPase compared to the native EF-Tu . No in vitro poly(U)-dependent poly(Phe) synthesis was detectable with a mixture of equimolar amounts of domains I/II and domain III, demonstrating the necessity of covalent linkage between the domains of EF-Tu for polypeptide synthesis . In contrast to native EF-Tu and EF-TuII/III, EF-TuI and, to a lesser extent the polypeptide consisting of domains I/II, are unstable at elevated temperatures . This indicates that domains II/III strongly contribute to the thermal stability of this T . thermophilus EF-Tu . Deletion of amino acid residues 181-190 from domain I of T . thermophilus EF-Tu decreases the thermostability to that of EF-Tu from E . coli, which does not have these residues . Interdomain interactions must be important for the stabilisation of the structure of domain I, since isolated T . thermophilus EF-TuI is thermolabile despite the presence of the 181-190 loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 14, 34(45), 14932 - 41
The single-ring Thermoanaerobacter brockii chaperonin 60 (Tbr-EL7) dimerizes to Tbr-EL14.Tbr-ES7 under protein folding conditions; Todd MJ et al.; Chaperone proteins assist in the folding of some newly synthesized proteins and inhibit protein aggregation . The Thermoanaerobacter brockii chaperonin proteins (Tbr-EL and Tbr-ES) have recently been purified and characterized {Truscott, W.N., Hoj, P . B., & Scopes, R . K . (1994) Eur . J . Biochem . 222, 277-284}; Tbr-EL was a single seven-membered toroid, unlike most GroELs which exist as double toroids . Using high-resolution gel filtration chromatography, we have resolved the purified Tbr-EL into single ringed (Tbr-EL7) and double ringed (Tbr-EL14) species . The latter contained tightly bound Tbr-ES co-chaperonin (Tbr-EL14.Tbr-ES7) . In the presence of Mg.ATP and either Escherichia coli GroES (Eco-ES) or Tbr-ES (i.e., under protein folding conditions), the isolated Tbr-EL7 rapidly dimerized to the Tbr-EL14.Eco-ES7 or Tbr-EL14.Tbr-ES7 complexes . The doubly toroidal species thus formed contained > or = 6 molecules tightly bound ADP and one GroES7 and are similar to the asymmetric chaperonin complex isolated from Thermus thermophilus {Taguch, H., Konishi, J., Ishii, N., & Yoshida, M . (1991) J . Biol . Chem . 266, 22411-22418} . The isolated Tbr-EL7 and Tbr-EL14.Tbr-ES7 hydrolyzed ATP at approximate to 2 and 1 min-1, respectively . Addition of a molar excess of Eco-ES7 to the isolated Tbr-EL7 reduced the ATPase activity to 1 min-1, consistent with the formation of Tbr-EL14.Eco-ES7 . Eco-ES7 failed to inhibit the Tbr-El14.Tbr-ES7 complex . The isolated Tbr-EL14.Tbr-ES7 complex did not support the folding of Rubisco under nonpermissive conditions . Only when the complex was supplemental with additional GroES was folding of Rubisco observed; i.e., one molar equivalent of GroES was not sufficient for folding . Both Tbr-EL7 and Tbr-EL14.Tbr-ES7 bound on unfolded {35S} Rhodospirillum rubrum Rubisco per mole particle . In contrast, Eco-EL14 bound 2 mol of protein per mole particle, consistent with each toroid having a peptide binding site . Eco-EL14.Eco-ES7 complex only bound one unfolded protein, thus GroES binding blocks one GroEL peptide binding site . Addition of Eco-ES7 to a Eco-EL14.Rubisco2 complex did not result in the displacement of one molecule of Rubisco but in the formation of a ternary Eco-EL14.Rubisco2.Eco-ES7 complex.

Antonie Van Leeuwenhoek, 1995 Nov, 68(4), 339 - 44
Granulation in thermophilic upflow anaerobic sludge blanket (UASB) reactors; Schmidt JE et al.; The state of the art for thermophilic UASB reactors is discussed focusing on the start-up of UASB reactors, the influence of the waste water composition and temperature on the development and maintenance of thermophilic granules, and the microbial composition and structure of thermophilic granules.

