Thermophile

Protein Expr Purif, 1995 Oct, 6(5), 707 - 15
High yield purification of a four subunit caa3-type cytochrome oxidase from the thermophilic bacterium Bacillus PS3 using fast protein liquid chromatography; Kirken RA et al.; The thermophilic bacterium, Bacillus PS3, was grown in a vigorously aerated nutrient broth at 65 degrees C with 100 mM glutamic acid serving as a supplemental carbon and nitrogen source . These growth conditions resulted in membranes highly enriched in cytochrome c oxidase (COX) {23.32 +/- 4.32 nmol heme a/g of cells (n = 5)}, which is nearly a threefold higher concentration of COX (heme caa3-type) than previously reported for this organism . A new high-yield purification of COX was performed by extracting the bacterial membranes with Triton X-100 (7 mg/mg protein), followed by ion-exchange fast liquid protein chromatography using a QAE (trimethyl ammonium) resin with subsequent hydroxyapatite chromatography and ammonium sulfate fractionation . This purification regime resulted in a 16% yield of cytochrome c oxidase with 20 mg of pure caa3-type COX (13 nmol heme a/mg protein) isolated from 100 g of cells . SDS-PAGE showed that the isolated enzyme had four subunits with apparent Mr of 68, 38, 23, and 13 kDa . In addition, a new 34-kDa peptide was also detected in this preparation, which may represent the ORF1 gene product for this organism . Subunit II (Mr = 38 kDa) of the isolated enzyme was shown to contain covalently bound heme c by using both heme-staining of SDS-PAGE and immunoreactivity with an anti-cytochrome c antibody . The purified enzyme also exhibited high electron transfer activity (340s-1) when assayed at pH 6.5 in the presence of the nonionic detergent, beta-dodecyl maltoside.

Protein Expr Purif, 1995 Oct, 6(5), 637 - 45
Overexpression and purification of Thermus thermophilus elongation factors G, Tu, and Ts from Escherichia coli; Blank J et al.; The translation elongation factors G (EF-G), Tu (EF-Tu), and Ts (EF-Ts) from the extreme thermophilic bacterium Thermus thermophilus were overproduced in Escherichia coli . The fus gene coding for EF-G and the tufA gene coding for EF-Tu were expressed under the control of a tac promoter, whereas EF-Ts was overproduced with the T7 RNA polymerase system . A detailed description for the purification of the three elongation factors from E . coli is presented . EF-G and EF-Tu are isolated by Q-Sepharose FF chromatography, heat treatment at 65 or 60 degrees C, respectively, and Sephacryl S200 gel permeation chromatography . For the purification of EF-Ts, a heat denaturation step is followed by DEAE-cellulose chromatography and a cation exchange EMD-SO-3 650 column . The overproduced factors show the same properties as those purified from T . thermophilus . As the crystal structures of T . thermophilus EF-Tu and EF-G have been solved recently, many questions concerning the function of particular residues or domains arise, which may be best addressed by studying the in vitro behavior and structure of altered recombinant constructs . The methods presented here should facilitate such studies.

J Appl Bacteriol, 1995 Oct, 79(4), 447 - 53
Purification and characterization of the major beta-1,4-endoglucanase from Thermomonospora curvata; Lin SB et al.; The major beta-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata, contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose . The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC . This monomeric enzyme had a specific activity of 750 IU mg(-1) when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70 degrees C, pH 6.0 . Highest activity was observed on CMC with a degree of polymerization of 3200 . The EG was stable for 48 h at 60 degrees C, pH 6.0 and had a half-life of 30 min at 80 degrees C; temperature and pH optima were 70-73 degrees C and 6.0-6.5, respectively . The mol . wt was 100,000 and the pI was 4.0 . The Km and Vmax values were 7.33 mg/ml(-1) and 833 microns min(-1), respectively . EG activity was inhibited by Fe(2+), Hg(2+), Ag(+) and Pb(2+), and enhanced by dithiothreitol and Zn(2+) . The first 12 amino acid residues at the N-terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser . Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.

Eur J Biochem, 1995 Oct 1, 233(1), 227 - 37
Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea . Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein . Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus; Hess D et al.; The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced . The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical M(r) of 46,195 . The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus {Fabry, S., Heppner, P., Dietmaier, W . & Hensel, R . (1990) Gene 91, 19-25} . By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms {Zwickl, P., Fabry, S., Bogedain, C., Haas, A . & Hensel, R . (1990) J . Bacteriol . 172, 4329-4338} . With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases . To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P . woesei and M . fervidus were expressed in Escherichia coli . Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E . coli indicate that the proteins expressed in the mesophilic host are folded correctly . Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects . (a) The 3-phosphoglycerate kinases from P . woesei and M . fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M . bryantii . (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+ . For the P . woesei enzyme, non-cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M . fervidus 3-phosphoglycerate kinase the K+ binding is rather complex: from the fitting of the saturation data, non-cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd = 270 mM) and at least three cooperative and highly specific K+ binding sites/subunit are deduced . At the optimum growth temperature of P . woesei (100 degrees C) and M . fervidus (83 degrees C), the 3-phosphoglycerate kinases show half-lives of inactivation of only 28 min and 44 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Appl Microbiol Biotechnol, 1995 Oct, 43(5), 856 - 60
Debranching of arabinoxylan: properties of the thermoactive recombinant alpha-L-arabinofuranosidase from Clostridium stercorarium (ArfB); Schwarz WH et al.; The gene arfB encoding alpha-L-arabinofuranosidase B of the cellulolytic thermophile Clostridium stercorarium was expressed in Escherichia coli from a 2.2-kb EcoRI DNA fragment . The recombinant gene product ArfB was purified by fast-performance liquid chromatography . It has a tetrameric structure with a monomeric relative molecular mass of 5200 . The optima for temperature and pH are 70 degrees C and 5.0 respectively . The enzyme appears to have no metal cofactor requirement and is sensitive to sulfhydryl reagents . It hydrolyzes aryl and alkyl alpha-L-arabinofuranosides and cleaves arabinosyl side-chains from arabinoxylan (oat-spelt xylan) and from xylooligosaccharides produced by recombinant endoxylanase XynA from the same organism . The identify of the N-terminal amino acid sequences indicates that ArfB corresponds to the major alpha-arabinosidase activity present in the culture supernatant of C . stercorarium.

Biotechnol Appl Biochem, 1995 Oct, 22 ( Pt 2), 203 - 14
Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions; Chang BS et al.; BSA stabilizes Streptococcus thermophilus beta-galactosidase against thermal inactivation and binds to the active enzyme subunits formed on heating . The mechanism of interaction and stabilization, however, is unknown, and it was investigated using different proteins . The results show that several proteins increased the enzyme half-life (t 1/2) at 64 degrees C in the presence of the substrate lactose . The best stabilizers were BSA (9-fold) and casein (6-fold) . There was a significant correlation between enzyme half-life (t 1/2) and surface hydrophobicity of the proteins (So), of the form t 1/2 varies; is directly proportional to S0.5o . The surface hydrophobicity of the enzyme increased upon heating, while that of BSA declined . Heating enzyme and BSA together caused a net loss in surface hydrophobicity, indicating hydrophobic interactions, but there was no change in the absence of heating . Stabilization of the enzyme by BSA was markedly affected by chaotropic and kosmotropic salts . Stabilization was increased by 1 M Na2SO4 and reduced by 1 M NaBr; 1 M NaCl had little effect . None of the three salts increased the stability of the enzyme itself, indicating that the effect was on the enzyme-protein interaction . The results indicate that BSA stabilized the enzyme by hydrophobic interactions with the heated enzyme and that surface hydrophobicity is a major determinant of the extent of stabilization by a protein.

Virology, 1995 Oct 1, 212(2), 632 - 40
Characterization of a temperate Streptococcus thermophilus bacteriophage and its genetic relationship with lytic phages; Brussow H et al.; The temperate Streptococcus thermophilus bacteriophage phi SFi21 showed an 38-kb-long double-stranded DNA genome with cohesive ends . A single integration site was used in lysogens established in three different S . thermophilus strains . The attP and attB sites were localized on the restriction map of phage DNA and by hybridization on pulsed field separated bacterial DNA . All laboratory-established lysogens showed in addition to integrated prophage DNA unintegrated monomer phage DNA with unligated cos sites . The genetic relatedness of phi SFi21 DNA with DNA from lytic phages was studied in dot blot and Southern blot hybridization by using individual restriction fragments of phiSFi21 DNA as probes . Lytic group I phages hybridized with fragments of the central and the right part of the phiSFi21 genome but failed to hybridize with a fragment joining both parts . Lytic group II phages showed hybridization with the right half of the phiSFi21 genome . In lytic group IV phages, biologically a heterogeneous group, many different combinations of cross hybridization were detected in accordance with the hypothesis of the modular evolution of phage genomes.

J Bacteriol, 1995 Oct, 177(19), 5473 - 9
Motility and thermotactic responses of Thermotoga maritima; Gluch MF et al.; Thermotoga maritima, a thermophilic eubacterium, is motile at temperatures ranging from 50 to 105 degrees C . The cells are propelled by a single flagellum which most of the time spins clockwise . Changes in the swimming direction ("tumbles") are achieved by short reversals of the direction of filament rotation . The average speed of swimming cells depends on the temperature, reaching a maximum value of about 60 microns/s at 85 degrees C . The cells show a thermotactic response to temporal temperature changes . When the temperature is raised, the rate of tumbles is increased, while decreasing temperature decreases the tumbling rate.

J Bacteriol, 1995 Oct, 177(19), 5460 - 6
Horizontal transference of S-layer genes within Thermus thermophilus; Fernandez-Herrero LA et al.; The S-layers of Thermus thermophilus HB27 and T . thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively . We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene . Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division . However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain . The addition of chromosomic DNA from T . thermophilus HB8 to growing cultures of T . thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein . Conversely, the addition of chromosomic DNA from T . thermophilus HB27 to growing cultures of T . thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein . The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments.

Dev Biol, 1995 Oct, 171(2), 497 - 506
The effects of lithium chloride on pattern formation in Tetrahymena thermophila; Jerka-Dziadosz M et al.; Lithium ions have long been known to exert dramatic effects on the specification of cell fates in multicellular systems . We have analyzed the effects of Li+ on intracellular patterning in a complex unicellular organism, the ciliate Tetrahymena thermophila . LiCl does not affect the locations of major structural landmarks in the cortical region of wild-type cells and does not modify the phenotype of pattern-mutant cells . However, in all strains studied LiCl differentially affects early stages of oral development . It initially triggers a slow regression of oral primordia, which is followed by an excessive proliferation of basal bodies that leads to a hypertrophy of the ciliature of the cell's feeding organelle . This hypertrophy mimics the effects of the membranellar-pattern-D mutation, the phenotype of which is enhanced in the presence of LiCl . These effects were partially reversed by myo-inositol; however, neomycin failed to mimic the effects of LiCl . Thus, although lithium ions have major cellular effects on Tetrahymena, they do not influence the specification of the body plan in a manner analogous to that observed in multicellular organisms and may work in part through mechanisms other than the now-classical inositol-phosphate cycle.

Int J Syst Bacteriol, 1995 Oct, 45(4), 811 - 9
Comparison of Mycobacterium 23S rRNA sequences by high-temperature reverse transcription and PCR; Stone BB et al.; We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium . We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus . A 70 degrees C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol% . The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with alpha-33P-labeled primers, again with extension at 70 degrees C . Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase . Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material . Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and Rhodococcus bronchialis as the outgroup . Our 23S rRNA trees were compared with previously published 16S rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously . Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed.

Int J Syst Bacteriol, 1995 Oct, 45(4), 783 - 9
Description of Thermoanaerobacter brockii subsp . lactiethylicus subsp . nov., isolated from a deep subsurface French oil well, a proposal to reclassify Thermoanaerobacter finnii as Thermoanaerobacter brockii subsp . finnii comb . nov., and an emended description of Thermoanaerobacter brockii; Cayol JL et al.; A strictly anaerobic, thermophilic, gram-positive, spore-forming cubacterium designated strain SERB 5268T (T = type strain) was isolated from an oil field at a depth of 2,100 m, where the temperature was 92 degrees C . The cells of this organism were gram-positive, straight, motile rods (0.5 by 2 to 3 microns) with peritrichous flagella . The cells occurred singly or in pairs during the logarithmic growth phase, but were pleomporphic and filamentous (length, 15 microns) in old cultures . Growth occurred at temperatures of 40 to 75 degrees C, and optimum growth occurred at temperatures between 55 and 60 degrees C . The fermentable substrates included glucose, fructose, galactose, mannose, cellobiose, maltose, sucrose, lactose, D-xylose, D-ribose, mannitol, pyruvate, and starch . The products of fermentation of glucose were lactate, acetate, ethanol, H2, and CO2 . The DNA base composition was 35 mol% G+C . The results of 16S rRNA sequence comparisons indicated that strain SEBR 5268T was closely related to Thermoanaerobacter brockii and Thermoanaerobacter finnii, and these three organisms exhibited levels of ribosomal DNA sequence homology of 98 to 99% . The results of DNA-DNA hybridization studies performed with the three organisms confirmed this close affiliation, and as base pairing values of > 70% were obtained, these organisms belong to the same species . Therefore, we propose that T . finnii should be reclassified as a subspecies of T . brockii, Thermoanaerobacter brockii subsp . finnii comb . nov . This automatically creates Thermoanaerobacter brockii subsp . brockii . We also propose that strain SEBR 5268T should be classified as a member of a new subspecies of T . brockii, Thermoanaerobacter brockii subsp . lactiehylicus.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Syst Bacteriol, 1995 Oct, 45(4), 676 - 81
Chloroflexus aggregans sp . nov., a filamentous phototrophic bacterium which forms dense cell aggregates by active gliding movement; Hanada S et al.; Two strains of thermophilic photosynthetic bacteria, designated MD-66T (T = type strain) and YI-9, were isolated from bacterial mats in two separate hot springs in Japan . These new isolates were phenotypically similar to Chloroflexus aurantiacus in some respects . They were thermophilic filamentous photosynthetic bacteria that grew well at 55 degrees C either anaerobically as photoheterotrophs or aerobically as chemoheterotrophs . They exhibited gliding motility, produced bacteriochlorophylls a and c, contained chlorosomes, and required thiamine and folic acid as growth factors . However, isolates MD-66T and YI-9 had the ability to rapidly form mat-like dense aggregates of filaments, an ability which has not been observed in any C . aurantiacus strain . Carbon source utilization tests revealed that unlike C . aurantiacus, the new isolates did not utilize acetate, citrate, ethanol, or glycylglycine . An analysis of the carotenoid components revealed that isolates MD-66T and YI-9 contained mainly gamma-carotene and OH-gamma-carotene glucoside fatty acid esters . These isolates also contained only trace amounts of beta-carotene, which is a major carotenoid component (28.4% of the total carotenoids) in C . aurantiacus . The results of DNA hybridization studies suggested that the new strains were genetically distinct from C . aurantiacus (levels of similarity, 9 to 18%), and 16S rRNA sequence comparisons showed that strain MD-66T was related to C . aurantiacus at a similarity level of 92.8% . On the basis of our data, we propose that a new Chloroflexus species should be created for our new isolates; the name of this new species is Chloroflexus aggregans, and the type strain is strain MD-66 (= DSM 9485).

Biochim Biophys Acta, 1995 Sep 27, 1252(1), 1 - 14
Understanding and increasing protein stability; Fagain CO; This review surveys the processes leading to loss of protein function and the types of molecular interaction that help stabilize proteins . It considers the effects of organic solvents on stability and the special features of thermophilic proteins . The deliberate manipulation of stability by protein engineering is discussed using the enzyme subtilisin as example . Both random and rational mutations of this protein have led to variants with greatly improved tolerances of high temperatures and organic solvents . One can also use chemical modification to modify protein stability and some of the main approaches are reviewed . The chemical and genetic strategies are complementary and have been combined to stabilize cytochrome c by metal-mediated cross-linking following site-specific mutagenesis . The article concludes by summarizing the beneficial effects of certain additives on protein stability.

Gene, 1995 Sep 22, 163(1), 109 - 13
Cloning of a gene from Thermus filiformis and characterization of the thermostable nuclease; Fomenkov A et al.; A gene coding for a thermostable nuclease was cloned from the thermophilic microorganism, Thermus filiformis (Tf), using an indicator strain containing a dinD::lacZ fusion . The gene, designated nuc17, has been mapped within a 2300-bp fragment . The 55-kDa Tf nuclease was purified to over 95% homogeneity . Single-stranded (ss) DNA is the preferred substrate for the Tf nuclease, although double-stranded (ds) DNA can also be digested . Nuclease activity increases with increasing temperature up to 80 degrees C and requires the metal ions Ca++ or Mg++ for catalysis . Tf nuclease is primarily an endonuclease that leaves 5' phosphates in the digested products . The ssDNA extensions remaining after exonuclease III digestion of dsDNA can be removed by the Tf nuclease, making it a useful reagent to generate unidirectional deletions.

Gene, 1995 Sep 22, 163(1), 103 - 7
A novel insertion sequence (IS)-like element of the thermophilic bacterium PS3 promotes expression of the alanine carrier protein-encoding gene; Murai N et al.; A novel insertion sequence (IS)-like element was found in the 5'-upstream region of the alanine carrier protein-encoding gene (acp) in the thermophilic bacterium PS3 chromosomal DNA . The sequence contained an open reading frame (ORF) encoding a polypeptide of 369 amino acids which revealed high similarity with ORFs from IS891 from the cyanobacterium Anabaena and IS1136 from Saccharopolyspora erythraea . The direction of transcription was the same as that of acp, and typical inverted and direct repeats characteristic of IS were found in both the 5' and 3' region of the ORF . Southern hybridization analysis of the chromosomal DNA revealed that multiple copies of the ORF sequence were contained in the PS3 genome . This element might well be a member of a new IS family including IS891 and IS1136, and we have designated this element IS1341 . The analysis of acp expression in Escherichia coli cells indicated that IS1341 promotes the expression of acp.

J Biol Chem, 1995 Sep 15, 270(37), 22037 - 43
Affinity purification, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation; Ryan MT et al.; Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host . In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation . By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro . In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver . The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.

J Biol Chem, 1995 Sep 15, 270(37), 21571 - 8
ATP synthesis by the F0F1-ATPase from the thermophilic Bacillus PS3 co-reconstituted with bacteriorhodopsin into liposomes . Evidence for stimulation of ATP synthesis by ATP bound to a noncatalytic binding site; Richard P et al.; F-type ATPase from the thermophilic Bacillus PS3, TF0F1, which was essentially free of bound nucleotides after isolation and purification, was co-reconstituted into liposomes with the light-driven proton pump bacteriorhodopsin . The time course of the light-induced ATP synthesis was biphasic; an initial slow phase accelerated to a final steady-state rate two to three times faster . Adding ATP before initiating the reaction suppressed the slow phase, suggesting that the state of occupancy of specific sites by ATP regulated the synthetic activity of TF0F1 . Incubating the purified TF0F1 with ADP and ATP revealed one ADP and two ATP binding sites that were stable to gel filtration . We analyzed the time courses of light-induced ATP synthesis for the enzyme with different nucleotide content, after co-reconstitution into liposomes with bacteriorhodopsin . The two ATP sites were identified to have regulatory function . A complex containing TF0F1.ADP, 1:1, was co-reconstituted with various quantities of ATP to obtain a range of molar ratios of TF0F1.ADP:ATP of between 1:0 and 1:1.7 . It was found that the initial rate of ATP synthesis increased with the level of ATP bound to the enzyme . After binding one ATP, a stimulation of ATP synthesis by a factor of 2 was observed . The second ATP site also exhibited regulatory properties . It stimulated ATP synthesis but to a much smaller extent; the stimulation did not exceed 20% . Binding of the photoreactive analogues 2-azido-{alpha-32P}ADP and 2-azido-{alpha-32P}ATP to the TF0F1 and their effects on the rate of ATP synthesis are described further.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 730 - 6
Gene of heat shock protein of sulfur-dependent archaeal hyperthermophile Desulfurococcus; Kagawa Y et al.; To elucidate thermoresistance, a gene of a hyperthermophilic heat shock protein (HHSP) was isolated from the hyperthermophile Desulfurococcus strain SY which grows at 95 degrees C . The molecular weight of HHSP deduced from the open reading frame was 59,137 (545 amino acid residues) . Sequence alignments of peptides reveal similarities (evolutionary distances) to the alpha (0.279) and beta (0.296) subunits of thermosome, TF55 (0.343) and human t-complex polypeptide 1 . The structure of a thermophilic heat shock protein TGroEL (Tamada et al . (1991) Biochem, Biophys . Res . Commun . 179, 565) was quite different from that of HHSP . TGroEL and HSP60 have sequences identical to HHSP at its equatorial domain, while those identical to the alpha subunit of F-type ATPase are at its apical domain.

Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 646 - 52
SaRD, a new protein isolated from the extremophile archaeon Sulfolobus acidocaldarius, is a thermostable ribonuclease with DNA-binding properties; Kulms D et al.; We have isolated the thermostable 9 kDa SaRD-protein from Sulfolobus acidocaldarius which exhibit RNase activity as well as DNA-binding properties (SaRD) . The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea . The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram . The temperature optimum for the RNase activity is 65 degrees C . The pH optimum ranges from 6.5-7.0 . DNA-binding properties were shown by gel-shift assays on agarose gels . In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated . The melting point (Tm) of genomic DNA was raised from 68 degrees C to 90 degrees C by addition of the SaRD-protein . CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85% C nor by addition of 8 M urea.

Biochim Biophys Acta, 1995 Sep 12, 1231(2), 139 - 46
Expression of the wild-type and the Cys-/Trp-less alpha 3 beta 3 gamma complex of thermophilic F1-ATPase in Escherichia coli; Matsui T et al.; The alpha, beta and gamma subunits of F1-ATPase from thermophilic Bacillus PS3 were expressed in Escherichia coli cells simultaneously in large amounts . Most of the expressed subunits assembled into a form of alpha 3 beta 3 gamma complex in E . coli cells and this complex was easily purified to homogeneity . The recombinant alpha 3 beta 3 gamma complex thus obtained showed similar enzymatic properties to the alpha 3 beta 3 gamma complex obtained by in vitro reconstitution from individual subunits (Yokoyama, K . et al . (1989) J . Biol . Chem . 264, 21837-21841) except that the former had several-fold higher ATPase activity than the latter . Using this expression system, a mutant alpha 3 beta 3 gamma complex with no Trp and Cys was generated by replacing alpha Cys193 and alpha Trp463 with Ser and Phe, respectively . This mutant complex was functionally intact, indicating both residues are not essential for catalysis . The Cys-/Trp-less complex is a convenient 'second wild type' enzyme from which one can generate mutants with Trp (as a fluorescent probe) or Cys (as an acceptor of a variety of probes) at desired positions without concern for 'background' Trp and Cys residues.

Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8715 - 8
High frequency of sex and equal frequencies of mating types in natural populations of the ciliate Tetrahymena thermophila; Doerder FP et al.; In ciliate protists, sex involves the temporary joining of two cells of compatible mating type, followed by meiosis and exchange of gametic nuclei between conjugants . Reproduction is by asexual binary fission following conjugation . For the many ciliates with fixed multiple mating types, frequency-dependent sex-ratio theory predicts equal frequencies of mating types, if sex is common in nature . Here, we report that in natural populations of Tetrahymena thermophila sexually immature cells, indicative of recent conjugation, are found from spring through fall . In addition, the seven mating types occur in approximately equal frequencies, and these frequencies appear to be maintained by interaction between complex, multiple mat alleles and environmental conditions during conjugation . Such genotype-environment interaction determining mating type frequency is rare among ciliates.

Biochim Biophys Acta, 1995 Sep 6, 1251(2), 170 - 6
Molecular properties of glutamate dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Facchiano AM et al.; This study is concerned with the structural characterization in solution of the glutamate dehydrogenase from the Archaeon Sulfolobus solfataricus . At neutral pH both alpha-helix and beta-sheet constitute the secondary structure of this enzyme, on the basis of circular dichroism . A complex, temperature dependent self-association equilibrium regulates the formation of the enzyme quaternary structure, which seems to be accompanied by a reversible structural change . At 25 degrees C the enzyme is mostly represented by monomeric subunits at concentrations lower than 0.02 mg/ml, while oligomers are predominant at concentrations higher than 0.12 mg/ml . The mid-point of the association curve shifts from 0.05 mg/ml at 25 degrees C to about 0.1 mg/ml at 45 degrees C . Only the oligomeric form appears to be temperature resistant . Monomeric and oligomeric enzyme show distinct behaviour on guanidine hydrochloride perturbation at neutral pH . The monomer denaturation, although complex, is reversible . Two fluorescent tryptophan classes are detectable in the monomer, monitoring the independent unfolding of two regions through a multistate transition . Instead, the oligomeric protein shows a complex denaturation pattern with the tendency to aggregate irreversibly at high denaturant concentration.

Protein Eng, 1995 Sep, 8(9), 905 - 13
Composition analysis of alpha-helices in thermophilic organisms; Warren GL et al.; We present a statistical comparison of the amino acid composition in a secondary structure element, the alpha-helix, of proteins stable at high temperatures with those which are less so . This study has shown that the temperature-dependent Zimm-Bragg helix propagation value s is not a good predictor for the helix-forming tendency of an amino acid in thermostable proteins . However, we have shown that delta s, the change in s from 20 to 60 degrees C, accurately predicts the direction of the probability shift for 15 amino acids in thermostable protein alpha-helices, although it does not predict the magnitude of that change . The residues tyrosine, glycine and glutamine show a significant increase in residency in alpha-helices for thermostable proteins over their non-thermostable counterparts . Significant decreases in alpha-helix residency occur for the residues valine, glutamic acid, histidine, cysteine and aspartic acid in proteins from thermophilic organisms . Aromatic interactions, hydrogen bonding and a reduction of charge may explain the increase observed for tyrosine and glutamine and the decrease in glutamic acid and aspartic acid, although packing considerations cannot be ruled out . The only physical explanation for the increase in glycine would seem to be its positive delta s value.

Rev Sci Tech, 1995 Sep, 14(3), 811 - 8
Reverse transcription combined with polymerase chain reaction as a detection method for pestiviral infections; Stadejek T et al.; An assay based on reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection of hog cholera virus (HCV) and bovine virus diarrhoea virus (BVDV) in cell culture . In this study, a precipitate of the supernatants derived from cell cultures infected with HCV and BVDV was used in RT reactions, in place of extracted viral RNA . Both RT and PCR were performed using recombinant Thermus thermophilus (rTth) DNA polymerase . The specificity of the RT-PCR products was confirmed by hybridisation with a digoxygenin-labelled DNA probe . The results not only show that the stage of RNA isolation can be bypassed, but also illustrate an easy and efficient means of obtaining templates suitable for identification and characterisation of HCV and BVDV in tissue culture by RT-PCR.

Hua Xi Yi Ke Da Xue Xue Bao, 1995 Sep, 26(3), 319 - 21
{Isolation and identification of extrem thermophilic bacteria from hot springs of Sichuan and Tibet}; Jia W et al.; Eleven strains of extrem thermophilic bacteria belonging to the Bacillus Stearothermophiles were isolated from the hot springs of Sichuan and Tibet . The cells were gram-positive, sporulating and motile . The optimum temperature for growth was between 65 degrees C to 70 degrees C; the maximum 94 degrees C, and minimum 40 degrees C . The colour of colony was yellow to bright orange . The G+C content of the DNA in strains has been found to be in the range 48.4-53.15 mol%.

Biokhimiia, 1995 Sep, 60(9), 1435 - 49
{Site-specific endonuclease and methylase from thermophilic bacteria of Bacillus species IS4}; Zelinskaia NV et al.; The site-specific endonuclease R . BspIS4I and methylase M . BspIS4I have been isolated and purified to functional purity from the thermophilic strain of Bacillus species IS4 . R . BspIS4I recognizes sequence {sequence: see text} on the DNA and cleaves it as indicated by the arrows to form single-stranded 4-nucleotide 5'-protruding termini . The enzyme is an isoschizomer of BbvII . M . BspIS4I is related to adenine-specific methylase.

J Bacteriol, 1995 Sep, 177(17), 4947 - 62
Structure of peptidoglycan from Thermus thermophilus HB8; Quintela JC et al.; The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented . The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported . The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide . In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid . This is the first time phenylacetic acid is described as a component of bacterial murein . Possible implications for murein physiology and biosynthesis are discussed . Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges . Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues . Neither reducing sugars nor murein-bound macromolecules were detected . Murein from T . thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms . The murein composition and peptide cross-bridges of T . thermophilus are typical for a gram-positive bacterium . However, the murein content, degree of cross-linkage, and glycan chain length for T . thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.

J Biol Chem, 1995 Sep 1, 270(35), 20345 - 58
Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8; Keightley JA et al.; Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3 . We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2 . Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J . A., Rusnak, F., and Munck, E . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 5779-5783) . By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly . These results suggested the presence of two polypeptides in this protein . Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB) . The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes . The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences . The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A . These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases . The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus . Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases . The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.

Mol Cell Biol, 1995 Sep, 15(9), 5173 - 9
Gene-specific signal transduction between microtubules and tubulin genes in Tetrahymena thermophila; Gu L et al.; Mammalian cells regulate tubulin mRNA abundance by a posttranscriptional mechanism dependent on the concentration of tubulin monomer . Treatment of mammalian cells with microtubule-depolymerizing drugs and microtubule-polymerizing drugs causes decreases and increases in tubulin mRNA, respectively (D . W . Cleveland, Curr . Opin . Cell Biol . 1:10-14, 1989) . In striking contrast to the case with mammalian cells, perturbation of microtubules in Tetrahymena thermophila by microtubule-depolymerizing or -polymerizing drugs increases the level of the single alpha-tubulin gene message by increasing transcription (L . A . Stargell, D . P . Heruth, J . Gaertig, and M . A . Gorovsky, Mol . Cell . Biol . 12:1443-1450, 1992) . In this report we show that antimicrotubule drugs preferentially induce the expression of one of two beta-tubulin genes (BTU1) in T . thermophila . In contrast, deciliation induces expression of both beta-tubulin genes . Tubulin gene expression was examined in a mutant strain created by transformation with an in vitro-mutagenized beta-tubulin gene that conferred resistance to microtubule-depolymerizing drugs and sensitivity to the polymerizing drug taxol and in a strain containing a nitrosoguanidine-induced mutation in the single alpha-tubulin gene that conferred the same pattern of drug sensitivities . In both cases the levels of tubulin mRNA expression from the drug-inducible BTU1 gene in the mutant cells paralleled the altered growth sensitivities to microtubule drugs . These studies demonstrate that T . thermophila has distinct, gene-specific mechanisms for modulating tubulin gene expression depending on whether ciliary or cytoplasmic microtubules are involved . They also show that the cytoplasmic microtubule cytoskeleton itself participates in a signal transduction pathway that regulates specific tubulin gene transcription in T . thermophila.

J Appl Bacteriol, 1995 Sep, 79(3), 302 - 7
Amino acid requirements and peptidase activities of Streptococcus salivarius subsp . thermophilus; Neviani E et al.; The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp . thermophilus strains . A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids . The strains showed different amino acid requirements . Some amino acids proved to be essential, some were required, while others did not affect growth . In the synthetic medium, only leucine and glutamic acid were essential for growth . In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth . Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed . Strains with higher amino acid requirements were also found to express a wider range of peptidases.

J Eukaryot Microbiol, 1995 Sep-Oct, 42(5), 510 - 5
A simple, efficient technique for freezing Tetrahymena thermophila; Cassidy-Hanley D et al.; We have developed a simple, efficient procedure for the long term freezing of Tetrahymena thermophila in liquid nitrogen . This technique yields excellent recovery of viable cells with all strains tested and does not require the use of a controlled rate low temperature freezer . To optimize the freezing technique, we have examined the effects of varying a number of parameters, including the physiological state of the cells prior to freezing, the time of exposure to cryoprotectant, and the rate of freezing and thawing . The frequency of viable cell recovery following freezing using this technique has been tested for a variety of different cell lines.

Enzyme Microb Technol, 1995 Sep, 17(9), 816 - 25
Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity; Wood AN et al.; An esterase activity obtained from a strain of Bacillus stearothermophilus was purified 5,133-fold to electrophoretic homogeneity with 26% recovery . The purified esterase had a specific activity of 2,032 mumol min-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30 degrees C . The apparent molecular mass was 50,000 +/- 2,000 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 3,000 daltons from gel filtration . Native polyacrylamide gels stained for esterase activity showed three bands . The isoelectric points were estimated to be 5.7, 5.8, and 6.0 . Forty amino acid residues were sequenced at the N-terminus . The sequence showed no degeneracy, suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids . The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0 . At this pH and 60 degrees C the half-life was 170 h . Esterase activity was totally inhibited by phenylmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and tosyl-L-phenylalanine, but not by ethylendiaminetetra acetic acid . The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both Vmax and KM were protein concentration-dependent . The esterase was able to hydrolyse a number of p-nitrophenyl derivatives (amino acid derivatives and aliphatic acids with different chain lengths).

EMBO J, 1995 Sep 1, 14(17), 4156 - 67
Crystal structure of glycyl-tRNA synthetase from Thermus thermophilus; Logan DT et al.; The sequence and crystal structure at 2.75 A resolution of the homodimeric glycyl-tRNA synthetase from Thermus thermophilus, the first representative of the last unknown class II synthetase subgroup, have been determined . The three class II synthetase sequence motifs are present but the structure was essential for identification of motif 1, which does not possess the proline previously believed to be an essential class II invariant . Nevertheless, crucial contacts with the active site of the other monomer involving motif 1 are conserved and a more comprehensive description of class II now becomes possible . Each monomer consists of an active site strongly resembling that of the aspartyl and seryl enzymes, a C-terminal anticodon recognition domain of 100 residues and a third domain unusually inserted between motifs 1 and 2 almost certainly interacting with the acceptor arm of tRNA(Gly) . The C-terminal domain has a novel five-stranded parallel-antiparallel beta-sheet structure with three surrounding helices . The active site residues most probably responsible for substrate recognition, in particular in the Gly binding pocket, can be identified by inference from aspartyl-tRNA synthetase due to the conserved nature of the class II active site.

EMBO J, 1995 Sep 1, 14(17), 4143 - 55
Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate; Arnez JG et al.; The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined . The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS) . The asymmetric unit is composed of two homodimers . Each monomer consists of two domains . The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A . The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization . The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His) . The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules.

Arch Microbiol, 1995 Sep, 164(3), 159 - 64
Thermococcus peptonophilus sp . nov., a fast-growing, extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents; Gonzalez JM et al.; Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean . These strains were cocci, obligately anaerobic Archaea about 0.7-2 microm in diameter . Optimum growth conditions for OG-1 and SM-2 were at 85-90 degrees C (range 60-100 degrees C), pH 6 (range pH 4-8), a NaCl concentration of 3% (range 1-5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005-5%) . Elemental sulfur stimulated the growth rate fourfold . Ammonium slightly stimulated growth . Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids . OG-1 showed the fastest growth rate within the genus Thermococcus . Growth was inhibited by rifampicin . The DNA G+C content was 52 mol% . Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus . OG-1 and SM-2 were different than the described Thermococcus species . We propose that OG-1 belongs to a new species: Thermococcus peptonophilus.

Microbiology, 1995 Sep, 141 ( Pt 9), 2033 - 40
alpha-D-glucuronidases from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum; Bronnenmeier K et al.; alpha-D-Glucuronidases were purified from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum . This enzyme activity was found to be intracellular in each organism, with T . saccharolyticum producing much greater total activity . The specific activities of the purified enzymes (10 U mg-1 T . saccharolyticum; 1.7 U mg-1 C . stercorarium) differed by a factor of approximately 5 . For the determination of enzyme activities, 4-O-methyl-alpha-D-glucuronosyl-xylotriose was used as a substrate and the glucuronic acid released by alpha-D-glucuronidase action was quantified by a colorimetric procedure . 4-O-Methyl-alpha-D-glucuronosyl-xylotriose was the hydrolysis product that accumulated after exhaustive degradation of 4-O-methyl-alpha-D-glucuronoxylan with xylanases of C . stercorarium . Hydrolysis of side chains in high-molecular-mass glucuronoxylan could not be detected . Neither of the enzymes was able to hydrolyse the chromogenic aryl-substrate p-nitrophenyl-alpha-D-glucuronoside . Both alpha-D-glucuronidases have a dimeric structure, with monomeric molecular masses of 72 and 76 kDa for C . stercorarium and of 71 kDa for T . saccharolyticum . The pI was estimated to be 4.3 for each enzyme . While both enzymes exhibited a similar pH optimum (pH 5.5-6.5) they differed in their thermostabilities . At 60 degrees C, half-lives of 14 and 2.5 h, respectively, were determined for the alpha-D-glucuronidases of C . stercorarium and T . saccharolyticum . This description of alpha-D-glucuronidase activity in thermophilic anaerobic bacteria extends our knowledge of these enzymes, previously purified and characterized only in fungi.

Gene, 1995 Aug 8, 161(1), 1 - 6
Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases; Thorbjarnardottir SH et al.; In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm) . We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig . The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da . Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues . Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures . Under our assay conditions the Rm Lig was active at 5-75 degrees C with apparent optimal activity above 55 degrees C . The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C . The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.

Biochemistry, 1995 Aug 8, 34(31), 10063 - 77
Gene cloning, expression, and characterization of the Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius; McAfee JG et al.; The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced . The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively . Southern blots indicate that these are the only two Sac7 protein genes in S . acidocaldarius, each present as a single copy . Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species . The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements . Potential ribosome binding sites have been identified downstream of the initiator codons . The sac7d gene has been expressed in E . coli, and various physical properties of the recombinant protein have been compared with those of native Sac7 . The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds . The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly{dGdC}.poly{dGdC} in 0.01 M KH2PO4 at pH 7.0 . The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein . Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C.

FEBS Lett, 1995 Aug 7, 369(2-3), 229 - 32
Ribosomal protein L22 from Thermus thermophilus: sequencing, overexpression and crystallisation; Davydova NL et al.; The gene for the ribosomal protein L22 from Thermus thermophilus has been sequenced and overexpressed in Escherichia coli . A multiple sequence alignment was carried out for all proteins of the L22 family reported so far . The recombinant protein was purified and crystallized . The crystals belong to the space group P2(1)2(1)2(1), with cell parameters of a = 32.6 A, b = 66.0 A, c = 67.8 A.

FEBS Lett, 1995 Aug 7, 369(2-3), 158 - 60
In vivo assembly of plasmid-expressed ribosomal protein S7 of Thermus thermophilus into Escherichia coli ribosomes and conditions of its overexpression; Karginov AV et al.; Researchers still have great difficulty in isolating individual ribosomal proteins from the ribosome in quantities high enough for structural research . To this end, when studying protein S7, we created an E . coli overproducer of the recombinant protein S7 of Thermus thermophilus . The vector for expression was pQE-32 having a strong promoter of E . coli phage T5 and six triplets of His at the 5'-end . This N-terminal six His tag of the fusion protein is responsible for binding to Ni-NTA-resin and allows purifying the protein in one step . The yield of the recombinant protein was 20% and more of the total cellular proteins . In addition we have shown that the recombinant thermophilic protein is incorporated in vivo into the ribosome of E . coli despite the fact that these proteins (thermophilic and mesophilic) have a rather low homology, only 52% . This fact provides a base for the system to study functions of individual proteins.