Antonie Van Leeuwenhoek, 1995 Nov, 68(4), 285 - 91
Isomerization of n- and i-butyrate in anaerobic methanogenic systems; Angelidaki I et al.; Studies of the degradation of the two isomeric forms of butyrate in different anaerobic environments showed isomerization between n- and i-butyrate . Degradation rates were similar for the different examined systems and degradation rates for n-butyrate degradation were generally higher than for i-butyrate . Degradation rates for n-butyrate ranged from 0.52 to 1.39 day-1, while the rates for i-butyrate were from 0.46 to 1.15 day-1 . Production of isomers was not observed when the volatile fatty acid degradation was inhibited by addition of bromoethane sulfonic acid, indicating that isomerization was coupled to the methanogenic degradation of the acid . The degree of isomerization observed during n-butyrate degradation was similar to the degree during i-butyrate degradation . Experiments indicated that the isomerization degree was higher for the thermophilic than for the mesophilic inocula.

Antonie Van Leeuwenhoek, 1995 Nov, 68(4), 273 - 80
Alanine as an end product during fermentation of monosaccharides by Clostridium strain P2; Orlygsson J et al.; The thermophilic Clostridium P2 was isolated from a semi-continuously fed reactor with high ammonium concentration . This bacterium formed substantial amounts of L-alanine as a major fermentation product from glucose, fructose and mannose . Low amounts of acetate, butyrate, carbon dioxide and hydrogen were also formed . A high partial pressure of hydrogen inhibited the degradation of the monosaccharides, whereas hydrogen removal, in the form of methanogenesis was found to be stimulatory . However, the amount of alanine produced per mole of hexose degraded did not change . Hexose degradation and alanine production were favoured by high ammonium concentrations . Nuclear magnetic resonance spectroscopy studies provided strong evidence that an active Embden-Meyerhof-Parnas pathway existed and that alanine was produced via an amination of pyruvate.

Antonie Van Leeuwenhoek, 1995 Nov, 68(4), 263 - 71
Extremely thermophilic cellulolytic anaerobes from Icelandic hot springs; Bredholt S et al.; Anaerobic enrichment cultures with Avicel as substrate and inoculated with biomat samples from Icelandic hot springs were cultured at 70 degrees C or 78 degrees C and examined for the presence of microorganisms that produce extracellular cellulolytic and xylanolytic enzymes . From four enrichments grown at 78 degrees C eighteen strains were isolated . Five of the strains were screened for their substrate utilization, and on the basis of differences in morphology and substrates used, the two most unique strains were selected for further characterization . All cellulolytic cultures were rod-shaped and non-sporeforming . Motility was not observed . Cells stained gram-negative at various stages of the growth phase . During growth on Avicel, most cultures produced acetate as the major fermentation product, with smaller amounts of lactic acid and ethanol . Carbon dioxide and hydrogen were also produced . The phenotypic characteristics of the enrichment cultures and of isolates are described and assessed in relation to temperature and pH in the hot spring environment . A comparison is made between Icelandic strains isolated in our laboratory and strains isolated from hot springs from other parts of the world . The biotechnological potential of this group of bacteria is briefly discussed.

Appl Microbiol Biotechnol, 1995 Nov, 43(6), 1001 - 5
Acetate treatment in 70 degrees C upflow anaerobic sludge-blanket (UASB) reactors: start-up with thermophilic inocula and the kinetics of the UASB sludges; Lepisto R et al.; This study focused on the use the thermophilic anaerobic granulae in the start-up of 70 degrees C acetate-fed upflow anaerobic sludge-blanket (UASB) reactors and the kinetics of granulae grown at 70 degrees C . In the UASB reactors, chemical oxygen demand removal commenced within 48 h of the start-up . The maximum reduction in chemical oxygen demand was 84% with the feed containing yeast and 71% without a yeast supplement . In the bioassays, the yeast-grown sludge converted 98% of the acetate consumed to methane as compared to 92% for the sludge grown without yeast . The highest initial specific methane production rate (mu-CH4) of the UASB sludges grown at 70 degrees C was 0.088 h(-1) at an acetate concentration of 4.6mM . The higher initial acetate concentration was found to prolong the lag-phase in methane production significantly and to decrease mu-CH4 . The half-saturation constant (Ks), the inhibition constant (Ki), the inhibition response coefficient (n) and the mu-CH4-max, calculated according to a modified Haldane equation, were 1.5 mM, 2.8 mM, 0.8 and 0.28 h(-1), respectively . The prolonged starvation of the 70 degrees C sludge (15 days) decreased the mu-CH4 from about 0.022 h(-1) to 0.011 h(-1) and increased the lag phase in methane production from 6 h to 24 h as compared with non-starved sludge.