J Dairy Sci, 1995 Aug, 78(8), 1657 - 64
Effect of milks inoculated with Lactobacillus acidophilus or a yogurt starter culture in lactose-maldigesting children; Montes RG et al.; Dairy products containing live bacteria that possess lactase activity are used for dietary management of lactose maldigestion . The efficacy of acidophilus milk and the effect of consuming unfermented milk that had been inoculated with yogurt bacteria have not been examined in children . We compared scores for breath H2 excretion and symptoms of 20 lactose-maldigesting children following ingestion of 250 ml of uninoculated milk with two identical milks inoculated with 10(10) cells of Lactobacillus acidophilus or with a commercial yogurt starter culture containing 10(8) cells of Lactobacillus lactis and 10(10) cells of Streptococcus thermophilus . Nine of 10 subjects who were symptomatic following ingestion of uninoculated milk experienced a reduction in symptoms following ingestion of milk inoculated with L . acidophilus, without a decline in H2 excretion . Five of 6 subjects who were symptomatic following uninoculated milk had decreased symptoms and a significant reduction in H2 excretion following milk inoculated with the yogurt culture . For lactose-maldigesting children, milks inoculated with L . acidophilus or with a yogurt culture were associated with decreased symptoms compared with those with uninoculated milk.

Protein Eng, 1995 Aug, 8(8), 763 - 7
The crystal structure of thermostable mutants of chimeric 3-isopropylmalate dehydrogenase, 2T2M6T; Sakurai M et al.; A chimeric 3-isopropylmalate dehydrogenase (IPMDH), 2T2M6T, was produced by replacing the amino acid sequences of the Thermus thermophilus enzyme with those of the Bacillus subtilis enzyme from residues 75 to 113 . Decreased thermostability of the chimeric enzyme was recovered by either evolutionary engineering (I93L) or site-directed mutagenesis (S82R) . The 3-D structures of the mutants have been determined by X-ray diffraction at 2.1 A resolution . Although S82R was refined routinely, I93L required the preliminary rigid-body refinement of each domain . The R-factors were reduced to 0.18 for both mutants . Removal of the unfavorable torsion angle at isoleucine 93 may have made I93L more thermostable than 2T2M6T . In the case of S82R, the replaced arginine residue contributed to the extra hydrogen bond with water molecules . The large replaced residue decreased the entropy of the solvent, which may have caused the improvement in enzyme thermostability . Denaturation by heating may be interpreted from these structural results.

Arch Microbiol, 1995 Aug, 164(2), 91 - 7
Thermotoga subterranea sp . nov., a new thermophilic bacterium isolated from a continental oil reservoir; Jeanthon C et al.; A thermophilic, strictly anaerobic bacterium, designated strain SL1, was isolated from a deep, continental oil reservoir in the East Paris Basin (France) . This organism grew between 50 and 75 degrees C, with an optimum at 70 degrees C . It was inhibited by elemental sulfur and was able to reduce cystine and thiosulfate to hydrogen sulfide . The G+C content (40 mol%), the presence of a lipid structure unique to the genus Thermotoga, and the 16S rRNA sequence of strain SL1 indicated that the isolate belongs to the genus Thermotoga . Based on DNA-DNA hybridization, isolate SL1 does not show species-level similarity with the recognized species T . maritima, T . neapolitana, and T . thermarum . Based on this description of strain SL1, we propose the recognition of a new species: Thermotoga subterranea.

J Biochem (Tokyo), 1995 Aug, 118(2), 347 - 54
Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8; Amada K et al.; The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced . The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins . The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells . The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E . coli . T.th cpn60 was expressed as a tetradecameric form, like GroEL of E . coli . Since chaperonin from T . thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before . T.th cpn60 tetradecamer tended to dissociate into monomers during storage . T.th cpn10 expressed in E . coli was purified as a stable oligomer, most likely a heptamer . The activity as holo-chaperonin was reconstituted by mixing both subunits . T.th cpn60 tetradecamer itself arrested refolding of other proteins . The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography . Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin.

J Biochem (Tokyo), 1995 Aug, 118(2), 319 - 24
Cloning and sequence analysis of the gene for phosphoenolpyruvate carboxylase from an extreme thermophile, Thermus sp; Nakamura T et al.; The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extreme thermophile, Thermus sp., was cloned and sequenced . The ppc gene had a high G+C content (69.2%) . An open reading frame for a 857-amino-acid polypeptide was found in the gene . The calculated molecular mass was 95,632 . The amino acid sequence of Thermus PEPC was 31-37% identical and 52-57% similar to those of 17 PEPCs from mesophilic organisms . No Cys residue was found in the polypeptide, demonstrating that this residue is not essential for the catalytic activity of PEPC . The cloned gene was expressed in Escherichia coli and thermostable PEPC was obtained.

Protein Sci, 1995 Aug, 4(8), 1516 - 27
The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions; Spassov VZ et al.; Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure . The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank . The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed . It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area . The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure . An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms . The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.

Eur J Biochem, 1995 Aug 1, 231(3), 773 - 8
Coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductase (Mer) from Methanobacterium thermoautotrophicum strain Marburg . Cloning, sequencing, transcriptional analysis, and functional expression in Escherichia coli of the mer gene; Vaupel M et al.; The gene encoding the F420-dependent N5,N10-methylenetetrahydromethanopterin reductase (Mer), which catalyzes an intermediate step in methanogensis, was cloned and sequenced from the thermophilic Methanobacterium thermoautotrophicum strain Marburg . The gene was identified on a 3.8-kbp BamHI fragment of M . thermoautotrophicum genomic DNA using a homologous probe . The mer gene encoded an acidic protein of 321 amino acids, corresponding to a calculated molecular mass of 33,492 Da . Sequence analysis revealed the presence of a ribosome binding site, a putative promoter, and a possible terminator structure . The size of the mer mRNA was estimated as 1 kb indicating monocistronic transcription . The mer gene was expressed in Escherichia coli yielding an active enzyme of 36 kDa consistent with the apparent molecular mass described for the enzyme from M . thermoautotrophicum . Sequence comparisons revealed similarities between the F420-dependent N5,N10-methylenetetrahydromethanopterin reductase and a F420-dependent reductase involved in lincomycin biosynthesis in Streptomyces lincolnensis.

J Bacteriol, 1995 Aug, 177(15), 4540 - 3
Molecular cloning of the pyrE gene from the extreme thermophile Thermus flavus; Vonstein V et al.; Mutants of the extreme thermophile Thermus flavus in the pyrimidine biosynthetic pathway (Pyr-) were isolated by resistance to 5-fluoroorotic acid . The pyrE gene, which codes for the orotate phosphoribosyltransferase, was cloned by recombination with one of the isolated Pyr- T . flavus mutant strains . It was subcloned by complementation of an Escherichia coli pyrE mutant strain and was sequenced . The deduced polypeptide sequence extends over 183 amino acids . Several independent Pyr- mutations were mapped within the pyrE locus by recombination with fragments of the cloned gene.

J Bacteriol, 1995 Aug, 177(15), 4417 - 26
A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating; Schleper C et al.; A plasmid of 45 kb, designated pNOB8, was found in high copy number in a new heterotrophic Sulfolobus isolate, NOB8H2, from Japan . Dissemination of the plasmid occurred in six cultures of nine different Sulfolobus strains when small amounts of the donor were added . These mixed cultures exhibited a high average copy number of the plasmid, between 20 and 40 per chromosome, and showed a marked growth retardation . Horizontal transfer of pNOB8 was proved by isolating transcipients from mating mixtures via single colonies . In these isolates, the copy number of the plasmid appeared to be subject to a control mechanism . Cell-free filtrates of donor cultures did not transmit the plasmid, and plating of the donor on lawns of recipients did not result in plaque formation, suggesting that the transfer was not mediated by a virus . Rapid formation of cell-to-cell contacts between differently stained donor and recipient partners was demonstrated after the two strains were mixed . Electron microscopic analysis of mating mixtures revealed many cell aggregates made up of 2 to 30 cells and intercellular cytoplasmic bridges connecting two or more cells . Cells that had been transformed with purified plasmid DNA as well as transcipients isolated from mating mixtures were shown to serve as donors for further transmission of pNOB8 . The plasmid undergoes extensive genetic variations, since deletions and insertions were frequently observed in plasmid preparations from the donor strain and from mating mixtures.

Curr Opin Biotechnol, 1995 Aug, 6(4), 370 - 4
Engineering thermostability: lessons from thermophilic proteins; Russell RJ et al.; As several groups begin to tap the rich pickings found in the Archaea--a vast kingdom that stretches the concept of life as we know it--the structures of proteins from hyperthermophiles are being elucidated . Certain features are beginning to emerge, such as compactness and hydrophobic clustering, but the ability to engineer these features into temperature-intolerant proteins is still some way off.

Biokhimiia, 1995 Aug, 60(8), 1318 - 25
{Site-specific endonuclease BspKT8 from the thermophilic strain KT8 of Bacillus species}; Chernov AV et al.; A site-specific endonuclease capable of recognizing the sequence 5'-AAGCTT-3' was detected and purified to homogeneity from the thermophilic strain of Bacillus species KT8 . The endonuclease has a molecular mass of 34 kDa and is found in solution in a monomeric form . The activity of BspKT8 does not depend on ATP and is not stimulated by S-adenosyl-L-methionine . The enzyme displays the highest activity with a broad range of temperatures (37 degrees-48 degrees C) . Since DNA cleavage occurs in accordance with the scheme: {formula: see text} the enzyme can be assigned to the class-II of restriction endonucleases and represents an isoschizomer of HindIII.

Biosci Biotechnol Biochem, 1995 Aug, 59(8), 1438 - 43
Expression of aqualysin I (a thermophilic protease) in soluble form in Escherichia coli under a bacteriophage T7 promoter; Sakamoto S et al.; The thermophilic protease aqualysin I (AQI) gene (aquI), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid . The plasmid was introduced into two strains of E . coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacIq gene . Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein . This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity . On the other hand, when the strain lacking an F' episome was used as a host cell for aquI expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form . This soluble protein could be processed into active AQI by heat treatment . Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.

Appl Microbiol Biotechnol, 1995 Aug-Sep, 43(4), 667 - 74
Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose; Healy FG et al.; Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years . Clones expressing cellulase and xylosidase were readily recovered from these libraries . Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranoside were characterized . All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment . The DNA sequence of the insert in one clone (plasmid pFGH1) was determined . This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues . CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity) . The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp . cellulosa (28% homology) . The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications.

RNA, 1995 Aug, 1(6), 598 - 609
Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA; Campbell TB et al.; Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library . Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product . Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU) . The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library . Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes . Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement . The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate . Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library.

Proteins, 1995 Aug, 22(4), 426 - 8
Crystallization of Thermus thermophilus histidyl-tRNA synthetase and its complex with tRNAHis; Yaremchuk AD et al.; Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus . The protein has been crystallized separately with histidine and with its cognate tRNAHis . Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant . The crystals of HisRS with histidine belong to the spacegroup P2(1)2(1)2 with cell parameters a = 171.3 A, b = 214.7 A, c = 49.3 A, alpha = beta = gamma = 90 degrees . A complete data set to a resolution of 2.7A with an Rmerge on intensities of 4.1% has been collected on a single frozen crystal . A partial data set collected on a crystal of HisRS in complex with tRNAHis shows that the crystals are tetragonal with cell parameters a = b = 232 A, c = 559 A, alpha = beta = gamma = 90 degrees and diffract to about 4.5 A resolution.

Mol Gen Genet, 1995 Jul 28, 248(2), 236 - 41
Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system in Salmonella typhimurium LT2; van der Vlag J et al.; In Enterobacteriaceae the nonphosphorylated form of IIAGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the MalK protein of the maltose transport system, respectively . In this report we show that the IIAGlc-like domain of the membrane-bound IIN-acetylglucosamine (IINag) of the PTS can replace IIAGlc in a Salmonella typhimurium crr mutant strain that lacks all soluble IIAGlc . The inhibition was most severe in cells which were partially induced for the glycerol or maltose uptake systems . The Streptococcus thermophilus lactose transporter LacS, which also contains a IIAGlc-like domain, could not replace IIAGlc . Neither IINag nor LacS could replace IIAGlc in activation of adenylate cyclase.

Nature, 1995 Jul 27, 376(6538), 344 - 8
Neural regulation of thermotaxis in Caenorhabditis elegans; Mori I et al.; Thermal stimulus is an important environmental factor influencing animal behaviour . However, the mechanisms underlying thermosensation and thermal adaptation are poorly understood . The nematode Caenorhabditis elegans can sense a range of environmental temperatures and migrate towards the cultivation temperature on a thermal gradient . This modifiable thermotactic response provides an ideal system for studying the cellular and molecular processes involved in thermosensation and thermal information storage . We have identified neurons critical for thermotaxis by killing individual cells in live animals . The results indicate that an amphid sensory neuron, AFD, is a major thermosensory neuron . Some of the genetically defined cryophilic and thermophilic mutant phenotypes were mimicked when amphid interneurons AIY and AIZ, respectively, were killed, indicating that AIY is responsible for thermophilic movement and AIZ for cryophilic movement . We propose a neural model in which regulation of the activities of the two interneurons in opposite directions, depending on the cultivation temperature, is essential for thermotaxis.

FEBS Lett, 1995 Jul 17, 368(2), 207 - 10
A minimum catalytic unit of F1-ATPase shows non-cooperative ATPase activity inherent in a single catalytic site with a Km 70 microM; Saika K et al.; F1-ATPase has three interacting catalytic sites and shows complicated kinetics . Here, we report reconstitution of a complex, most likely composed of one alpha subunit and one beta subunit, with a single catalytic site from thermophilic Bacillus PS3 F1-ATPase on the solid surface . The complex has an ATPase activity which obeys a simple non-cooperative kinetics with a Km(ATP) of 70 microM and a Vmax of 0.1 unit/mg . Different from F1-ATPase, the complex is not inactivated by 7-chrolo-4-nitrobenzofrazan . Thus, the inherent activity attributable to a single catalytic site unaffected by other catalytic sites of F1-ATPase is characterized.

Biochem J, 1995 Jul 15, 309 ( Pt 2), 595 - 9
Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC); Welch SG et al.; Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases . Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA . Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined . Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA . Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3'-OH overhang . Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) {Lee, Clanton and Chirikjiam (1979) Fed . Proc . 28, 294}, generating fragments with a three base 3'-OH overhang . However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C . Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.

Cell, 1995 Jul 14, 82(1), 47 - 56
Linker histones are not essential and affect chromatin condensation in vivo; Shen X et al.; We have (separately) disrupted all of the expressed macronuclear copies of the HHO gene encoding macronuclear histone H1 and of the micronuclear linker histone (MLH) gene encoding the protein MicLH in Tetrahymena thermophila . These disruptions are shown to eliminate completely the expression of each protein . Strains without either linker histone grow at normal rates and reach near-normal cell densities, demonstrating that linker histones are not essential for cell survival . Histone H1 knockout (delta H1) cells have enlarged DAPI-stained macronuclei and normal-sized micronuclei, while MicLH knockout (delta MicLH) cells have enlarged micronuclei and normal-sized macronuclei . delta MicLH cells undergo mitosis normally . However, the micronuclear mitotic chromosome structure is less condensed . These studies provide evidence that linker histones are nonessential and are involved in chromatin packaging and condensation in vivo.

FEBS Lett, 1995 Jul 10, 368(1), 132 - 4
Three-dimensional crystals of cytochrome-c oxidase from Thermus thermophilus diffracting to 3.8 A resolution; Soulimane T et al.; The ba3-type cytochrome-c oxidase from Thermus thermophilus has been crystallized in its native form . Crystallization was achieved by the batch and the vapour diffusion sitting drop methods using polyethylene glycol monomethyl ether 2000 as precipitating agent in the presence of octyl-beta-D-thioglucoside as detergent . The crystals diffract to 3.8 A, belong to the space group P2 or P2(1) and have unit cell dimensions of a = 80.7 A; b = 116.0 A; c = 156.9 A and beta = 104.4 degrees . The asymmetric unit contains two ba3-type oxidase molecules.

FEBS Lett, 1995 Jul 10, 368(1), 117 - 21
Redox properties of the sulfhydrogenase from Pyrococcus furiosus; Arendsen AF et al.; The sulfhydrogenase from the extreme thermophile Pyrococcus furiosus has been re-investigated . The alpha beta gamma delta heterotetrameric enzyme of 153.3 kDa was found to contain 17 Fe, 17 S2-, and 0.74 Ni . The specific activity of the purified protein was 80 U/mg . Three EPR signals were found . A rhombic S = 1/2 signal (g = 2.07, 1.93, 1.89) was observed reminiscent in its shape and temperature dependence of spectra from {4Fe-4S}(2+; 1+) clusters . However, in reductive titrations the spectrum appeared at the unusually high potential Em,7.5 = -90 mV . Moreover, the signal disappeared again at Em7.5 = -328 mV . Also, two other signals appear upon reduction: a near-axial (g = 2.02, 1.95, 1.92) S = 1/2 spectrum (Em,7.5 = -303 mV) indicative for the presence of a {2Fe-2S}(2+; 1+) cluster, and a broad spectrum of unknown origin with effective g-values 2.25, 1.89 (Em,7.5 = -310 mV) . We hypothesize that the latter signal is caused by magnetic interaction of the rhombic signal and a third cluster.

Biochem Biophys Res Commun, 1995 Jul 6, 212(1), 77 - 83
Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus; Fee JA et al.; We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T . thermophilus . The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00 . The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure . Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent {Cu(II)/Cu(I)} complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

Biochim Biophys Acta, 1995 Jul 3, 1250(1), 60 - 8
Steady state kinetics of the glutamate dehydrogenase from an archaebacterial extreme thermophile, isolate AN1; Hudson RC et al.; A steady state kinetic study was carried out with the glutamate dehydrogenase from the thermophilic, archaebacterial isolate AN1 . Initial velocity studies of the oxidative deamination reaction showed the mechanism is sequential and indicated that the order of substrate addition is random, while inhibition studies with products and substrate analogues suggested a strong preference for NADP+ to bind first . Initial velocity studies of the reductive amination reaction showed that the mechanism is sequential and indicated that the order of substrate addition is random, while product inhibition studies and the effect of substrate saturation on the initial velocity suggested that the preferred order of substrate addition is NADPH, 2-ketoglutarate, ammonia.

Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6364 - 8
An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei; Brownell JE et al.; Macronuclei of the ciliated protozoan Tetrahymena thermophila possess a histone acetyltransferase activity closely associated with transcription-related histone acetylation . Nothing definitive is known concerning the polypeptide composition of this activity in Tetrahymena or any comparable activity from any cellular source . An acetyltransferase activity gel assay was developed which identifies a catalytically active subunit of this enzyme in Tetrahymena . This activity gel assay detects a single polypeptide of 55 kDa (p55) in crude macronuclear extracts, as well as in column-purified fractions, which incorporates {3H}acetate from {3H}acetyl-CoA into core histone substrates polymerized directly into SDS polyacrylamide gels . p55 copurifies precisely with acetyltransferase activity through all chromatographic steps examined, including reverse-phase HPLC . Gel-filtration chromatography of this activity indicates a molecular mass of 220 kDa, suggesting that the native enzyme may consist of four identical subunits of 55 kDa . Furthermore, p55 is tightly associated with di- and greater polynucleosomes and therefore may be defined as a component of histone acetyltransferase type A--i.e., chromatin associated.