Genetics, 1995 Nov, 141(3), 925 - 36
Developmental DNA rearrangements and micronucleus-specific sequences in five species within the Tetrahymena pyriformis species complex; Huvos P; In Tetrahymena thermophila, the development of a transcriptionally active macronucleus from a transcriptionally inert micronucleus includes the elimination of many segments of DNA, the bulk of which belong to repetitive sequence families . Two approaches were used to study the interspecies variations in developmentally eliminated DNA segments . First, the occurrence of restriction fragments crosshybridizing to developmentally eliminated DNA segments isolated from T . thermophila was examined in other species of Tetrahymena . Most micronucleus-specific sequence families examined showed large differences in numbers and intensities of crosshybridizing bands in different species, indicating the possibility of gain or loss of repeats within each of the sequence families . Second, the presence of developmentally excisable DNA segments, i.e., of rearrangement sites, was examined in the same set of species at a number of unique loci . This was carried out by comparing the hybridization patterns of seven unique macronucleus-retained sequences in the micro- and macronuclei of each of the species . Essentially all of the loci displayed variability with respect to the presence of rearrangement sites among the species examined . Results from the two approaches indicate that generation or loss of developmental rearrangements can occur among the species examined here.

J Clin Microbiol, 1995 Nov, 33(11), 2826 - 32
Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni; Burnens A et al.; We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428 . The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed . The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18 . Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E . coli . Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum . All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18 . The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C . jejuni . PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C . jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species . In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues . Expression of this construct in E . coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography . Sera from patients with past C . jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen . Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C . jejuni and is highly immunogenic . It is therefore a good candidate as an antigen for the serological diagnosis of past C . jejuni infections.

Arch Microbiol, 1995 Nov, 164(5), 331 - 6
Thermodesulforhabdus norvegicus gen . nov., sp . nov., a novel thermophilic sulfate-reducing bacterium from oil field water; Beeder J et al.; A novel gram-negative, thermophilic, acetate-oxidizing, sulfate-reducing bacterium, strain A8444, isolated from hot North Sea oil field water, is described . The rod-shaped cells averaged 1 micron in width and 2.5 microns in length . They were motile by means of a single polar flagellum . Growth was observed between 44 and 74 degrees C, with an optimum at 60 degrees C . Spores were not produced . Sulfate and sulfite were used as electron acceptors . Sulfur, thiosulfate, nitrate, fumarate, and pyruvate were not reduced . In the presence of sulfate, growth was observed with acetate, lactate, pyruvate, butyrate, succinate, malate, fumarate, valerate, caproate, heptanoate, octanoate, nonadecanoate, decanoate, tridecanoate, pentadecanoate, palmitate, heptadecanoate, stearate, and ethanol . Pyruvate, lactate, and fumarate did not support fermentative growth . Cytochromes of the c-type were present . Desulfoviridin, desulforubidin, P582, and desulfofuscidin were not present . The G+C content of the DNA was 51 mol% . Sequence analysis of 16S rDNA showed that phylogenetically strain A8444 belongs to the delta subdivision of the Proteobacteria . The closest relatives are Desulfacinum infernum and Syntrophobacter wolinii: Strain A8444 is described as the type strain of the new taxon Thermodesulforhabdus norvegicus gen . nov., sp . nov.

Protein Sci, 1995 Nov, 4(11), 2429 - 32
Structural similarities in the noncatalytic domains of phenylalanyl-tRNA and biotin synthetases; Safro M et al.; Detailed comparison between the structures of the Escherichia coli biotin synthetase/repressor protein (BirA) and the recently solved Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) reveals significant similarities outside their respective catalytic domains . These comprise a DNA-binding alpha+beta domain and an Src-homology 3 (SH3)-like domain that were observed in both enzymes . This similarity provides a novel example in which all domains of one multidomain protein appear to be constituents of the other multidomain protein and supports a concept of a common ancestor for two different synthetase families.

Enzyme Microb Technol, 1995 Nov, 17(11), 992 - 7
Expression and extensive characterization of a beta-glycosidase from the extreme thermoacidophilic archaeon Sulfolobus solfataricus in Escherichia coli: authenticity of the recombinant enzyme; Moracci M et al.; The gene coding for the beta-glycosidase from the archaeon Sulfolobus solfataricus has been overexpressed in Escherichia coli . The enzyme was purified to homogeneity with a rapid purification procedure employing a thermal precipitation as a crucial step . The final yield was 64% and the purification from the thermal precipitation was 5.4-fold . The expressed enzyme shows the same molecular mass, thermophilicity, thermal stability, and broad substrate specificity, with noticeable exocellobiase (glucan 1,4-beta-D-glucosidase) activity, of the enzyme purified from S . Solfataricus . We provide evidence that the beta-glycosidase can assume its functional state in E . coli without the contribution of N-epsilon-methylated lysine residues.

Eur J Biochem, 1995 Nov 1, 233(3), 800 - 8
The phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase genes from the thermophilic archaeon Sulfolobus solfataricus overlap by 8-bp . Isolation, sequencing of the genes and expression in Escherichia coli; Jones CE et al.; The overlapping genes encoding phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) from the hyperthermophilic archaeon Sulfolobus solfataricus have been cloned and sequenced . PCR primers based on highly conserved regions of different PGK sequences were used to isolate an internal region of the pgk gene . This was then used to screen a genomic library to isolate the full length pgk gene . A 2.5-kb BglII fragment of S . solfataricus DNA contained both the pgk gene and the gap gene immediately downstream . Unexpectedly, the pgk and gap genes were found to overlap by 8 bp, with the initiation codon of the gap gene preceding the termination codon of the pgk gene . Evidence that the two genes are co-transcribed was obtained by Northern-blot analysis . The S . solfataricus PGK amino acid sequence shows 43% and 45% identity to the PGK sequences of the Archaea Methanobacterium bryantii and Methanothermus fervidus, respectively . High level expression of the S . solfataricus PGK and GraP-DH in Escherichia coli was achieved, with heat treatment at 80 degrees C proving an effective first step in the purification of these recombinant enzymes from extracts of the E . coli host . Purified recombinant S . solfataricus PGK and GraP-DH showed half lives of 39 min and 17 h, respectively, at 80 degrees C . Unlike bacterial GraP-DH enzymes, S . solfataricus GraP-DH was able to use both NAD+ and NADP+ as cofactors, but exhibited a marked preference for NADP+.

J Eukaryot Microbiol, 1995 Nov-Dec, 42(6), 742 - 8
Deciliation induces phosphorylation of a 90-kDa cortical protein in Tetrahymena thermophila; Gitz DL et al.; We have used the anti-phosphoprotein antibody MPM-2 to examine changes in phosphorylation of cortical proteins during cilia regeneration in Tetrahymena thermophila . Although numerous cortical proteins are phosphorylated in both nondeciliated and deciliated cells, deciliation induces a dramatic increase in the phosphorylation of a 90-kDa cortical protein . The 90-kDa protein remained phosphorylated during cilia regeneration and then gradually became dephosphorylated . The 90-kDa protein was phosphorylated and dephosphorylated normally in Tetrahymena mutants that assemble short cilia, suggesting that achievement of full length is not the signal that triggers dephosphorylation of the 90-kDa protein . When initiation of cilia assembly is blocked, the 90-kDa protein becomes phosphorylated and remains phosphorylated for an extended period of time, suggesting that initiation of cilia elongation triggers eventual dephosphorylation of the 90-kDa protein, regardless of how long the cilia actually become.

Curr Microbiol, 1995 Nov, 31(5), 270 - 8
16S-23S and 23S-5S intergenic spacer regions of Streptococcus thermophilus and Streptococcus salivarius, primary and secondary structure; Nour M et al.; The 16S-23S intergenic spacer region (spacer region 1) of Streptococcus salivarius, S . thermophilus, and Lactococcus lactis subsp . cremoris and the 23S-5S intergenic spacer region (spacer region 2) of S . salivarius and L . lactis subsp . cremoris were sequenced and compared with the spacer regions 1 and 2 of other streptococci . A high degree of intraspecific conservation was observed for S . thermophilus and L . lactis, and very similar sequences were found for S . salivarius and S . thermophilus . Whereas spacer region 1 is highly conserved in the genus Streptococcus sensu-stricto, only the tRNA gene and the rRNA processing stems are highly conserved in the three genera: Streptococcus sensu-stricto, Lactococcus, and Enterococcus . The presence of a unique tRNA(Ala) gene without the 3' terminal CCA sequence seems to be a general feature of the streptococci spacer region 1 . A secondary structure model was built to show the interaction between the spacer regions 1 and 2 of S . thermophilus and S . salivarius . The rapid evolution of spacer region 1 in streptococci is in part due to insertions and deletions of small RNA stem/loop structures.