Int J Syst Bacteriol, 1995 Jul, 45(3), 495 - 9
DNA relatedness of Thermus strains, description of Thermus brockianus sp . nov., and proposal to reestablish Thermus thermophilus (Oshima and Imahori)
Williams RA, Smith KE, Welch SG, Micallef J, Sharp RJ.
Aerobic, thermophilic, gram-negative bacteria obtained from Yellowstone National Park that were placed in the genus Thermus on the basis of phenotypic data were examined by chemotaxonomic techniques to determine their peptidoglycan compositions, their respiratory quinones, their mean DNA base compositions, and their levels of DNA-DNA homology as determined by both the filter hybridization and reassociation rate methods . These isolates from hot springs included Thermus aquaticus strains and strains of a new genospecies . We propose the name Thermus brockianus for this new genospecies; strain YS38 is the type strain of this taxon . A collection of 10 strains, including the type strain of "Thermus thermophilus", which were isolated from widely separated geothermal sites, exhibited high levels of DNA-DNA homology with each other and had similar physiological properties . Therefore, we propose that the species Thermus thermophilus (Oshima and Imahori) should be reestablished, with strain HB8 as the type strain.

Int J Syst Bacteriol, 1995 Jul, 45(3), 454 - 61
Isolation and characterization of a novel alkalitolerant thermophile, Anaerobranca horikoshii gen . nov., sp . nov; Engle M et al.; Nine moderately alkalitolerant thermophilic bacteria with similar properties were isolated from water and soil samples obtained from Yellowstone National Park . These Gram-type-positive, rod-shaped bacteria produce cells with primary branches . The cells are peritrichous and exhibit only slight tumbling motility . At 60 degrees C the pH range for growth is 6.9 to 10.3, and the optimum pH is 8.5 . At pH 8.5 the temperature range for growth is 34 to 66 degrees C, with an optimum temperature of 57 degrees C . The strains are mainly proteolytic . The fermentation products from yeast extract are acetate, CO2, and H2 . Fumarate added to minimal medium containing yeast extract is stoichiometrically converted to succinate, indicating that it is used as an alternative electron acceptor . The DNA G + C content is 33 to 34 mol% . On the basis of its unique properties, such as branch formation, growth at alkaline pH values at elevated temperatures, and the relative distance of its 16S rRNA sequence from those of other known bacteria, we propose that strain JW/YL-138T (T = type strain) and eight similar strains represent a new genus and species, Anaerobranca horikoshii . Strain JW/YL-138 is designated the type strain of the type species, A . horikoshii, which was named in honor of Koki Horikoshi, a pioneer in the field of alkaliphilic bacteria.

Int J Syst Bacteriol, 1995 Jul, 45(3), 441 - 8
Bacillus infernus sp . nov., an Fe(III)- and Mn(IV)-reducing anaerobe from the deep terrestrial subsurface; Boone DR et al.; Bacillus infernus sp . nov . was isolated from ca . 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia . B . infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO2, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose . Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61 degrees C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8) . A phylogenetic analysis of its 16S rRNA indicated that B . infernus should be classified as a new species of the genus Bacillus . B . infernus is the only strictly anaerobic species in the genus Bacillus.

Anal Biochem, 1995 Jul 1, 228(2), 330 - 5
Purification of aminoacyl-tRNA by affinity chromatography on immobilized Thermus thermophilus EF-Tu.GTP; Ribeiro S et al.; Elongation factor Tu from Thermus thermophilus containing six histidine residues on its C-terminus, EF-Tu(CHis6), was used for purification of aminoacyl-tRNA isoacceptors, from aminoacylated bulk tRNA, by affinity chromatography . Preformed aminoacyl-tRNA.EF-Tu(CHis6).GTP ternary complexes were immobilized on Ni(2+)-nitriloacetic acid agarose and the aminoacyl-tRNA was eluted at high ionic strength or with buffers containing GDP . Compared to alternative methods, the reported immobilization by C-terminal histidine residues does not lead to loss of EF-Tu's affinity for aminoacyl-tRNA . The method is well suited for preparative isolation of aminoacylated tRNA isoacceptors and as an analytical tool to test tRNA composition and aminoacylation of tRNA in crude cellular extracts.

Genetics, 1995 Jul, 140(3), 989 - 1005
Effects of nullisomic chromosome deficiencies on conjugation events in Tetrahymena thermophila: insufficiency of the parental macronucleus to direct postzygotic development; Ward JG et al.; Conjugation fails postzygotically after mating of Tetrahymena cells that have wild-type parental macronuclei but harbor noncomplementing nullisomic parental germline deficiencies . Failures begin shortly after formation of the new macronuclear precursor (anlage) and completion of the first step in elimination of the parental macronucleus (pycnosis) . Conjugants fail to complete pair separation, to eliminate one new micronucleus, and to amplify anlage DNA, and they eventually die . Some deficiencies block resorption of the pycnotic parental macronucleus, but we find no evidence for its regeneration . Some deficiencies cause aberrant anlage DNA loss . Those that do not cause DNA loss are epistatic to those that do, indicating that normal anlage development requires the dependent function of at least two types of genes . The possibility that these genes are involved in developmentally regulated anlage DNA rearrangements is discussed . Each observed conjugation defect indicates insufficiency of the parental macronucleus to direct postzygotic development and can be explained by the deficiency of essential conjugation genes that are expressed from the anlage . The failure of nullisomic conjugants to complete pair separation indicates a requirement for gene products, expressed from the early anlage or its precursors, soon after anlage first differentiate.

Nat Struct Biol, 1995 Jul, 2(7), 537 - 47
Structure of phenylalanyl-tRNA synthetase from Thermus thermophilus; Mosyak L et al.; The crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus, solved at 2.9 A resolution, displays (alpha beta)2 subunit organization . Unexpectedly, both the catalytic alpha- and the non-catalytic beta-subunits comprise the characteristic fold of the class II active-site domains . The alpha beta heterodimer contains most of the building blocks so far identified in the class II synthetases . The presence of an RNA-binding domain, similar to that of the U1A spliceosomal protein, in the beta-subunit is indicative of structural relationships among different families of RNA-binding proteins . The structure suggests a plausible catalytic mechanism which explains why the primary site of tRNA aminoacylation is different from that of the other class II enzymes.

J Ind Microbiol, 1995 Jul, 15(1), 39 - 44
Expression of Streptomyces melC and choA genes by a cloned Streptococcus thermophilus promoter STP2201; Solaiman DK et al.; Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected in Escherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3 . Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone STP2201 as a strong promoter in E . coli . Subcloning of a STP2201-melC DNA fragment into the pMEU-series S . thermophilus-E . coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel+ phenotype to E . coli . The pEU5aML2201a was further shown to afford a high level of tyrosinase pro-anti-tyrosinase antiserum in S . thermophilus . Substituting melC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to an E . coli transformant at a level of (1.06 +/- 0.15) x 10(-7) units mg-1 protein . Introduction of this plasmid into S . thermophilus by electrotransformation yielded ChoA+ transformant that produced the enzyme at about 25% of the level found in E . coli.

Int J Biochem Cell Biol, 1995 Jul, 27(7), 729 - 39
Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp; Prescott M et al.; An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography . A pI of 3.8 was determined by isoelectric focussing . The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase) . Optimal proteolytic activity was observed at pH 3.0 (at 60 degrees C) . The Leu15-Tyr16 bond was the major site of hydrolysis for the oxidized B chain of insulin . Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases . The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity . Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme . Proteinase activity was inhibited by diazoacetyl-DL-norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases . Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases . The thermal stability at pH 3.0 and 60 degrees C increased 2.1-fold (t1/2, 4.5-9.7 hr) in the presence of 5 mM Ca++ . An increase in both pH (3.0-4.5) and Ca++ concentration (0-30 mM) resulted in a 15-fold increase (t1/2, 15-230 min) in thermal stability at 75 degrees C . The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

Plant Mol Biol, 1995 Jul, 28(4), 759 - 66
Cloning and sequencing of the nitrate transport system from the thermophilic, filamentous cyanobacterium Phormidium laminosum: comparative analysis with the homologous system from Synechococcus sp . PCC 7942; Merchan F et al.; A genomic region from the filamentous, thermophilic non-N2-fixing cyanobacterium Phormidium laminosum was cloned and sequenced . It includes the nitrite reductase gene (nirA) and three other genes (nrtA, B and C) located downstream of nirA, which are related to the nitrate transport system on the basis of a comparison with the homologous system from Synechococcus sp . PCC 7942 . No additional nitrate assimilation-related genes were identified in about 5 kb sequenced downstream of nrtC . All four genes are arranged as an operon with a promoter-like region upstream of the nirA gene . Transcripts of these nitrate assimilation genes accumulated after long periods of nitrogen starvation . This operon also contains inverted repeat sequences in the intercistronic regions which might be involved in mRNA processing or stability.

J Eukaryot Microbiol, 1995 Jul-Aug, 42(4), 422 - 7
Monoclonal antibodies reveal complex structure in the membrane skeleton of Tetrahymena; Williams NE et al.; Twelve monoclonal antibodies were raised that are specific for the membrane skeleton of Tetrahymena . Five were directed against T . pyriformis and seven were directed against T . thermophila . Some cross-reactivity between species was found . Each monoclonal antibody recognized one of the three major components of epiplasm, i.e . the bands A, B, and C identified in electrophoretic separations of epiplasmic proteins . It was found, using these antibodies, that the epiplasmic proteins A, B and C have overlapping but independent distributions within the cell.

Appl Environ Microbiol, 1995 Jul, 61(7), 2798 - 801
Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species; Farrelly V et al.; In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification . The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T . thermophilus." The values for the pair of Bacillus subtilis and "T . thermophilus" showed greater deviations from the predicted value . The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present.

Appl Environ Microbiol, 1995 Jul, 61(7), 2762 - 4
Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii; Konisky J et al.; The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens . Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D . J . Hei and D . S . Clark, Appl . Environ . Microbiol . 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.

Appl Environ Microbiol, 1995 Jul, 61(7), 2566 - 71
Microbial degradation and humification of the lawn care pesticide 2,4-dichlorophenoxyacetic acid during the composting of yard trimmings; Michel FC Jr et al.; The fate of the widely used lawn care herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) during the composting of yard trimmings consisting of primarily leaves and grass is an important unexplored question . In this study, we determined the extent of 2,4-D mineralization, incorporation into humic matter, volatilization, and sorption during the composting of yard trimmings . Yard trimmings (2:1 {wt/wt} leaves-grass) were amended with 14C-ring-labeled 2,4-D (17 mg/kg of dry weight) and composted in a temperature-controlled laboratory scale compost system . During composting, thermophilic microbes were numerically dominant, reaching a maximum of 2 x 10(11)/g . At the end of composting, 46% of the organic matter (OM) present in the yard trimmings was lost and the compost was stable, with an oxygen uptake rate of 0.09 mg of O2 per g of OM per h, and was well humified (humification index, 0.39) . Mineralization of the OM temporally paralleled mineralization of 2,4-D . In the final compost, 47% of the added 2,4-D carbon was mineralized, about 23% was complexed with high-molecular-weight humic acids, and about 20% was not extractable (humin fraction) . Less than 1% of the added 14C was present in water expressed from the finished compost, suggesting a low potential for leaching of 2,4-D . Very little volatilization of 2,4-D occurred during composting . It is of interest that our results indicate active mineralization of 2,4-D at composting temperatures of 60 degrees C because microbial 2,4-D degradation at thermophilic temperatures has not been previously documented.

J Bacteriol, 1995 Jul, 177(13), 3668 - 72
Pressure effects on the composition and thermal behavior of lipids from the deep-sea thermophile Methanococcus jannaschii; Kaneshiro SM et al.; The deep-sea archaeon Methanococcus jannaschii was grown at 86 degrees C and under 8, 250, and 500 atm (1 atm = 101.29 kPa) of hyperbaric pressure in a high-pressure, high-temperature bioreactor . The core lipid composition of cultures grown at 250 or 500 atm, as analyzed by supercritical fluid chromatography, exhibited an increased proportion of macrocyclic archaeol and corresponding reductions in aracheol and caldarchaeol compared with the 8-atm cultures . Thermal analysis of a model core-lipid system (23% archaeol, 37% macrocyclic archaeol, and 40% caldarchaeol) using differential scanning calorimetry revealed no well-defined phase transition in the temperature range of 20 to 120 degrees C . Complementary studies of spin-labeled samples under 10 and 500 atm in a special high-pressure, high-temperature electron paramagnetic resonance spectroscopy cell supported the differential scanning calorimetry phase transition data and established that pressure has a lipid-ordering effect over the full range of M . jannaschii's growth temperatures . Specifically, pressure shifted the temperature dependence of lipid fluidity by ca . 10 degrees C/500 atm.

Cell Mol Biol (Noisy-le-grand), 1995 Jul, 41(5), 625 - 38
Detection and identification of Campylobacter spp . using the polymerase chain reaction; Giesendorf BA et al.; Since Campylobacters have fastidious growth requirements and conventional detection and identification requires at least 4-6 days, the development of fast but reliable detection procedures is needed . Although methods based on DNA probe technology have been developed, these are not sensitive enough for the detection of Campylobacter spp . in food products . Therefore a PCR procedure based on the amplification of the 16S rRNA gene was developed that specifically detects the thermophilic Campylobacter species . This assay provides an excellent tool for the rapid and sensitive isolation and identification of Campylobacter spp . from chicken samples . In order to further identify the different Campylobacter spp., which are difficult to distinguish by conventional methods, PCR mediated approximately DNA typing was used to select species-specific DNA probes . This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information . PCR mediated DNA typing was also used to study the epidemiology of diarrheal diseases caused by Campylobacter spp . Using primers complementary to dispersed repetitive DNA sequences and arbitrarily chosen DNA motifs PCR fingerprinting has proven to be a fast, highly discriminative and relatively simple method that can be applied in epidemiological investigations on Campylobacter infections . Besides this application of PCR fingerprinting for typing of Campylobacter spp . this method can also be used for the development of specific DNA probes.

Microbiology, 1995 Jul, 141 ( Pt 7), 1731 - 8
A gene encoding a thermophilic alkaline serine proteinase from Thermus sp . strain Rt41A and its expression in Escherichia coli; Munro GK et al.; The extreme thermophile Thermus sp . strain Rt41A produces an extracellular alkaline serine proteinase during growth . This enzyme is stable for more than 24 h at 70 degrees C and has a pH optimum of 8.0 . The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene . The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase . Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus . The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein . The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present.

Bioorg Khim, 1995 Jul, 21(7), 492 - 7
{Structural properties of ribosomal protein S8 from the extreme thermophile Thermus thermophilus}; Vysotskaia VS et al.; The gene of ribosomal protein S8 from the extreme thermophile Thermus thermophilus was expressed in E . coli using the strain BL21(DE3) and vector pET3-1 . A method of isolating this protein from the super producing strain was developed, which makes it possible to obtain 8-12 mg of product from 11 of culture . The secondary structure of protein S8 was determined by using CD spectroscopy . The protein was shown to be highly resistant to denaturants.

Yeast, 1995 Jul, 11(9), 873 - 83
A 37.5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP-1-related gene, an open reading frame similar to the DAL80 gene, and a tRNA(Arg); Rasmussen SW; The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromosome X was determined from an ordered set of subclones . The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons . Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding gamma-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation . The deduced amino acid sequence of A550 is 63% identical to the Cc eta subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family . Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein . In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI . Finally, the sequence contained a tRNA(Arg3) (AGC) gene.

Mol Biol (Mosk), 1995 Jul-Aug, 29(4), 942 - 9
{Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction for detecting CD4 receptor mRNA}; Glukhov AI et al.; The RT/PCR method was applied to study a possible use of Tth DNA-polymerase for coupled reaction of reverse transcription and polymerase chain reaction (RT/PCR) on the CD-4 receptor mRNA template in the total cellular RNA . The conditions for detecting the CD-4 receptor mRNA were optimized . The pH-optimum for RT reaction was 8.8 . The influence of Mn2+, Cu2+, Co2+, and Cd2+ cations in RT and PCR reaction was investigated . The efficiency of the RT reaction was shown to be the highest in the presence of Mn2+ (optimal concentration 1 mM) . At Mn2+ concentration > or = 3 mM complete inhibition of RT/PCR was observed . The Tth DNA polymerase in RT/PCR was shown to be more effective than Taq DNA polymerase . The Tth DNA polymerase allows observation of the specific product in the gel containing ethidium bromide using 20 ng of the total RNA . High sensitivity and specificity of RT/PCR performed with the Tth DNA polymerase allow its wide application in the detection, quantitative analysis and cloning of cellular and viral RNAs.

Mol Biol (Mosk), 1995 Jul-Aug, 29(4), 930 - 41
{Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1)}; Grebennikova TV et al.; According to our data native Tth DNA polymerase displays higher reverse transcription activity than Taq DNA polymerase . This allows one to use Tth DNA polymerase in the complete reaction of reverse transcription and amplification (RT/PCR) . We used this enzyme to synthesize the interleukine (IL-2 alpha) RNA template synthesized by the RT/PCR method in vitro . The conditions for RNA IL-2 alpha detection were optimized . The maximum yield of the specific product was obtained at pH 8.5-9.0 . The influence of bivalent cations on the efficiency of RT reaction of coupled RT/PCR can be expressed as: Mn2+ > or = Cu2+ > Mg2+ > Cd2+ >> Co2+ . The optimal ratio is 1.25-1.88 for Mn2+/dNTPs and 1.88-2.5 for Cu2+/dNTPs and Cd2+/dNTPs . The maximum yield of the RT/PCR product is found at Mg2+/dNTPs = 3.75 . When Mn2+ is used instead of Mg2+ in the PCR reaction the efficiency of RT/PCR decreases . The RT/PCR method embracing thermostable Tth DNA-polymerase provides detection of 10(3) copies of RNA IL-2 alpha . An efficient method of the express-diagnostics of MDR-1 gene expression by coupled RT/PCR using Tth DNA polymerase is described.

Mol Microbiol, 1995 Jul, 17(1), 1 - 12
glmS of Thermus thermophilus HB8: an essential gene for cell-wall synthesis identified immediately upstream of the S-layer gene; Fernandez-Herrero LA et al.; A 30 kbp chromosomal region containing the S-layer gene (slpA) from Thermus thermophilus HB8 was cloned from a lambda phage gene library . DNA sequence analysis of the region upstream to the slpA gene revealed the presence of an open reading frame (ORF) which coded for a 604-amino-acid protein highly homologous to the glucosamine-6-P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin . The identification of this ORF as the glucosamine-6-P synthase gene from T . thermophilus (glmSth) has been carried out using three different strategies: (i) complementation of an Escherichia coli glmS mutant; (ii) in vivo insertional inactivation of the gene; and (iii) in vitro synthesis of glucosamine-6-P at 60 degrees C by a cytoplasmic extract of an overproducing E . coli strain . The glmSth gene is transcribed divergently from slpA in a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3' extreme . As the products of both the glmSth and the slpA genes are main components of the cell envelope of T . thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their coordinate expression.

J Biol Chem, 1995 Jun 9, 270(23), 13593 - 5
DNA enzymology above 100 degrees C . Topoisomerase V unlinks circular DNA at 80-122 degrees C; Kozyavkin SA et al.; The widespread application of polymerase chain reaction and related techniques in biology and medicine has led to a heightened interest in thermophilic enzymes of DNA metabolism . Some of these enzymes are stable for hours at 100 degrees C, but no enzymatic activity on duplex DNA at temperatures above 100 degrees C has so far been demonstrated . Recently, we isolated topoisomerase V from the hyperthermophile Methanopyrus kandleri, which grows up to 110 degrees C . This novel enzyme is similar to eukaryotic topoisomerase I and acts on duplex DNA regions . We now show that topoisomerase V catalyzes the unlinking of double-stranded circular DNA at temperatures up to 122 degrees C . In this in vitro system, maximal DNA unlinking occurs at 108 degrees C and corresponds to complementary strands being linked at most once . These results further imply that in the presence of sufficient positive supercoiling DNA can exist as a double helix even at 122 degrees C.

J Biol Chem, 1995 Jun 2, 270(22), 12995 - 3003
Kinetic analysis of lactose and proton coupling in Glu379 mutants of the lactose transport protein of Streptococcus thermophilus; Poolman B et al.; The role of Glu379 in the lactose-H+ symport protein (LacS) of Streptococcus thermophilus was studied by analyzing the kinetic mechanism of transport of wild-type and Ala379, Asp379, and Gln379 mutant proteins . Glu379 forms part of the sequence motif Lys-X-X-His-X-X-Glu that is present in a number of sugar transport proteins, including LacY of Escherichia coli . The E379A and E379Q mutants were defective in the uptake of lactose against a concentration gradient and lactose-dependent proton uptake, but catalyzed facilitated influx of lactose down a concentration gradient and equilibrium exchange with rates similar to that of the wild-type enzyme . The E379D mutant was partially defective in the coupled transport of lactose and protons . These results suggest that an acidic residue at position 379 is required for the coupled uptake of lactose and protons and are consistent with a mechanism in which lactose transport in the E379A and E379Q mutants occurs by uniport rather than proton symport . Lactose efflux down a concentration gradient in wild-type LacS and LacS-E379D increased with pH with apparent pK (pKa) values of > or = 8.5 and 8.0, respectively, whereas efflux in the E379Q mutant increased sigmoidally with a pKa of about 6.0 . Imposition of an artificial membrane potential (inside negative) in membrane vesicles bearing wild-type LacS or LacS-E379Q not only inhibited the lactose efflux mediated by wild-type but also that of the mutant enzyme . To associate the role of Glu379 with specific step(s) in the translocation cycle of LacS, the properties of wild-type LacS and the Glu379 mutants have been evaluated by numerical analysis of simple kinetic schemes for translocation catalysis by solute H+ symport proteins . The properties of the wild-type enzyme are consistent with a mechanism in which the order of ligand binding on the inside is substrate first and proton last, whereas the order is random (or proton first, substrate last) at the outer surface of the membrane . The wild-type enzyme is asymmetric with regard to proton binding; the pK for proton binding on the outside is at least 4 units higher than the pK on the inside . The properties of the Glu379 mutants correspond with a lowering of the pK on the outside (pKOUT approximately pKIN), and the induction of a leak pathway in which the binary enzyme-substrate complex becomes mobile.

Zentralbl Hyg Umweltmed, 1995 Jun, 197(5), 387 - 97
Survival of E . coli and Salmonella populations in aerobic thermophilic composts as measured with DNA gene probes; Droffner ML et al.; Aerobic, thermophilic composting is a widely practiced method for disposal of organic wastes . The wastes which are composted include biosolids from waste water treatment plants (WWTP), and biowastes (food scraps and yardwaste) . Important hygiene issues are involved in composting since many potential pathogens may be present in the fresh wastes . In this study, the survival of Salmonella and Escherichia coli is examined during aerobic composting of municipal solid wastes, municipal wastewater sludge and biowastes . A laboratory compost was prepared by inoculating with 10(7) Salmonella typhimurium Q and Escherichia coli B . In both industrial and laboratory trials, gene probes were used to determine at what time during the composting and at what temperature these bacteria became undetectable . It was observed that Salmonella and E . coli survived for 59 days at about 60 degrees C in an industrial compost . The bacteria became undetectable after the temperature decreased from 62 degrees C to about 40 degrees C in the compost curing . The bench scale trials showed that E . coli B survived for at least 9 days at 60-70 degrees C in a biowaste (food waste) compost or a waste water sludge compost . Salmonella typhimurium Q survived for at least 9 days over 60 degrees C in the food biowaste compost and at least 5 days in the waste water sludge compost . Data collected show that the temperature or the time of high temperature is difficult to correlate to the destruction of the pathogen, Salmonella, or the pathogen indicator, E . coli . These results suggest that the mechanism for removal of these microorganisms during aerobic composting is complex and not simply the result of a thermal physical environment.

Zentralbl Hyg Umweltmed, 1995 Jun, 197(5), 370 - 86
Polymerase chain reaction assay for detection of Campylobacter coli and Campylobacter jejuni in poultry meat; Manzano M et al.; Primers of 16S rRNA and flaA genes were used to optimise a PCR technique for detecting thermotolerant campylobacters in poultry meat . Different methods for crude DNA extraction were also evaluated . The use of flaA primers and extraction of nucleic acid by boiling and proteinase K gave good results in the detection of Campylobacter either in artificially or naturally contaminated foodstuffs . The lowest sensitivity limit for the PCR reaction was 10(1)-10(2) thermophilic Campylobacter cells either in pure cultures or in artificially and naturally contaminated poultry skins, corresponding to a concentration of 10(2)-10(3) Campylobacter/ml or g product . The PCR method we devised had a high sensitivity and specificity . It appears to give better results than conventional methods and is very easy and fast, requiring only eight hours to detect thermotolerant Campylobacter from poultry meat . In contrast, conventional methods require almost 4 days.

J Am Coll Nutr, 1995 Jun, 14(3), 299 - 303
Composition and preliminary evaluation of a hydrolyzed rice-based oral rehydration solution for the treatment of acute diarrhea in children; Lebenthal E et al.; OBJECTIVE: The purpose of this study was to experimentally develop and clinically evaluate the safety and potential usefulness of a rice-based, short glucose polymer oral rehydration solution (ORS), Amylyte, in the treatment of acute diarrhea . Amylyte has a similar osmolality but a higher caloric density than the WHO ORS . METHODS: Different amounts of rice were cooked in 500 ml of water containing salts (1.5 g NaCl, 600 mg KCl, and 150 mg CaCl2) with varying amounts of thermophilic amylase (252,500 modified Wohlgemuth units) . Amylase (25 mg) thinned the gluey rice water when 100 g of rice was cooked in 500 ml of water for 10 minutes . The volume of the resultant supernatant (Amylyte) was approximately 250 ml . A two-part, clinical case study was performed . In study 1, 12 children with diarrhea and mild dehydration were studied to determine the safety of Amylyte . In study 2, Amylyte and the WHO ORS were given to 24 and 31 male children with acute diarrhea and moderate to severe dehydration, respectively . RESULTS: 92-96% of the rice amylose and amylopectin were converted to short polymers of glucose (3-9 molecules of glucose) . The osmolality of 7,994 packages used to make the Amylyte solution ranged between 277-340 mOsm/kg . The mean electrolyte composition was Na+ = 68 mEq/L, K+ = 20 mEq/L, Cl = 73 mEq/L, the caloric density 425 kcal/L and rice proteins 0.7 g/L . In study 1, 12 children with diarrhea and mild dehydration were rehydrated successfully with Amylyte ORS and the diarrhea ceased within 48 hours . None developed clinical features of carbohydrate intolerance . In study 2, an open-label clinical case study, children with acute diarrhea given Amylyte ORS had significantly less stool output than children given the WHO ORS . CONCLUSIONS: Amylyte ORS has the advantages of a higher caloric density than the WHO ORS and shares a simple preparation of appropriate osmolality and electrolyte composition . It can safely and effectively rehydrate children with acute diarrhea and dehydration.

Protein Eng, 1995 Jun, 8(6), 583 - 92
Citrate synthase from the hyperthermophilic Archaeon, Pyrococcus furiosus; Muir JM et al.; The gene encoding the enzyme citrate synthase has been cloned and sequenced from the hyperthermophilic Archaeon Pyrococcus furiosus, and the derived amino acid sequence has been phylogenetically compared with citrate synthases from archaeal, bacterial and eukaryal organisms . The gene has been over-expressed in Escherichia coli to produce an active enzyme that has then been characterized with respect to its kinetic, oligomeric and hyperthermostable properties . A structurally-based sequence alignment was made to the citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum, the crystal structure of which we have determined recently . From this alignment, a homology-modelled structure for the P.furiosus citrate synthase was generated and analysed.

Res Microbiol, 1995 Jun, 146(5), 371 - 83
Characterization of the gor gene of the lactic acid bacterium Streptococcus thermophilus CNRZ368; Pebay M et al.; Cloning and characterization of the gor gene of the lactic acid bacterium Streptococcus thermophilus, encoding glutathione reductase, are described in this paper . This enzyme is a part of the enzymatic defences against oxidative stress in eukaryotic cells and in Gram-negative bacteria, but was never found in Gram-positive bacteria before this study . The amino acid sequence shares extensive similarities with glutathione reductases from other organisms, e.g . 62% amino acid identity with Escherichia coli protein . Northern blot analysis and glutathione reductase enzyme assays gave evidence that the gene is expressed in aerobically growing cells.

Appl Environ Microbiol, 1995 Jun, 61(6), 2071 - 8
Identification and epidemiological typing of Naegleria fowleri with DNA probes; Kilvington S et al.; Naegleria fowleri is a small free-living amoeboflagellate found in warm water habitats worldwide . The organism is pathogenic to humans, causing fatal primary amoebic meningoencephalitis . When monitoring the environment for the presence of N . fowleri, it is important to reliably differentiate the organism from other closely related but nonpathogenic species . To this end, we have developed species-specific DNA probes for use in the rapid identification of N . fowleri from the environment . Samples were taken from the thermal springs in Bath, England, and cultured for amoebae . Of 84 isolates of thermophilic Naegleria spp., 10 were identified as N . fowleri by probe hybridization . The identity of these isolates was subsequently confirmed by their specific whole-cell DNA restriction fragment length polymorphisms (RFLPs) . One DNA clone was found to contain a repeated element that detected chromosomal RFLPs that were not directly visible on agarose gels . This enabled the further differentiation of strains within geographically defined whole-cell DNA RFLP groups . N . fowleri DNA probes represent a specific and potentially rapid method for the identification of the organism soon after primary isolation from the environment.

Dev Biol, 1995 Jun, 169(2), 644 - 61
Cellular polarity in ciliates: persistence of global polarity in a disorganized mutant of Tetrahymena thermophila that disrupts cytoskeletal organization; Jerka-Dziadosz M et al.; Much of the cell surface on the ciliate Tetrahymena thermophila is covered by a polarized lattice of cytoskeletal structures that are associated with basal bodies of the ciliary rows . Unique structural landmarks, including an oral apparatus and contractile vacuole pores, develop before cell division in localized domains located, respectively, posterior and anterior to the transverse fission zone . All of these structures can be visualized by specific monoclonal antibodies . A single-locus recessive mutation, disorganized-A (disA), primarily affects the striated rootlets of the ciliary-row basal bodies and brings about a severe disorganization in the positioning and orientation of these basal bodies and associated cytoskeletal elements . Nonetheless, the new oral apparatus, contractile vacuole pores, and other unique structures appeared at or near their normal sites along the anteroposterior axis of disA cells, indicating that the positioning of these localized structures is not dependent on the integrity of the ciliary rows . Abnormalities were present in the details of construction of some of the localized structures and in aspects of cell shape that may be influenced by these details . In the main, however, analysis of disA mutant cells indicates that intracellular domains near the cell poles develop independently of the vectorial polarity of the ciliary rows.

J Cell Biol, 1995 Jun, 129(5), 1301 - 10
Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila; Gaertig J et al.; In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta-tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes . Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes . In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated . We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position . No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40 . Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells . Thus, acetylation of alpha-tubulin at lysine 40 is non-essential in Tetrahymena . In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.

J Bacteriol, 1995 Jun, 177(11), 2977 - 81
The adenylate kinases from a mesophilic and three thermophilic methanogenic members of the Archaea; Rusnak P et al.; Adenylate kinase has been isolated from four related methanogenic members of the Archaea . For each, the optimum temperature for enzyme activity was similar to the temperature for optimal microbial growth and was approximately 30 degrees C for Methanococcus voltae, 70 degrees C for Methanococcus thermolithotrophicus, 80 degrees C for Methanococcus igneus, and 80 to 90 degrees C for Methanococcus jannaschii . The enzymes were sensitive to the adenylate kinase inhibitor P1, P5-di(adenosine-5')pentaphosphate, a property that was exploited to purify the enzymes by CIBACRON Blue affinity chromatography . The enzymes had an estimated molecular mass (approximately 23 to 25 kDa) in the range common for adenylate kinases . Each of the enzymes had a region of amino acid sequence close to its N terminus that was similar to the canonical P-loop sequence reported for all adenylate kinases . However, the methanogen sequences lacked a lysine residue that has previously been found to be invariant in adenylate kinases, including an enzyme isolated from the archaeon Sulfolobus acidocaldarius . If verified as a nucleotide-binding domain, the methanogen sequence would represent a novel nucleotide-binding motif . There was no correlation between amino acid abundance and the optimal temperature for enzyme activity.

Mol Cell Biol, 1995 Jun, 15(6), 3372 - 81
Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter; Pan WJ et al.; In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer . The replication of this rDNA minichromosome is under both cell cycle and copy number control . We addressed the effect on origin function of transcription through this origin region . A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome . Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J . Pan and E . H . Blackburn, Nucleic Acids Res, in press) . We present direct evidence that the +G mutation inactivates this rRNA promoter . It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript . Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter . Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance . Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region . We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

Orig Life Evol Biosph, 1995 Jun, 25(1-3), 251 - 64
The effects of heavy meteorite bombardment on the early evolution--the emergence of the three domains of life; Gogarten-Boekels M et al.; A characteristic of many molecular phylogenies is that the three domains of life (Bacteria, Archaea, Eucarya) are clearly separated from each other . The analyses of ancient duplicated genes suggest that the last common ancestor of all presently known life forms already had been a sophisticated cellular prokaryote . These findings are in conflict with theories that have been proposed to explain the absence of deep branching lineages . In this paper we propose an alternative scenario, namely, a large meteorite impact that wiped out almost all life forms present on the early Earth . Following this nearly complete frustation of life on Earth, two surviving extreme thermophilic species gave rise to the now existing major groups of living organisms, the Bacteria and Archaea . {The latter also contributed the major portion to the nucleo-cytoplasmic component of the Eucarya} . An exact calibration of the molecular record with regard to time is not yet possible . The emergence of Eucarya in fossil and molecular records suggests that the proposed late impact should have occurred before 2100 million years before present (BP) . If the 3500 million year old microfossils {Schopf, J . W . 1993: Science 260: 640-646} are interpreted as representatives of present day existing groups of bacteria (i.e., as cyanobacteria), then the impact is dated to around 3700 million years BP . The analysis of molecular sequences suggests that the separation between the Eucarya and the two prokaryotic domains is less deep then the separation between Bacteria and Archaea . The fundamental cell biological differences between Archaea and Eucarya were obtained over a comparatively short evolutionary distance (as measured in number of substitution events in biological macromolecules) . Our interpretation of the molecular record suggests that life emerged early in Earth's history even before the time of the heavy bombardment was over . Early life forms already had colonized extreme habitats which allowed at least two prokaryotic species to survive a late nearly ocean boiling impact . The distribution of ecotypes on the rooted universal tree of life should not be interpreted as evidence that life originated in extremely hot environments.

Microbiology, 1995 Jun, 141 ( Pt 6), 1443 - 9
Discovery of a ptsHI operon, which includes a third gene (ptsT), in the thermophile Bacillus stearothermophilus; Lai X et al.; The discovery of ptsHI operon in Bacillus stearothermophilus XL-65-6 coupled with our previous report of a cel operon (Lai & Ingram, J Bacteriol 175, 6441-6450, 1993) demonstrates that this thermophilic organism contains all of the genes required for cellobiose uptake by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) . Genes encoding the two general PTS proteins, HPr (ptsH) and enzyme I (ptsI), were cloned and sequenced . These form an operon which includes a third small gene (ptsT) of unknown function (encoded product M(r) 18428) . Both ptsH and ptsI were expressed at high levels from a single plasmid in Escherichia coli and complemented corresponding host mutations . Although the translated sequences for these genes were similar to homologues from Gram-positive mesophiles (64-77% identity), the B . stearothermophilus gene products were unusual in having a higher predicted pI and fewer negatively charged amino acid residues . Enzyme I also contained more alanine and leucine than mesophilic counterparts . Interestingly, ptsT inhibited the growth of E . coli ptsI mutants at 37 degrees C . No such inhibition was observed during incubation at a lower temperature (30 degrees C) or in E . coli DH5 alpha, which is wild-type for ptsI . The predicted translation product from ptsT contained a high proportion of basic amino acids (27%) and had a high predicted pI (pH 11.7), properties similar to bacterial histone-like proteins, but did not exhibit homology to any sequences in the current database . Regions upstream and downstream from the ptsHI operon contain genes with homology to Bacillus subtilis ptsG and wapA (wall-associated protein), respectively.

Biochem Mol Biol Int, 1995 Jun, 36(2), 401 - 10
Molecular cloning and characterization of the xylose isomerase gene from a thermophilic Bacillus species; Liao WX et al.; The gene (xylA) encoding a thermostable xylose isomerase has been isolated and characterized from a thermophilic Bacillus species for the first time . The xylA open reading frame of 1323 bp encoded a protein containing 441 amino acids with a calculated molecular weight of 50,176 . The amino acid sequence of this protein showed 76% homology to xylose isomerase isolated from Bacillus subtilis and contained all the important catalytic domains of the enzyme . The gene complemented the xyl-5 mutation and produced a functional enzyme constitutively in Escherichia coli . The crude cell-free extract of E . coli recombinants exhibited xylose isomerase activity over a wide range of temperatures from 60 to 100 degrees C with an optimal enzyme activity of 10.4 Units/mg protein at 85 degrees C . This optimal temperature was one of the highest reported so far for thermostable xylose isomerases . The recombinant enzyme was found to be a tetramer with each subunit having molecular weight of 50,000.

J Mol Evol, 1995 Jun, 40(6), 551 - 8
Self-incorporation of coenzymes by ribozymes; Breaker RR et al.; RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes . Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities . The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function . In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNA-catalyzed incorporation of various coenzymes and coenzyme analogues . The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme . Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH . Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

Biochim Biophys Acta, 1995 Jun 1, 1230(1-2), 31 - 7
Thermostability of respiratory terminal oxidases in the lipid environment; Elferink MG et al.; The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined . Cytochrome-c oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively . Quinol oxidase from the archaeon Sulfolobus acidocaldarius represented the model for a extreme thermoacidophilic enzyme . All three integral membrane proteins were tested for their thermal inactivation in detergent and after reconstitution in liposomes composed of phospholipids of Escherichia coli or tetraether lipids from S . acidocaldarius . When preincubated at 0 degrees C, all three enzymes exhibited biphasic thermal inactivation curves . Data could be analysed according to a two-state model that defines two conformations of the enzyme, differing in their thermostability . Monophasic inactivation curves were observed when the enzymes were preincubated at higher temperatures prior to thermal inactivation . Lipids rendered the beef-heart cytochrome-c oxidase and S . acidocaldarius quinol oxidase more thermostable as compared to detergent solution . In contrast, the B . stearothermophilus oxidase, an intrinsically thermostable enzyme, was as thermostable in detergent as in the reconstituted state . These data suggest that the lipid environment can be an important factor in the thermostability of membrane proteins.