FEBS Lett, 1995 Oct 23, 374(1), 110 - 2
Crystallization of threonyl-tRNA synthetase from Thermus thermophilus and preliminary crystallographic data; Cura V et al.; Threonyl-tRNA synthetase from Thermus thermophilus (ttTRS) has been overproduced in Escherichia coli, purified and crystallized in solutions containing ammonium sulfate and glycerol . The crystals grew in the orthorhombic space group C222(1) with unit cell dimensions a = 119.5 A, b = 120.0 A, c = 317.5 A . The asymmetric unit is constituted of two monomers and the crystals contain 66% solvent . This paper reports the first crystals of ttTRS and preliminary crystallographic results since the presumed crystals of ttTRS described in a previous paper {1} were crystals of aspartyl-tRNA synthetase {2}.

Biochim Biophys Acta, 1995 Oct 17, 1264(1), 53 - 62
Structure and evolution of Paramecium hemoglobin genes; Yamauchi K et al.; Hemoglobin (Hb) genes have been cloned from three different species of ciliated protists, P . multimicronucleatum, P . triaurelia and P . jenningsi . Southern blotting of the genomic DNAs using the P . caudatum Hb cDNA showed both intraspecies variation in different stocks of P . caudatum and interspecies variation within the genus Paramecium . The isolated Hb genes were composed of 118, 117 and 117 codons, and interrupted by a short intron with 27, 29 and 29 bp at the same position, in P . multimicronucleatum, P . triaurelia and P . jenningsi, respectively . This suggests that the one-intron and two-exon structure has been conserved in the Hb genes in this genus . The amino acid sequences of the Paramecium Hbs were more than 87% identical to one another and homologous to those from the other ciliated protists Tetrahymena thermophila and T . pyriformis, the green alga Chlamydomonas eugametos, and the cyanobacterium Nostoc commune Hbs, all of which consist of about 120 amino acid residues (120-aa group) . In particular, the amino acid sequences of the P . triaurelia and P . jenningsi Hbs were the same, although there were 20 nucleotide differences between the coding regions in the two genes . A maximum likelihood inference as to the phylogenetic relationships among these genes suggests that the Paramecium Hbs genes have evolved more rapidly than the other genes in the 120-aa group, and that P . triaurelia and P . genningsi are sibling species and the P . aurelia complex became a small cell after it separated from P . jenningsi.

Gene, 1995 Oct 16, 164(1), 163 - 6
Sequence, codon usage and cysteine periodicity of the SerH1 gene and in the encoded surface protein of Tetrahymena thermophila; Deak JC et al.; The temperature-regulated SerH1 gene coding for an immunodominant surface glycoprotein (i-Ag H1) of Tetrahymena thermophila has been sequenced . The gene is reproducibly rearranged during macronuclear development and steady state mRNA levels are present at < 36 degrees C . The deduced i-Ag H1 amino acid (aa) sequence is rich in Ser, Thr and Cys, and contains three periods each consisting of 85 aa punctuated by eight Cys with the general formula, CX6CX17CX2CX18CX2CX11CX2CX19 (where X = any aa) . Such Cys periodicity is common to ciliate i-Ag . Codon usage in Tt, Paramecium primaurelia and P . tetraurelia i-Ag encoding genes is similar, with approx . 80% A+T in the 3' position which is in marked contrast to the approx . 54% 3' A+T in other ciliate genes.

Eur J Biochem, 1995 Oct 15, 233(2), 677 - 82
Functional and structural changes of the photosystem II complex induced by high irradiance in cyanobacterial cells; Komenda J et al.; A gradual disintegration of the photosystem II (PSII) complex, initiated by a release of the chlorophyll-protein CP43, was identified during low-temperature illumination of Synechococcus cells . This process was slower compared to the decline of the PSII primary charge separation activity, and much slower than the photoinactivation of oxygen evolution . All three processes were slowed down in the presence of diuron . The results indicate that when the PSII repair was blocked, the inactivation of charge separation activity and the release of CP43 preceded the degradation of the D1 protein . In contrast, a much faster degradation of D1 connected to its rapid exchange was triggered by inactivation of oxygen evolution, and no disassembly of PSII was needed . We propose the existence of two different mechanisms of D1 degradation in the cells of the thermophilic cyanobacterium Synechococcus elongatus.