Cell Struct Funct, 1995 Jun, 20(3), 239 - 44
The vegetative micronucleus has a critical role in maintenance of cortical structure in Tetrahymena thermophila; Haremaki T et al.; The vegetative role of the germinal micronucleus of Tetrahymena thermophila was studied . Amicronucleate cells that had spontaneously arisen in star strains (very aged cones with defective micronuclei) were observed in B* as well as in A* and in C* and were found to lack the oral apparatus and to have disordered ciliary rows . To reduce uncertainty given the very small micronucleus and possible effects of aging factors, we induced amicronucleate cells in a young clone by treatment with the antitubulin drug, nocodazole, and observed their cortical structure and nuclei . Amicronucleate cells gradually lost their oral apparatus and then their ciliary rows became disordered, even without cell division, and they became crinkled cells . It can be concluded that the vegetative micronucleus appears to play a critical role in the maintenance of the cortical structure, especially of the oral apparatus.

Electrophoresis, 1995 Jun, 16(6), 1060 - 6
Separation of heat-stable proteins from Thermus thermophilus HB8 by two-dimensional electrophoresis; Kawaguchi S et al.; Thermostable proteins from Thermus thermophilus HB8, an extremely thermophilic bacterium, were separated by two-dimensional gel electrophoresis . About 1200 spots were detected with silver staining on the gel between pH 3 and 10 . According to the genome size of T . thermophilus, we consider that more than half of the proteins in the cell are visualized on a two-dimensional gel . Using comigrated standard marker proteins, the molecular weight and isoelectric point of each protein spot were calculated . The average molecular weight and isoelectric point values were estimated to be 30 000 and 5.2, respectively . The average size and isoelectric point of detected protein from T . thermophilus were smaller and more acidic than those from Escherichia coli . After the protein spots had been electroblotted onto a polyvinylidene difluoride membrane and stained with Coomassie Brilliant Blue, the N-terminal amino acid sequences were determined for about twenty protein spots . Few proteins had blocked N-termini . Some spots were identified as proteins whose sequences had been reported previously from T . thermophilus . Others had amino acid sequences homologous with those of various proteins from other organisms . The amino acid sequence information of this report will be useful for obtaining stable proteins and for identifying open reading frames determined from the genome DNA sequence . Considering its small genome size and protein stability, T . thermophilus will be an excellent candidate for studying the molecular biology of an autotrophic living cell at the atomic level.

RNA, 1995 Jun, 1(4), 363 - 74
Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA; Zaug AJ et al.; Dimethyl sulfate modification of RNA in living Tetrahymena thermophila allowed assessment of RNA secondary structure and protein association . The self-splicing rRNA intron had the same methylation pattern in vivo as in vitro, indicating that the structures are equivalent and suggesting that this RNA is not stably associated with protein in the nucleolus . Methylation was consistent with the current secondary structure model . Much of telomerase RNA was protected from methylation in vivo, but the A's and C's in the template region were very reactive . Thus, most telomerase is not base paired to telomeres in vivo . Protein-free telomerase RNA adopts a structure different from that in vivo, especially in the template and pseudoknot regions . The U2 snRNA showed methylation protection at the Sm protein-binding sequence and the mRNA branch site recognition sequence . For both telomerase RNA and U2 snRNA, the in vivo methylation pattern corresponded much better to the structure determined by comparative sequence analysis than did the in vitro methylation pattern . Thus, as expected, comparative analysis gives the structure of the RNA in vivo.

Scand J Work Environ Health, 1995 Jun, 21(3), 223 - 8
Prevalence of microfungi in Finnish cow barns and some aspects of the occurrence of Wallemia sebi and Fusaria; Hanhela R et al.; OBJECTIVES: The occurrence of microfungi in the air and in feeding and bedding materials was studied on 32 Finnish dairy farms . METHODS: Air samples for determining viable and total spore concentrations were collected on membrane filters and with a cascade impactor . Genera of mesophilic, xerophilic, and thermophilic fungi were identified in four culture media . Total spore counts were done with the aid of an epifluorescence microscope . To identify fungal flora in agricultural materials, feeding and bedding material samples were also taken from the farms . RESULTS: The airborne spore concentrations varied for viable mesophilic, xerophilic, and thermophilic fungi from 10(1) to 10(7) colony-forming units per cubic meter, and for total spores from 10(5) to 10(7) spores per cubic meter . Aspergillus, Penicillium, Cladosporium, Absidia species, Wallemia sebi and yeasts were the predominant fungi in the air, as well as in the material samples . CONCLUSIONS: In general, the airborne spore concentrations were high although the variation in the concentrations of different fungal groups was large between the farms . Along with using new growth media, two fungi whose prevalence was earlier poorly known in Finland were detected . W sebi proved to be the most abundant xerophilic fungi in the air and hay samples, while Fusarium spp were very common in grain and straw but rare in air.

Mol Microbiol, 1995 Jun, 16(5), 1031 - 6
Screening of stable proteins in an extreme thermophile, Thermus thermophilus; Tamakoshi M et al.; The leuB gene codes for 3-isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus . The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene . The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained . A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant . The resultant amino acid residue coincided with the corresponding residue of the T . thermophilus enzyme . It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme . This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.

Gene, 1995 May 26, 158(1), 147 - 8
Cloning and sequencing of the neutral protease-encoding gene from a thermophilic strain of Bacillus sp; Vecerek B et al.; The neutral protease-encoding gene (npr) from the thermophilic strain Bacillus sp . BT1 was cloned and sequenced . A possible means of regulation of npr expression is suggested.

Gene, 1995 May 26, 158(1), 101 - 5
The dihydrofolate reductase-encoding gene dyrA of the hyperthermophilic bacterium Thermotoga maritima; Van de Casteele M et al.; The structural gene (dyrA) encoding dihydrofolate reductase (DHFR) of Thermotoga maritima has been cloned, sequenced and expressed in Escherichia coli . The dyrA gene, located immediately upstream from the gene encoding aspartate carbamoyltransferase (pyrB), encodes a highly thermostable enzyme with a distinct thermophilic activity profile . Important structural features are conserved among all bacterial DHFR, yet the DHFR of T . maritima appears unique in a number of insertions and deletions, some of which are reminiscent of eukaryotic DHFR.

Nucleic Acids Res, 1995 May 25, 23(10), 1766 - 74
Modular structural elements in the replication origin region of Tetrahymena rDNA; Du C et al.; Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS {Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res . 22, 2479-2489} . This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions . In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses . Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels . Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo . The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.

Nucleic Acids Res, 1995 May 25, 23(10), 1737 - 43
Genetic and molecular analysis of the tRNA-tufB operon of the myxobacterium Stigmatella aurantiaca; Bremaud L et al.; The tufB gene, encoding elongation factor Tu (EF-Tu), from the myxobacterium Stigmatella aurantiaca was cloned and sequenced . It is preceded by four tRNA genes, the first ever described in myxobacteria . The tRNA synthesized from these genes and the general organization of the locus seem identical to that of Escherichia coli, but differences of potential importance were found in the tRNA sequences and in the intergenic regions . The primary structure of EF-Tu was deduced from the tufB DNA sequence . The factor is composed of 396 amino acids, with a predicted molecular mass of 43.4 kDa, which was confirmed by expression of tufB in maxicells . Sequence comparisons between S.aurantiaca EF-Tu and other bacterial homologues from E.coli, Salmonella typhimurium and Thermus thermophilus displayed extensive homologies (75.9%) . Among the variable positions, two Cys residues probably involved in the temperature sensitivity of E.coli and S.typhimurium EF-Tu are replaced in T.thermophilus and S.aurantiaca EF-Tu . Since two or even three tuf genes have been described in other bacterial species, the presence of multiple tuf genes was sought for . Southern and Northern analysis are consistent with two tuf genes in the genome of S.aurantiaca . Primer extension experiments indicate that the four tRNA genes and tufB are organized in a single operon.

Biochem Biophys Res Commun, 1995 May 25, 210(3), 733 - 7
His273 of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 is involved in the coenzyme binding; Yaoi T et al.; The coenzyme binding site of NAD(+)-dependent 3-isopropylmalate dehydrogenase from Thermus thermophilus was analyzed by chemical modification and site-directed mutagenesis, and His273 of the enzyme was identified to be involved in the coenzyme binding.

Biochemistry, 1995 May 23, 34(20), 6628 - 39
Pulsed EPR studies of the binuclear Mn(III)Mn(IV) center in catalase from Thermus thermophilus; Ivancich A et al.; The nature of possible protein ligands to the binuclear metal core in manganese catalase from Thermus thermophilus has been addressed by EPR and ESEEM (pulsed EPR) spectroscopies . The three-pulse ESEEM spectrum of the superoxidized Mn(III)Mn(IV) enzyme obtained at 3429 G shows a frequency pattern with peaks at 0.60, 1.45, 2.06, and 5.03 MHz that is assigned to the magnetic coupling in the exact cancellation regime of one 14N atom that coordinates the Mn dimer, with magnetic parameters e2Qq = 2.34 MHz, eta = 0.51, and Aiso = 2.45 MHz . When the enzyme is chemically modified by reductive methylation, dramatic effects are detected both in the CW-EPR spectrum and in the ESEEM data . Spectral simulations of the CW-EPR signal suggest that the alterations in the spectra are related to the properties of the hyperfine coupling tensors of the Mn ions and of the g tensor, which changes from axial symmetry (gparallel - gperpendicular = 0.018) in the untreated catalase to a nearly isotropic symmetry (gparallel - gperpendicular = 0.002) in the modified enzyme . The three-pulse ESEEM spectrum of the catalase is also completely altered after the reductive methylation, with a rather different frequency pattern at 1.57, 2.35, 3.88, and 6.00 MHz . These data are interpreted as indicating that the hyperfine interaction from the coupled 14N donor is profoundly modified by the methylation treatment, changing from Aiso = 2.45 MHz to a larger value . The spectra are compared with ESEEM data obtained on two polynuclear Mn systems with 14N donors: the Mn cluster of Photosystem II inhibited by 14NH4Cl, and the model compound {Mn2(bipy)4(mu-O)2}(ClO4)3 . It is found that the ESEEM data measured on the untreated Mn(III)Mn(IV) catalase resemble those on the Photosystem II manganese site, suggesting that the coupled 14N coordinates the Mn dimer in an analogous fashion . By analogy to the mode of binding of ammonia in Photosystem II proposed by Britt et al . {Britt, R . D., Zimmermann, J . L., Sauer, K., & Klein, M . P . (1989) J . Am . Chem . Soc . 111, 3522-3532}, it is proposed that a 14N atom bridges the two Mn ions in Mn(III)Mn(IV) catalase . By contrast, comparison of the data obtained on the methylated enzyme with those on the model compound suggests that the 14N couplings are similar in both systems; this is indicative of a terminal 14N ligand in the modified catalase.(ABSTRACT TRUNCATED AT 400 WORDS)

FEBS Lett, 1995 May 22, 365(1), 30 - 4
Two molecules of cytochrome c function as the electron donors to P840 in the reaction center complex isolated from a green sulfur bacterium, Chlorobium tepidum; Oh-oka H et al.; A photoactive reaction center complex was isolated from a thermophilic green sulfur bacterium, Chlorobium tepidum under anaerobic conditions . The electron transfer occurred from heme c to the photo-oxidized reaction center chlorophyll, P840+, with a half time (t1/2) of 110 or 340 microseconds at 24 or 12 degrees C, respectively . Optical measurements under multiflash excitations indicated that two hemes function as the immediate electron donors to P840+ . SDS-PAGE analysis of the RC complex in combination with the N-terminal amino acid sequence analyses revealed five subunit bands; a core protein (65 kDa), the light harvesting bacteriochlorophyll alpha protein (41 kDa), a protein with 2{4Fe-4S} clusters (31 kDa), monoheme cytochrome c (22 kDa), and a 18-kDa protein whose function is unknown . The reaction center complex, thus, contains two molecules of cytochrome c per P840.

Science, 1995 May 19, 268(5213), 1036 - 9
Requirement of a small cytoplasmic RNA for the establishment of thermotolerance; Fung PA et al.; Thermotolerance is an inducible state that endows cells with an enhanced resistance to thermal killing . Heat shock proteins are believed, and in a few instances have been shown, to be the agents conferring this resistance . The role of a small cytoplasmic RNA (G8 RNA) in developing thermotolerance in Tetrahymena thermophila was investigated by creating a strain devoid of all functional G8 genes . These G8 null cells mounted an apparently normal heat shock response, but they were unable to establish thermotolerance.

Gene, 1995 May 19, 157(1-2), 321 - 2
New restriction endonucleases from thermophilic soil bacteria; Repin VE et al.; Using the most effective rapid method for the detection of restriction endonucleases (ENases) in microorganisms, 32 thermophilic producers have been isolated . All strains belong to the genus Bacillus . Thermostable isoschizomers of ENases, such as AvaI, BbvI, BbvII, BclI, BsaBI, BsiYI, BsrI, BstEII, BstNI, Cfr10I, ClaI, FspI, HaeIII, HpaII, Ksp632I and SfeI, were isolated.

Gene, 1995 May 19, 157(1-2), 103 - 4
M.BssHII: a new multispecific C5-DNA-methyltransferase; Schumann J et al.; M.BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets . As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

Biochemistry, 1995 May 16, 34(19), 6504 - 12
The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme; Banerjee AR et al.; L-21 ScaI ribozyme is a linear form of the self-splicing intron from the precursor of the Tetrahymena thermophila LSU intron . The time scales for tertiary folding of L-21 ScaI were investigated in two ways after bringing it from a partially denatured state at 60 degrees C and 1 mM Mg2+ to a renatured state of 15 degrees C and 10 mM Mg2+ . First, formation of a catalytically active structure was monitored by measuring the kinetics of the reaction: p*CUCUA3 + G<==>p*CUCU + GA3 . This reaction mimics the first step of splicing . After 1 min of folding time, the catalytic rate is roughly 10% of the rate attained after 8 h of folding . This indicates that much of the structure refolds quickly . Also, at least two time scales of folding are observed, separated by a lag time of about 30 min . To define the regions folding on various time scales, all the guanosines of L-21 ScaI were probed with kethoxal at 15 degrees C while folding was in progress . Based on folding time scales, the guanosines can be placed into at least four classes . These are guanosines that (1) are already protected at 60 degrees C in 1 mM Mg2+ or which fold immediately, (2) fold during the lag time, (3) continue to fold after 1 h, and (4) never fold . These results give insight into the folding pathway of a group I ribozyme at nucleotide resolution . This provides useful information on the regions whose foldings are important for catalytic function of the ribozyme . The method may also provide a general way to suggest regions of an RNA that may interact with each other to form tertiary structure.

Eur J Biochem, 1995 May 15, 230(1), 3 - 16
The chaperonin containing t-complex polypeptide 1 (TCP-1) . Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol; Kubota H et al.; Many proteins in the cell require assistance from molecular chaperones at stages in their life cycles in order to attain correctly folded states and functional conformations during protein synthesis or during recovery from denatured states . A recently discovered molecular chaperone, which is abundant in the eukaryotic cytosol and is called the chaperonin containing TCP-1 (CCT), has been shown to assist the folding of some proteins in cytosol . This chaperone is a member of the chaperonin family which includes GroEL, 60-kDa heat shock protein (Hsp60), Rubisco subunit binding protein (RBP) and thermophilic factor 55 (TF55), but is distinct from the other members in several respects . Presently the most intriguing feature is the hetero-oligomeric nature of the CCT; at least eight subunit species which are encoded by independent and highly diverged genes are known . These genes are calculated to have diverged around the starting point of the eukaryotic lineage and they are maintained in all eukaryotes investigated, suggesting a specific function for each subunit species . The amino acid sequences of these subunits share approximately 30% identity and have some highly conserved motifs probably responsible for ATPase function, suggesting this function is common to all subunits . Thus, each subunit is thought to have both specific and common functions . These observations, in conjunction with biochemical and genetic analysis, suggest that CCT functions as a very complex machinery for protein folding in the eukaryotic cell and that its chaperone activity may be essential for the folding and assembly of various newly synthesized polypeptides . This complex behaviour of CCT may have evolved to cope with the folding and assembly of certain highly evolved proteins in eukaryotic cells.

Nucleic Acids Res, 1995 May 11, 23(9), 1561 - 9
Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation; Pan WJ et al.; The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA) . We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene . A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions . As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene {Yu, G.-L . and Blackburn, E . H . (1990) Mol . Cell . Biol., 10, 2070-2080}, origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes . The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold . However, the rRNA gene dosage was unchanged . Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number.

Arch Biochem Biophys, 1995 May 10, 319(1), 149 - 56
Purification, cloning, and sequencing of archaebacterial pyrophosphatase from the extreme thermoacidophile Sulfolobus acidocaldarius; Meyer W et al.; Cytoplasmic pyrophosphatases are indispensible for the function of cellular bioenergetics . From the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius, situated at one of the lowest branches of the phylogenetic tree, a cytosolic pyrophosphatase has been isolated and purified 200-fold to electrophoretic homogeneity by combining ion-exchange and gel-exclusion chromatography . The native enzyme consists of a homotetramer of 71 kDa apparent molecular mass; the subunit displays an apparent molecular mass of 17 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme has an absolute requirement for divalent cations (Mg2+) and a temperature optimum of 75 degrees C coinciding with the growth optimum of the organism; the apparent estimated activation energy is 79.5 kJ/mol . A large variety of cytosolic extracts from other archaebacteria has been probed with a polyclonal antiserum raised against the purified protein; surprisingly, except for an extremely weak signal with S . solfataricus none of the other organisms showed any cross-reactivity . Also, Escherichia coli PPase does not cross-react . Based on N-terminal sequencing the gene has been cloned and sequenced . It codes for a 173-amino-acid protein with a calculated molecular mass of 19,365 kDa . Alignment with known eucaryotic and procaryotic PPases reveals invariant conservation of all residues presently assumed to be involved in metal and substrate binding . Unexpectedly, the highest similarity is found with the enzyme from the phylogenetically extremely distant eubacterium E . coli, but immunological cross-reactivity is absent . Similarity to the only known other archaebacterial PPase is much weaker . Using the 3D structure of the Thermus thermophilus enzyme as a scaffold an energy-minimized structural model is presented, deviating only minimally from the former . The structural features are discussed . The enzyme provides an excellent model for studies of thermostability and folding dynamics since heterologous overexpression has been achieved and genetically mutated forms become accessible.

J Biol Chem, 1995 May 5, 270(18), 10612 - 7
Thermophilic bacilli have split cytochrome b genes for cytochrome b6 and subunit IV . First cloning of cytochrome b from a gram-positive bacterium (Bacillus stearothermophilus); Sone N et al.; The genes of Bacillus stearothermophilus K1041 encoding cytochrome b(6) (Bacillus cytochrome b is referred to as cytochrome b(6) for its resemblance to plastid b6) and subunit IV of the quinol:cytochrome c oxidoreductase (bc1 complex) were cloned and sequenced . For preparation of the probe for cloning, polymerase chain reaction was carried out using oligonucleotide mixtures targeting for N-terminal regions of cytochrome bc and subunit IV of the thermophilic Bacillus PS3 . The deduced amino acid sequences contained 224 residues of 25,425 daltons for cytochrome b(6) and 173 residues of 19,371 daltons for subunit IV, and both open reading frames were separated by 67 base pairs . Cytochrome b and subunit IV contained 4 and 3 hydrophobic transmembrane segments, respectively, indicating that the fourth segment of subunit IV (eighth segment of cytochrome b) is lacking . Four histidine residues supposed to ligand two protohemes were conserved, but the two His in the fourth segment were separated by 14 amino acid residues like cytochrome b6, not like mitochondrial cytochrome b . The residues that might have conferred the two quinol-binding sites were mostly conserved, but especially the third His residue in the fourth segment of mitochondrial cytochrome b was replaced by Arg in Bacillus cytochrome b6 as in cytochrome b6 . These characteristics and quantitative comparison of the protein sequences indicate that this Bacillus sequence is unique and meanwhile rather close to the cyanobacteria-plastids type than the purple bacteria-mitochondria type.

Br Vet J, 1996 May, 152(3), 283 - 306
The pathogenesis of chronic obstructive pulmonary disease of horses; Robinson NE et al.; Present evidence suggests that chronic obstructive pulmonary disease (COPD) of horses is a delayed hypersensitivity response to inhaled antigens, particularly the thermophilic moulds and actinomycetes that grow in damp hay . Within several hours of exposing COPD-susceptible horses to such hay, neutrophils invade the lung and accumulate in the lumens of airways, particularly bronchioles . The inflammatory response is accompanied by increased levels of histamine in bronchoalveolar lavage fluid, increased plasma levels of the inflammatory mediators thromboxane and 15-hydroxyeicosatetraenoic acid (15-HETE), and a decrease in the production of prostaglandin (PG) E2 by the airway mucosa . During acute exacerbations of COPD, airways exhibit nonspecific hyperresponsiveness and become obstructed as a result of bronchospasm and the accumulation of mucus and exudates . Bronchospasm is due largely to activation of smooth muscle muscarinic receptors by acetylcholine (ACh) . Because the in vitro response of smooth muscle to ACh is unaltered, the increase in airway smooth muscle tone is probably a result of activation of airway reflexes by inflammatory mediators and decreases in inhibitory mechanisms such as the intrapulmonary nonadrenergic noncholinergic nervous system and the production of PGE2 in affected horses . The diffuse airway obstruction leads to uneven distribution of ventilation, ventilation/perfusion mismatching, and hypoxaemia . As a result of the increased respiratory drive caused by hypoxaemia and the presence of airway obstruction, horses adopt a characteristic breathing strategy in which very high peak flows at the start of exhalation rapidly diminish as exhalation proceeds.

Vet Med (Praha), 1995 May, 40(5), 157 - 62
Devitalization of bacterial and parasitic germs in sewage sludge during aerobic digestion under laboratory conditions; Juris P et al.; The survival of 8 bacterial species (Pseudomonas sp., Salmonella sp., Enterobacteriae, Streptococcus sp., Escherichia coli) was detected in municipal sewage sludge up to 37 hours of mesophilic aerobic digestion under laboratory conditions . The model strain Enterococcus faecium CCM 4231 survived almost twice as long as the above-mentioned isolates . Similar findings, regarding the viability of the microorganisms studied, were also determined during thermophilic aerobic digestion of municipal sewage sludges . The final reduction in the total count of bacteria was not directly dependent on the temperature during aerobic digestion . It may be supposed that E . faecium CCM 4231 strain as a bacteriocin-producing strain with a broad antimicrobial spectrum, inoculated into the sludges, could inhibit the growth of microorganisms in the sludges by the way of its bacteriocin activity . Studying the effect of aerobic digestion on the viability of helminth eggs, the observed negative effect of higher temperatures was more expressive in comparison with bacterial strains . During thermophilic digestion process all helminth eggs (Ascaris suum, Toxocara canis) were devitalized . All eggs of T . canis were killed in experiments under mesophilic temperature . However, 32% of nonembryonated A . suum eggs remained viable.

Biosci Biotechnol Biochem, 1995 May, 59(5), 917 - 9
Purification and characterization of membrane-bound hydrogenase from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1; Ono T et al.; A membrane-bound hydrogenase was purified aerobically by one step using a hydroxyapatite column after solubilization by acetone treatment from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1 . The enzyme consists of two polypeptides of 63 and 31 kDa, respectively . The amino-terminal amino acid sequences of both subunits were homologous to membrane-bound type {Ni-Fe} hydrogenases from other origins . The thermostability under a hydrogen gas atmosphere is highly stable at 50 degrees C, which is the optimum temperature for the cell growth.

Eur J Biochem, 1995 May 1, 229(3), 688 - 95
Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis; Britton KL et al.; In the light of the solution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from the mesophile Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequences for the thermophilic glutamate dehydrogenases from Thermococcus litoralis and Pyrococcus furiosus against the sequence and the molecular structure of the glutamate dehydrogenase from C . symbiosum, to provide insights into the molecular basis of their thermostability . This homology-based modelling is simplified by the relatively small number of amino acid substitutions between the two thermophilic glutamate dehydrogenase sequences . The most frequent amino acid exchanges involve substitutions which increase the hydrophobicity and sidechain branching in the more thermostable enzyme; particularly common is the substitution of valine to isoleucine . Examination of the sequence differences suggests that enhanced packing within the buried core of the protein plays an important role in maintaining stability at extreme temperatures . One hot spot for the accumulation of exchanges lies close to a region of the molecule involved in its conformational flexibility and these changes may modulate the dynamics of this enzyme and thereby contribute to increased stability.

Eur J Biochem, 1995 May 1, 229(3), 596 - 604
Site-directed mutagenesis of Thermus thermophilus elongation factor Tu . Replacement of His85, Asp81 and Arg300; Zeidler W et al.; His85 in Thermus thermophilus elongation factor Tu (EF-Tu) was replaced by glutamine, leucine and glycine residues, leading to {H85Q}EF-Tu, {H85L} EF-Tu and {H85G}EF-Tu, respectively . Asp81 was replaced by alanine leading to {D81A}EF-Tu, and replacement of Arg300 provided {R300I}EF-Tu . Glycine in position 85 of domain I induces a protease-sensitive site in domain II and causes complete protein degradation in vivo . A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine . Degradation is probably due to disturbed interactions between the domains of EF-Tu.GTP, inducing a protease-sensitive cleavage site in domain II . {H85Q}EF-Tu, which can be effectively overproduced in Escherichia coli, is slower in poly(U)-dependent poly(Phe) synthesis, has lower affinity to aminoacyl-tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein . The GTPase of this protein variant is not efficiently stimulated by aminoacyl-tRNA and ribosomes . The slow GTPase of {H85Q}EF-Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)-dependent ribosomal translation . Replacement of His85 in T . thermophilus EF-Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl-tRNA binding . {H85L}EF-Tu is inactive in poly(U)-dependent poly(Phe)-synthesis . The rate of nucleotide dissociation is highest for {H85L}EF-Tu, followed by {H85Q}EF-Tu and native T . thermophilus EF-Tu . Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF-Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.

FEMS Microbiol Lett, 1995 May 1, 128(2), 127 - 34
Screening and analysis of DNA fragments that show promoter activities in Thermus thermophilus; Maseda H et al.; We have constructed a Thermus promoter probe vector pPP11 containing the promoterless heat-stable kanamycin resistance (Kmr) gene . Total DNA of T . thermophilus HB27 was randomly sheared into 100-200 bp fragments and inserted into the EcoRI site of pPP11 . About 1000 clones which showed Kmr were obtained, and they were classified into three groups according to their Kmr levels . The DNA sequences of the inserted fragments of fifteen clones (Kmr levels were high for seven, medium for three and low for five clones) were determined . Kanamycin nucleotidyl transferase (KNTase) activities of the cell-free extracts of the clones were measured . Transcriptional starting points were determined for ten clones which showed a high or medium level of Kmr . Amounts of Kmr mRNA were also estimated for the ten clones . Amounts of Kmr mRNA synthesized in the clones were in good agreement, and KNTase activities were in fairly good agreement with the Kmr levels of the clones, respectively . From these results, we propose a possible consensus promoter sequence for T . thermophilus.

Biochem J, 1995 May 1, 307 ( Pt 3), 783 - 9
A pepstatin-insensitive aspartic proteinase from a thermophilic Bacillus sp; Toogood HS et al.; Bacillus sp . strain Wp22.A1 produced a cell-associated aspartic proteinase which was purified to homogeneity using phenyl-Sepharose (hydrophobic and affinity chromatography) and Mono Q . The proteinase has a molecular mass of 45 kDa by SDS/PAGE and a pI of 3.8 . It is insensitive to pepstatin, but is sensitive to the other aspartic proteinase-specific inhibitors diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)propane . Inactivation by DAN was only partial, suggesting that it had non-specifically modified an aspartate residue at a site other than the active site . The enzyme was not inhibited by any of the serine or cysteine proteinase inhibitors tested . Maximum proteolytic activity was observed at pH 3.5 . The proteinase had a higher activity with haemoglobin, but was more specific (Vmax./Km) for cytochrome c . Substrate inhibition was observed with both these substrates . The cleavage of oxidized insulin B chain tended to occur at sites where the P1 amino acid was bulky and non-polar, and the P1' amino acid was bulky and polar, such as its primary cleavage site of Val2-Asn3 . The proteinase was stable in the pH range 2.5-5.5 . Thermostability was increased in the presence of Ca2+, although to a lesser extent at higher temperatures . The thermostabilities at 60, 70, 80 and 90 degrees C were 45 h, 102, 21 and 3 min respectively in the presence of Ca2+.

J Bacteriol, 1995 May, 177(9), 2583 - 8
Genetic transfer by conjugation in the thermophilic green sulfur bacterium Chlorobium tepidum; Wahlund TM et al.; The broad-host-range IncQ group plasmids pDSK519 and pGSS33 were transferred by conjugation from Escherichia coli into the thermophilic green sulfur bacterium Chlorobium tepidum . C . tepidum exconjugants expressed the kanamycin and ampicillin-chloramphenicol resistances encoded by pDSK519 and pGSS33, respectively . Ampicillin resistance was a particularly good marker for selection in C . tepidum . Both pDSK519 and pGSS33 were stably maintained in C . tepidum at temperatures below 42 degrees C and could be transferred between C . tepidum and E . coli without modifications . Conjugation frequencies ranged from 10(-1) to 10(-4) exconjugants per donor cell, and frequencies of 10(-2) to 10(-3) were consistently obtained when ampicillin resistance was used as a selectable marker . Methods for growth of C . tepidum on agar, isolation of plating strains and antibiotic-resistant mutants of wild-type C . tepidum cells, and optimum conditions for conjugation were also investigated.

J Bacteriol, 1995 May, 177(9), 2576 - 82
Sulredoxin: a novel iron-sulfur protein of the thermoacidophilic archaeon Sulfolobus sp . strain 7 with a Rieske-type {2Fe-2S} center; Iwasaki T et al.; A novel pink {2Fe-2S} protein has been purified from the cytosol fraction of the thermoacidophilic archaeon Sulfolobus sp . strain 7 (originally named Sulfolobus acidocaldarius 7) and called "sulredoxin." Its absorption, circular dichroism, and electron paramagnetic resonance spectra suggest the presence of a Rieske-type {2Fe-2S} cluster (g-factors of 2.01, 1.91, and 1.79; average g-factor {gav} = 1.90) which is remarkably similar to that of Thermus thermophilus respiratory Rieske FeS protein (J . A . Fee, K . L . Findling, T . Yoshida, R . Hille, G . E . Tarr, D . O . Hearshen, W . R . Dunham, E . P . Day, T . A . Kent, and E . Munck, J . Biol . Chem . 259:124-133, 1984) and distinctively different from those of the plant-type ferredoxins (gav = 1.96) . Sulredoxin, which is the first Rieske-type {2Fe-2S} protein isolated from an archaeal species, does not function as an electron acceptor of the cognate 2-oxoacid:ferredoxin oxidoreductase . Whether sulredoxin is derived from the archaeal membrane-bound respiratory Rieske-type FeS center (gy = 1.91) is the subject of further investigation.

Mikrobiologiia, 1995 May-Jun, 64(3), 366 - 74
{Determination of the phylogenetic position of Sulfobacillus thermosulfidooxidans on the basis of analysis of the 5S and 16S ribosomal RNA}; Turova TP et al.; The nucleotide sequence of the 16S rRNA gene of the type strain Sulfobacillus thermosulfidooxidans VKM-1269 was determined . This microorganism belongs to the group of thermophilic acidophilic chemolithotrophic bacteria capable of utilizing elemental sulfur and iron ions as the sole energy sources . The phylogenetic position of the bacterium studied among Gram-positive bacteria was estimated by a comparative analysis of 16S rRNA nucleotide sequences . It was shown that Sulfobacillus forms a coherent group with species of a new genus Alicyclobacillus . The Sulfobacillus-Alicyclobacillus group is suggested to represent a separate evolutionary branch within the clostridial subdivision of Gram-positive bacteria.

Appl Environ Microbiol, 1995 May, 61(5), 1867 - 75
xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana; Vieille C et al.; The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli . The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892 . The native enzyme was a homotetramer with a molecular weight of 200,000 . This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus) . The enzyme was extremely thermostable, with optimal activity above 95 degrees C . The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range . The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km . The T . neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases . Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved . Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.

J Dairy Sci, 1995 May, 78(5), 989 - 97
Cloning and characterization of the galactokinase gene from Streptococcus thermophilus; Mustapha A et al.; The objective of this research was to clone and characterize the galactokinase gene (galK) from Streptococcus thermophilus F410 . Partially digested genomic DNA was cloned into pBR322 and transformed into galK Escherichia coli, and a galactose-fermenting transformant was isolated . Restriction analysis revealed that the transformant resulted from a Sau3A-HindIII 4.0-kb fragment . Galactokinase activity in the recombinant was 10 times that of the parent strain . Analysis of the DNA sequence showed the presence of a 1.3-kb open reading frame that had high homology with the galK gene from other organisms . A putative ribosome-binding site, start and stop codons, and -10 and -35 sequences were identified . The predicted protein had a molecular mass of 49 kDa, which corresponded to the estimated size of a band apparent by SDS-PAGE . Amino acid sequence homologies with other galactokinases ranged from 50 to 62% similarity . Northern blots were performed between the galK gene and mRNA from S . thermophilus . No hybridization signals were observed for cells grown in glucose, but cells grown in lactose or galactose gave moderate and strong signals . The results suggest that repression of the galK gene by glucose may be responsible for the galactose-releasing phenotype in these strains.

Appl Microbiol Biotechnol, 1995 May-Jun, 43(2), 330 - 5
Characterization of a bacteriocin produced by Streptococcus thermophilus ST134; Ward DJ et al.; A pH-dependent adsorption/desorption technique was used to screen Streptococcus thermophilus strains for the production of bacteriocins . Agar-diffusion tests with S . thermophilus strains as targets identified 13 out of 41 strains as producers of antibacterial activity . Thermophilin A, the bacteriocin-like substance present in the culture supernatant of S . thermophilus ST134 was purified to homogeneity by ammonium sulfate precipitation and ion-exchange chromatography, followed by ultrafiltration . Thermophilin A is a relatively heat-stable and apparently glycosylated bacteriocin with a bactericidal mode of action against sensitive cells.

Appl Microbiol Biotechnol, 1995 May-Jun, 43(2), 291 - 6
celA, another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum; Te'o VS et al.; Caldocellum saccharolyticum is an extremely thermophilic anaerobic bacterium capable of growth on cellulose and hemicellulose as sole carbon sources . Cellulase and hemicellulase genes have been found clustered together on its genome . The gene for one of the cellulases (celA) was isolated on a lambda genomic library clone, sequenced and found to comprise a large open-reading frame of 5253 base pairs that could be translated into a peptide of 1751 amino acids . To date, it is the largest cellulase gene sequenced . The translated product is a multidomain structure composed of two catalytic domains and two cellulose-binding domains linked by proline-threonine-rich regions (PT linkers) . The N-terminal domain of celA encodes for an endoglucanase activity on carboxymethylcellulose, consistent with its high homology to the sequences of several other endo-1, 4-beta-D-glucanases . The carboxyterminal domain shows sequence homology with a cellulase from Clostridium thermocellum (CelS), which is known to act synergistically with a second component to hydrolyze crystalline cellulose . In the absence of a Caldocellum homologue for this second protein, we can detect no activity from this domain.

Appl Microbiol Biotechnol, 1995 May-Jun, 43(2), 285 - 90
Expression of cho and melC operons by a Streptococcus thermophilus synthetic promoter in Escherichia coli; Solaiman DK et al.; A 63-base-pair synthetic promoter, sP1, was synthesized on the basis of the nucleotide sequence of a putative Streptococcus thermophilus promoter . When inserted upstream from the Streptomyces cho operon in a recombinant plasmid, pUCO195P-36, sP1 activated the expression of the cho genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants . The sP1-driven cholesterol oxidase production in pUCO195P-36-transformed cells was estimated to be 40% of that produced by P(lac)-mediated cho expression in a pUCO193-containing host . The recombinant pUCO195P-36 appeared to be segregationally less stable in E . coli DH5 alpha than in HB101 . Its non-expressing counterpart, pUCO195P-1, was stable in both E . coli strains . The activity of sP1 was further demonstrated in E . coli by the expression of a Streptomyces melC operon . When placed upstream from the test operon in the pMCU22aPa construct, sP1 activated the melC expression as shown by the production of tyrosinase at (3.0 +/- 0.3) x 10(-3) U/mg and (16.0 +/- 1.0) x 10(-3) U/mg protein equivalent of cell extract in the absence and presence of isopropyl beta-D-thiogalactopyranoside, respectively . The presence of a counter-oriented P(lac) at the 3' end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.

Nippon Rinsho, 1995 May, 53(5), 1081 - 6
{Molecular mechanisms of the expression of cytosolic and mitochondrial isozyme genes}; Setoyama C; We isolated the mouse cytosolic and mitochondrial malate dehydrogenase (cMDH and mMDH) and the mouse cytosolic and mitochondrial aminotransferase (cAspAT and mAspAT) genes functioning in the malate-aspartate shuttle, and localized the DNA regions required for the promoter activity of these isozyme genes . We also characterized nuclear proteins binding to the promoter regions, and found that a transcription factor, CTF/NFI may contribute to the regulation of cMDH, mMDH and cAspAT levels, and that another transcription factor, Sp1 is probably linked to the regulation of mAspAT level . Comparison of the amino acid sequences among the mammalian and bacterial MDHs revealed that the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and E . coli MDH, markedly exceeds the intraspecies sequence homology between cMDH and mMDH from mice . Moreover, structural organizations of the two-pairs of isozyme genes indicated that introns antedate the divergence of these cytosolic and mitochondrial isozyme genes.

Mol Microbiol, 1995 May, 16(3), 575 - 86
Characterization of the pilF-pilD pilus-assembly locus of Neisseria gonorrhoeae; Freitag NE et al.; Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host . Several N . gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems . We report here the isolation and characterization of transposon insertion mutants in N . gonorrhoeae whose phenotypes indicate that the N . gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis . Mutants lacking the pilD gene product, a pre-pilin peptidase, were unable to process the pre-pilin subunit into pilin and thus were non-piliated . pilF mutants processed pilin but did not assemble the mature subunit . Both classes of mutants released S-pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly . In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype . Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression . orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide-binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans . Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili . Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.