J Mol Biol, 1995 Oct 6, 252(5), 583 - 95
Dissecting and analyzing the secondary structure domains of group I introns through the use of chimeric intron constructs; Tanner NK et al.; The mitochondrial genes of the yeast Saccharomyces cerevisiae are often interrupted by introns defined as either group I or group II . Some of the introns contained within the precursor RNAs of these genes will self splice in vitro . The fourth introns of apocytochrome b (bi4) and cytochrome oxidase (ai4) are group I introns that do not self splice in vitro, even though they can fold into the same RNA secondary structures that are characteristic of the self-splicing introns . They require an intron-encoded maturase protein and a nuclear-encoded protein (a tRNALeu synthetase) for splicing in vivo . We have divided these introns into several sequence or structural elements and assayed them individually for their ability to support self-splicing activity . This was done by replacing the equivalent elements from the self-splicing intron from Tetrahymena thermophila with the mitochondrial elements . These intron chimeras show that peripheral sequences and the elements that define the splice sites are adequate for self-splicing activity but that the central portions containing the catalytic cores of ai4 and bi4 are deficient; these cores are the likely targets of the splicing proteins . In addition, the catalytic activity of the Tetrahymena intron is remarkably resistant to the structural alterations that we have introduced; this suggests that this technique will be of general utility for studying the structural and functional relationships of elements contained within different RNAs.

Biochemistry, 1995 Oct 3, 34(39), 12535 - 42
Interaction of guanosine nucleotides and their analogs with elongation factor Tu from Thermus thermophilus; Wagner A et al.; Transient kinetic experiments on the interaction of nucleotide-free EF-Tu from Thermus thermophilus with nucleotides using intrinsic protein fluorescence, extrinsic nucleotide fluorescence and fluorescence resonance energy transfer show that nucleotide binding is in general at least a two-step process . The first step is a weak initial binding, which is followed by a relatively slow isomerization of the protein-nucleotide complex in which changes of both intrinsic and extrinsic fluorescence, as well as energy transfer, occur . The values obtained for the equilibrium and kinetic constants confirm the earlier observation that EF-Tu has a higher affinity for GDP than GTP . This is mainly due to a lower dissociation rate constant for GDP, in combination with a somewhat higher effective association rate constant . Modifications of the triphosphate moiety of GTP are quite well tolerated by EF-Tu, with GTP gamma S displaying the same affinity as GTP and with GppNHp and GppCH2p being only ca . 2-3-fold less strongly bound . Caged GTP is bound about 6-fold more weakly than GTP . These results suggest that the binding of GppNHp and GppCH2p is likely to be similar to that of GTP . The photolytic protecting group of caged GTP (or the loss of one of the negative charges on the gamma-phosphate group) appears to interfere to a certain extent with the interaction with the protein, but the affinity is high enough to permit generation of 1:1 complexes for dynamic structural studies . Discrimination between GDP and ADP is dramatic, with a difference of 6 orders of magnitude in affinity.

Protein Eng, 1995 Oct, 8(10), 1039 - 47
Stability of native and covalently modified papain; Rajalakshmi N et al.; Covalent modification of enzymes with large polymers can produce modified enzymes which retain considerable biological activity and at the same time display resistance to denaturation by high temperatures and chaotropic agents . The cysteine protease, papain, with potential applications in industry, was covalently coupled to polymeric sucrose (mol . wt 400 kDa) at different ratios . The derivatives retained > 80% intrinsic catalytic activity with no change in pH optima and kinetic constants, indicating that the gross tertiary structure was not altered by modification . However, they displayed better thermotolerance than native papain, as indicated by their higher T50 values (6-10 degrees C) and their temperature optima being shifted by 10 degrees C . The half-life of modified papain, calculated from the rate of thermoinactivation, was prolonged by 2- to 30-fold over the native depending on the temperature and proportion of polymeric sucrose in the adducts . The increases in activation free energy of inactivation (1-10 kJ/mol) and activation enthalpy (4-78 kJ/mol) indicate stabilization of the protein and lesser inactivation due to spontaneous unfolding . In the presence of urea, modified papain showed activation, which may be due to a loosening of the 'rigid' structure, reminiscent of the property of thermophilic enzymes.

Int J Food Microbiol, 1995 Oct, 27(2-3), 253 - 61
Rapid enzymatic method for biotyping and control of lactic acid bacteria used in the production of yogurt and some cheeses; Bianchi-Salvadori B et al.; Determination of enzymatic patterns of 30 strains of Streptococcus thermophilus and 18 strains of Lactobacillus delbrueckii subsp . bulgaricus with a rapid APIZYM method was carried out . Alkaline and acid phosphatase, naphthol-AS-BI-phosphohydrolase, 10 esterases, 20 glycosidases, 61 peptidases and 2 proteases (trypsin and chymotrypsin) were included . The strains investigated were isolated from yogurt and from different starters used for different Italian cheeses . For S . thermophilus, all strains were positive for 3 glycosidases, 4 monopeptidases, 9 dipeptidases, 1 tripeptidase and all were negative for 2 esterases; 9 glycosidases; 8 peptidases and trypsin . For L . delbrueckii subsp . bulgaricus it was observed that all strains were positive for 2 esterases; 2 glycosidases; 11 monopeptidases, 9 dipeptidases, 2 tripeptidases and 1 tetrapeptidase and all were negative for alkaline-phosphatase; 3 esterases, 7 glycosidases, 5 monopeptidases, 2 dipeptidases . The defined enzymatic pattern of starter cultures can be used for predicting their suitability for dairy fermentations and for monitoring their stability as well as for typing.

Med Microbiol Immunol (Berl), 1995 Oct, 184(3), 147 - 53
Binding of outer membrane preparations of Campylobacter jejuni to INT 457 cell membranes and extracellular matrix proteins; Moser I et al.; Binding of outer membrane (OM) preparations of the thermophilic Campylobacter species C . jejuni to epithelial cell membranes and extracellular matrix proteins were studied in an in vitro model system using enzyme-linked immunosorbent assay . The OM preparations exhibited significant binding to INT 407 intestinal cell membranes . The process of adhesion was modulated by enzymatic, chemical or immunological pretreatment of the bacteria . Following oxidation of the lipopolysaccharide (LPS) with sodium meta-periodate, the OM preparations essentially retained their binding properties . After pretreatment with proteinase K, the OM preparations lost their binding capacity and the apparent molecular mass of the major OM protein shifted from 42 to 24 kDa . Preincubation of C . jejuni bacteria with C . jejuni-specific antiserum reduced adhesion significantly; preincubation with LPS-specific monoclonal antibodies only to a minimal extent . The OM preparations also bound significantly to the extracellular matrix proteins collagen and fibronectin; however, they bound virtually no bovine serum albumin or horse serum.

J Biochem (Tokyo), 1995 Oct, 118(4), 745 - 52
Ligand-induced changes in the conformation of 3-isopropylmalate dehydrogenase from Thermus thermophilus; Kadono S et al.; The structures of 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus in complexes with its substrate, cofactor, and a cofactor analog were investigated by X-ray diffraction in a crystalline state and by small-angle X-ray scattering (SAXS) in solution . The structures at 2.8 A resolution of the complexes with the substrate, 3-isopropylmalate (IPM), and with an analog of NAD, ADP-ribose, were both very close to the structure of the free enzyme, which adopts an open conformation . However, the binding of a ligand induced a small conformational change near the binding site . This result contrasts with results for NADP(+)-bound and isocitrate-bound isocitrate dehydrogenase (ICDH) from Escherichia coli, which adopts a closed conformation . The SAXS analysis in solution clearly showed that IPMDH without a ligand adopts two distinct intermediate conformations, between the open and closed states, upon binding of NADH and IPM respectively, and adopts a fully closed conformation when in a ternary complex with NADH and IPM together.

J Biochem (Tokyo), 1995 Oct, 118(4), 679 - 80
Crystallization and preliminary X-ray studies of isocitrate dehydrogenase from Thermus thermophilus HB8; Ohzeki M et al.; Isocitrate dehydrogenase from the thermophilic bacterium, Thermus thermophilus HB8, was crystallized by the vapor diffusion hanging drop method with polyethylene glycol 6000 as the precipitant . Pillar-like crystals of about 0.6 x 0.6 x 0.3 mm3 were obtained . Analysis of a series of oscillation photographs indicated that the orthorhombic crystals belonged to the I222 or I2(1)2(1)2(1) space group with unit cell dimensions of a = 100.1 A, b = 150.4 A, and c = 87.4 A . Intensity data were collected up to 2.5 A resolution.

Genet Anal, 1995 Oct, 12(2), 119 - 21
T-vector cloning and high performance PCR with SuperTth from Thermus thermophilus; Leal JF et al.; The SuperTth DNA polymerase from Thermus thermophilus exhibits template-independent terminal transferase (extendase) activity . This enzyme is proposed as a cheap alternative for both high performance PCR as well as quick T-vector cloning of amplicons, including reverse transcription and cDNA cloning.

J Clin Microbiol, 1995 Oct, 33(10), 2643 - 6
Inhibition of PCR by aqueous and vitreous fluids; Wiedbrauk DL et al.; The detection of viral nucleic acids in intraocular fluids and tissues by PCR has become increasingly important in clinical ophthalmology . While much attention has been directed toward minimizing false-positive reactions resulting from specimen contamination or amplicor carryover, relatively little attention has been given to the causes of false-negative PCRs . This report describes a PCR inhibitor in normal aqueous and vitreous fluids that can produce false-negative PCR results . As little as 0.5 microliter of vitreous fluid and 20 microliters of aqueous fluid can completely inhibit DNA amplification in a 100-microliters PCR mixture . This inhibition was not primer specific, nor was it due to chelation of Mg2+ ions or DNase activity in the ocular fluid . The inhibitor was completely resistant to boiling for 15 min . However, the inhibitory effects were completely removed by a single chloroform-isoamyl alcohol (24:1) extraction . The extent of PCR inhibition depended upon the type of thermostable DNA polymerase used in the reaction . Taq DNA polymerase was very sensitive to the inhibitor, while thermostable DNA polymerases from Thermus thermophilus HB-8 (Tth) and Thermus flavus (Tfl) were completely resistant . Thus, the inhibitory effects of intraocular fluids on PCRs can be removed by diluting the specimen, by chloroform extraction, or by using Tth or Tfl DNA polymerases.

Biokhimiia, 1995 Oct, 60(10), 1720 - 30
{Preparative isolation of proteins from 30S ribosomal subparticles from Thermus thermophilus under nondenaturing conditions}; Eliseikina IA et al.; A procedure for isolation in preparative amounts of 15 individual proteins from ribosomal 30S subparticles of Thermus thermophilus under non-denaturing conditions, has been developed . The amino acid composition and molecular masses of the proteins have been determined and the UV absorption spectra and extinction coefficients measured . A homology of 13 proteins to corresponding ribosomal proteins of E . coli has been established.

Protein Sci, 1995 Oct, 4(10), 2156 - 67
The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase; Dean AM et al.; The kinetic mechanism of the oxidative decarboxylation of 2R,3S-isopropylmalate by the NAD-dependent isopropylmalate dehydrogenase of Thermus thermophilus was investigated . Initial rate results typical of random or steady-state ordered sequential mechanisms are obtained for both the wild-type and two mutant enzymes (E87G and E87Q) regardless of whether natural or alternative substrates (2R-malate, 2R,3S-tartrate and/or NADP) are utilized . Initial rate data fail to converge on a rapid equilibrium-ordered pattern despite marked reductions in specificity (kcat/Km) caused by the mutations and alternative substrates . Although the inhibition studies alone might suggest an ordered kinetic mechanism with cofactor binding first, a detailed analysis reveals that the expected noncompetitive patterns appear uncompetitive because the dissociation constants from the ternary complexes are far smaller than those from the binary complexes . Equilibrium fluorescence studies both confirm the random binding of substrates and the kinetic estimates of the dissociation constants of the substrates from the binary complexes . The latter are not distributed markedly by the mutations at site 87 . Mutations at site 87 do not affect the dissociation constants from the binary complexes, but do greatly increase the Michaelis constants, indicating that E87 helps stabilize the Michaelis complex of the wild-type enzyme . The available structural data, the patterns of the kinetics results, and the structure of a pseudo-Michaelis complex of the homologous isocitrate dehydrogenase of Escherichia coli suggest that E87 interacts with the nicotinamide ring.

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