Biochim Biophys Acta, 1995 Apr 26, 1229(2), 225 - 32
F1-ATPase alpha-subunit made up from two fragments (1-395, 396-503) is stabilized by ATP and complexes containing it obey altered kinetics; Miyauchi M et al.; Inferred from the crystal structure of mitochondrial F1-ATPase (Abrahams, J.P . et al . (1994) Nature 370, 621-628), the proteinase-sensitive region around Phe-395 of thermophilic F1-ATPase alpha-subunit corresponds to the loop which connects main part of the carboxyl-terminal helical bundle domain with the ATP binding domain . This loop is in contact with the gamma- and adjacent beta-subunits . Two polypeptides corresponding to the sequence 1-395 and 396-503 of the alpha-subunit were expressed in Escherichia coli cells and they were copurified as an apparently functional alpha-subunit (alpha(395/396)) made up of two polypeptides . The isolated alpha(395/396) was stabilized by ATP-Mg, but not by ADP-Mg, although it bound both ATP-Mg and ADP-Mg with similar affinities (Kd, 11 microM and 14 microM, respectively) . The alpha(395/396) was reconstitutable into alpha(395/396)3 beta 3 and alpha(395/396)3 beta 3 gamma complexes . Different from the intact the ATP-Mg-induced dissociation into alpha 1 beta 1 heterodimers . ATP hydrolysis by the alpha(395/396)3 beta 3 gamma complex underwent a slow initial phase, whereas the intact alpha 3 beta 3 gamma complex exhibited an accelerated initial phase . Steady-state ATPase activity at various ATP concentrations showed negative cooperativity for the intact alpha 3 beta 3 gamma complex but apparently positive cooperativity for the alpha(395/396)3 beta 3 gamma complex . The ATPase activities at a saturating ATP concentration of the complexes containing the alpha(395/396) were 180% of those containing intact alpha-subunits . These results indicate that a loop around Phe-395 is involved in intersubunit interaction in F1-ATPase.

Biochemistry, 1995 Apr 18, 34(15), 4994 - 5002
Properties of the 5'-->3' exonuclease/ribonuclease H activity of Thermus thermophilus DNA polymerase; Auer T et al.; The recombinant 94 kDa Thermus thermophilus DNA polymerase (rTth pol) was found to release {33P}UMP when incubated with a RNA.DNA hybrid containing a {33P}UMP-labeled RNA strand . The RNase H activity was optimally active in the presence of low monovalent salt concentrations and when Mn2+ was used as the divalent cation activator . RNase H activity also was observed when Mg2+ replaced the Mn2+, but to a much lesser extent . A 60 nucleotide long, 5'- or 3'-radiolabeled RNA or DNA oligomer hybridized to a complementary DNA oligomer was used to determine the mode of digestion . The radiolabeled RNA.DNA hybrid or DNA.DNA duplex was incubated with rTth pol using various metal ion conditions and different incubation times . The DNA.DNA duplex showed very little enzymatic cleavage by rTth pol regardless of the Mn2+ or Mg2+ concentration . However, nearly complete digestion of the RNA.DNA hybrid was observed over a wide Mn2+ concentration range, thus demonstrating a preferential degradation of the RNA.DNA hybrid vs the DNA.DNA duplex . Time course reactions of the enzymatic digestion of the 3'-labeled RNA.DNA hybrid or DNA.DNA duplex by rTth pol indicated that digestion of the substrates occurred exonucleolytically in the 5'-->3' direction.

Structure, 1995 Apr 15, 3(4), 341 - 52
The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase; Belrhali H et al.; BACKGROUND: Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthetase that specifically charges cognate tRNAs with serine . In the first step of this two-step reaction, Mg.ATP and serine react to form the activated intermediate, seryl-adenylate . The serine is subsequently transferred to the 3'-end of the tRNA . In common with most other aminoacyl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap4A) from the enzyme-bound adenylate intermediate and a second molecule of ATP . Understanding the structural basis for the substrate specificity and the catalytic mechanism of aminoacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis in all living cells . RESULTS: Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermophilus are described . The first complex is of the enzyme with ATP and Mn2+ . The ATP is found in an unusual bent conformation, stabilized by interactions with conserved arginines and three manganese ions . The second complex contains seryl-adenylate in the active site, enzymatically produced in the crystal after soaking with ATP, serine and Mn2+ . The third complex is between the enzyme, Ap4A and Mn2+ . All three structures exhibit a common Mn2+ site in which the cation is coordinated by two active-site residues in addition to the alpha-phosphate group from the bound ligands . CONCLUSIONS: Superposition of these structures allows a common reaction mechanism for seryl-adenylate and Ap4A formation to be proposed . The bent conformation of the ATP and the position of the serine are consistent with nucleophilic attack of the serine carboxyl group on the alpha-phosphate by an in-line displacement mechanism leading to the release of the inorganic pyrophosphate . A second ATP molecule can bind with its gamma-phosphate group in the same position as the beta-phosphate of the original ATP . This can attack the seryl-adenylate with the formation of Ap4A by an identical in-line mechanism in the reverse direction . The divalent cation is essential for both reactions and may be directly involved in stabilizing the transition state.

J Biol Chem, 1995 Apr 14, 270(15), 8893 - 901
Gel shift and UV cross-linking analysis of Tetrahymena telomerase; Harrington L et al.; Telomerase is an unusual ribonucleoprotein that synthesizes new telomeres onto chromosome ends . The enzyme has been most extensively characterized in ciliates, where the RNA component has been cloned from several species, and its elongation properties have been characterized in detail . To understand the substrate specificity and protein composition of telomerase, we have used gel shift and UV cross-linking to characterize the enzyme from the ciliate Tetrahymena thermophila . In a mobility shift assay, a complex was identified that contained telomerase RNA, co-purified with telomerase activity, and was sensitive to nuclease treatment . The mobility shift complexes specifically formed using several different single-stranded, telomeric sequences but not non-telomeric primers . These results suggest that the specificity of telomerase for G-rich primer sequences occurs at least in part at the level of primer binding . UV cross-linking analysis identified a 100-kDa cross-linked protein that may be a telomerase component.

Biochemistry, 1995 Apr 11, 34(14), 4610 - 6
Visualization of nucleosomal substructure in native chromatin by atomic force microscopy; Martin LD et al.; Intact rDNA minichromosomes from Tetrahymena thermophila were isolated as native chromatin and imaged by atomic force microscopy (AFM) . AFM measurements of condensed rDNA chromatin were consistent with a 30 nm fiber that frequently (87% of molecules observed) contained stretches of nucleosome cores arranged in a zig-zag conformation . Examination of rDNA chromatin in a dispersed conformation by tapping mode AFM in low humidity resulted in high resolution images of partially dissociated nucleosome cores and associated linker DNA . A majority of these nucleosome cores contained six to eight smaller particles with dimensions consistent with those of individual histones . Many of the nucleosome cores showed a striking resemblance to the wedge (35%), axial (15%), and front (6%) views of the nucleosome histone octamer modeled by Arents et al . {Arents, G., Burlingame, R . W., Wang, B.-C., Love, W.E., & Moudrianakis, E . N . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 10148-10152} . This direct visualization of histone subunits and nucleosomal substructure in native chromatin illustrates the potential use of AFM to localize individual proteins in condensed cellular chromatin.

Biochemistry, 1995 Apr 11, 34(14), 4583 - 92
A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA; Hou Z et al.; Type I repeat sequences are evolutionarily conserved sequence elements found in the replication origin and transcriptional promoter region of the rRNA genes (rDNA) in Tetrahymena thermophila . An abundant single-stranded DNA binding protein, ssA-TIBF, specifically interacts with the A-rich strand of the Type I repeat sequence . Quantitative binding competition experiments performed with purified ssA-TIBF demonstrate that the binding site for ssA-TIBF includes sequences both within the conserved 33 nt element and in a 3' flanking region: addition of the 3' flanking sequence to the Type I repeat oligonucleotide increases the binding affinity of ssA-TIBF by nearly 100-fold (apparent Kd = 3.0 x 10(-10) M) . A mutation in the ssA-TIBF binding site previously shown to be the determinant of an rDNA replication defect in vivo results in a 25-fold decrease in ssA-TIBF binding affinity in vitro . ssA-TIBF also binds with high affinity to a copy of the Type I repeat sequence within the essential promoter region defined by in vitro transcription assays . The affinity of ssA-TIBF for the promoter repeat, which differs from other copies of the repeat at 8 out of 33 positions, is at least equal to its affinity for the Type I repeat sequences in the origin region . The biochemical properties of ssA-TIBF in vitro suggest that it could play a role in both replication and transcription of Tetrahymena rDNA in vivo.

J Chromatogr B Biomed Appl, 1995 Apr 7, 666(1), 188 - 93
Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms; Gey MH et al.; Two thermostable enzymes synthesized by thermophilic microorganisms were isolated and purified . A thermostable beta-galactosidase was produced in a continuous fermentation process by Bacillus stearothermophilus TP 32 as an intracellular enzyme . After applying different concentration procedures the raw extract enzyme was prepurified on a Sephadex G-200 size exclusion column . The isolated beta-galactosidase fraction was then separated with HPLC on a TSK G 3000 SW size exclusion column to determine the molecular mass based on calibration curves of standard proteins . The other enzyme, a thermostable protease, was synthesized by Bacillus stearothermophilus TP 26 as an extracellular enzyme . After its concentration, the enzyme was purified on a classical size exclusion column (Sephacryl S-200) and on a HPLC size exclusion column (BIO-SIL TSK-250) . The micropreparatively isolated fraction was separated again on this HPLC column to determine its molecular mass . The optimum temperature of both enzymes was approximately 75 degrees C.

FEBS Lett, 1995 Apr 3, 362(2), 121 - 5
Equatorial split of holo-chaperonin from Thermus thermophilus by ATP and K+; Ishii N et al.; Holo-chaperonin molecule from Thermus thermophilus is a bullet-shaped particle whose cylinder part and round top are composed of two stacked rings of the cpn60 heptamer and a single ring of the cpn10 heptamer, respectively . We found that it splits at the plane between two cpn60 rings into two halves under physiological conditions, that is, in the presence of ATP (but not AMP-PNP, ADP) + K+ (but not Na+) at 60 degrees C . This equatorial split could be functionally important although it has not been considered in any current mechanistic model of chaperonin functioning.

J Bacteriol, 1995 Apr, 177(7), 1699 - 702
Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila; Singh-Wissmann K et al.; Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate in Methanosarcina thermophila . The genes encoding these enzymes (pta and ack) have been cloned and sequenced . They are arranged on the chromosome with pta upstream of ack (M.T . Latimer, and J . G . Ferry, J . Bacteriol . 175:6822-6829, 1993) . The activities of phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine . Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level . Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence . S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats . Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA . A primer extension product was detected with an ack-specific primer; the 5' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence . This result, as well as detection of an additional 1.4-kb mRNA species, suggests processing of the polycistronic 2.4-kb transcript.

Mutat Res, 1995 Apr, 334(2), 213 - 24
Antimutagenicity of an acetone extract of yogurt; Nadathur SR et al.; Reconstituted non-fat dry milk powder, fermented by a mixture of Streptococcus thermophilus CH3 and Lactobacillus bulgaricus 191R to produce yogurt, was freeze-dried and extracted in acetone . After evaporation of the acetone, the extract was dissolved in dimethyl sulfoxide (DMSO) and tested for antimutagenicity . In the Ames test, significant dose-dependent activity was observed against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-quinoline-N-oxide (4NQO), 3,2'-dimethyl-4-aminobiphenyl (DMAB), 9,10-dimethyl-1,2-benz{a}anthracene (DMBA), and 3-amino-1-methyl-5H-pyrido{4,3-b}indole acetate (Trp-P-2) . Weak activity was observed against 1,2,7,8-diepoxyoctane (DEO), and no activity was observed against methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), or aflatoxin B1 (AFB1) . In a related assay (Saccharomyces cerevisiae D7), significant antimutagenic activity was detected against MNNG and 4NQO . Activity against the experimental colon carcinogens MNNG and DMAB was examined further, as assayed in the Ames test (Salmonella typhimurium TA100) . Compounds responsible for both activities were less soluble in aqueous solutions than in DMSO . Adjustment of yogurt pH to 3, 7.6, or 13 prior to freeze-drying and acetone extraction did not significantly alter the amount of anti-MNNG activity recovered . In contrast, extractability of anti-DMAB activity was significantly greater at acidic pH . Conjugated linoleic acid, a known dairy anticarcinogen, failed to inhibit mutagenesis caused by either mutagen, suggesting that other yogurt-derived compound(s) are responsible . Unfermented milk was treated with lactic acid, yogurt bacteria without subsequent growth, or both, to determine if formation of antimutagenic activity required bacterial growth . Extracts of the milk treatments exhibited the same weak antimutagenicity observed in unfermented milk, approximately 2.5-fold less than in the yogurt extracts, suggesting that antimutagenic activity is associated with bacterial growth.

Appl Microbiol Biotechnol, 1995 Apr, 43(1), 1 - 6
The lysins of bacteriophages infecting lactic acid bacteria; Sable S et al.; This short review highlights the complete absence of literature on lysins of bacteriophages infecting species like S . salivarius subsp . thermophilus, Pediococcus and Leuconostoc species, L . helveticus, L . acidophilus, L . plantarum and L . brevis, which are also widely used in the dairy industry . The lysins described share some similar biochemical characteristics: optimal pH and temperature, site of hydrolysis inside the peptidoglycan, and some activators and inhibitors . The cloning of the genes encoding these lysins only began in the last few years and four of them have been completely sequenced . In the future, these lysin genes could be interestingly compared to the host autolysin(s) gene(s) . By contrast, the passage of phage lysins through the cytoplasmic membrane of the host cell in order to reach the peptidoglycan (via a signal sequence or the presence of a holin) seems not to be clearly resolved . The presence of a second open-reading frame upstream from the gene of the lysin, enabling a putative holin to be encoded, has already been suggested . No doubt our ever increasing knowledge about bacteriophage genome organization will help to elucidate this question . Meanwhile the obtention of a Lactococcus strain with an autolytic phenotype, using a bacteriophage lysin gene, as well as the successful use of purified PL1 lysin to obtain protoplasts of L . casei encourage us to continue to explore the field of bacteriophage lysins.

Appl Environ Microbiol, 1995 Apr, 61(4), 1497 - 501
Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus; Fischer L et al.; A new inducible intracellular beta-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography . The first 22 amino acid residues were determined by N-terminal sequencing . Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5 . The native beta-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique . The beta-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9 . It was completely stable at pH 6.8 and 47 degrees C for 2 h . After 191 h at 50 degrees C, the remaining beta-galactosidase activity of an enzyme fraction after salt precipitation was 58% . The beta-galactosidase hydrolyzed p- and o-NO2-phenyl-beta-D-galactopyranoside, lactose, lactulose, MeOH-beta-D-galactopyranoside, phenyl-beta-D-galactopyranoside, and p-NO2-phenyl-alpha-L-arabinopyranoside . The kinetic constants (Km) measured for p- and o-NO2-phenyl-beta-D-galactopyranoside and beta-lactose were 4.8, 11.3, and 18.2 mM, respectively.

Appl Environ Microbiol, 1995 Apr, 61(4), 1252 - 6
Characterization of the Bacillus stearothermophilus BR219 phenol hydroxylase gene; Kim IC et al.; The catabolic genes pheA and pheB, coding for the conversion of phenol to catechol and catechol to 2-hydroxymuconic semialdehyde, respectively, have been cloned from Bacillus stearothermophilus BR219 into Escherichia coli . Following its localization on the 11-kb B . stearothermophilus DNA insert by deletion and expression analysis, the phenol hydroxylase gene pheA was subcloned as a 2-kb HindIII fragment, whose transformant expressed the enzyme after phenol induction and even more strongly after o-, m-, and p-cresol induction . In vitro transcription-translation experiments indicated that the phenol hydroxylase and catechol 2,3-dioxygenase enzymes are constituted of single subunits with molecular weights of 44,000 and 33,000, respectively . Nucleotide sequencing of the pheA gene revealed a strong similarity to flavin hydroxylases from Rhodococcus and Streptomyces species . Hybridization experiments indicated that the fragment containing PheA and PheB is located on a 66-kb plasmid in the parental thermophile.

Int J Syst Bacteriol, 1995 Apr, 45(2), 218 - 21
Isolation and characterization of a thermophilic sulfate-reducing bacterium, Desulfotomaculum thermosapovorans sp . nov; Fardeau ML et al.; Strain MLFT (T = type strain), a new thermophilic, spore-forming sulfate-reducing bacterium, was characterized and was found to be phenotypically, genotypically, and phylogenetically related to the genus Desulfotomaculum . This organism was isolated from a butyrate enrichment culture that had been inoculated with a mixed compost containing rice hulls and peanut shells . The optimum temperature for growth was 50 degrees C . The G+C content of the DNA was 51.2 mol% . Strain MLFT incompletely oxidized pyruvate, butyrate, and butanol to acetate and presumably CO2 . It used long-chain fatty acids and propanediols . We observed phenotypic and phylogenetic differences between strain MLFT and other thermophilic Desulfotomaculum species that also oxidize long-chain fatty acids . On the basis of our results, we propose that strain MLFT is a member of a new species, Desulfotomaculum thermosapovorans.

J Bacteriol, 1995 Apr, 177(8), 2164 - 77
The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases; Uemori T et al.; We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum . The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases . Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized . Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin . Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities . In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak . A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C . However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus . In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A . These findings suggest that both enzymes from P . occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.

Biochem J, 1995 Apr 1, 307 ( Pt 1), 151 - 8
Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria; Fontes CM et al.; A genomic library of Clostridium thermocellum DNA constructed in lambda ZAPII was screened for xylanase-expressing clones . Cross-hybridization experiments revealed a new xylanase gene isolated from the gene library, which was designated xyn Y . The encoded enzyme, xylanase Y (XYLY), displayed features characteristic of an endo-beta1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and was active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates . The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinant XYLY, expressed by Escherichia coli had a maximum Mr of 116000 . The nucleotide sequence of xyn Y contained an open reading frame of 3228 bp encoding a protein of predicted Mr 120 105 . The encoded enzyme contained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity . Downstream of domain A was a 351-residue xylanase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28% sequence identity with a thermostable domain of Thermoanaerobacterium saccharolyticum xylanase A . The C-terminal portion of XYLY comprised the 23-residue duplicated docking sequence found in all other C . thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, followed by a 286-residue domain which exhibited 32% sequence identity with the N-terminal region of C . thermocellum xylanase Z . The enzyme did not contain linker sequences found in other C . thermocellum plant cell wall hydrolases . Analysis of truncated forms of XYLY and hybrid proteins, comprising segments of XYLY fused to the E . coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and an adjacent thermostable domain . The C-terminal domain did not bind to cellulose or xylan . Western blot analysis using antiserum raised against XYLY showed that the xylanase was located in the cellulosome and did not appear to be extensively glycosylated . The non-catalytic domains of XYLY are discussed in relation to the general stability of thermophilic xylanases.

Mol Microbiol, 1995 Apr, 16(1), 69 - 78
Characterization and distribution of two insertion sequences, IS1191 and iso-IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co-cultures?
Guedon G, Bourgoin F, Pebay M, Roussel Y, Colmin C, Simonet JM, Decaris B.
A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191 . This is the first IS element characterized in this species . This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8 bp . The single large open reading frame of IS1191 encodes a 391-amino-acid protein which displays homologies with transposases encodes by IS1201 from Lactobacillus helveticus (44.5% amino-acid sequence identity) and by the other ISs of the IS256 family . One of the copies of IS1191 is inserted into a truncated iso-IS981 element . The nucleotide sequences of two truncated iso-IS981s from S . thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity . The distribution of these insertion sequences in L . lactis and S . thermophilus strains suggests that intergeneric transfers occur during cocultures used in the manufacture of cheese.

C R Acad Sci III, 1995 Apr, 318(4), 423 - 9
{Demonstration of thermostable enzymes in thermophilic micro-organisms of hydrothermal origin}; Ladrat C et al.; During 3 cruises in the Pacific ocean, hydrothermal samples have been collected and some thermophilic bacteria and archaea have been purified . Four enzymatic activities have been screened on 77 chemo-organoheterotrophic thermophilic microorganisms . Forty-two isolates exhibited intracellular beta-glucosidase activity whereas only 7 (including only one archaeon) showed alcohol dehydrogenase one . Protease activity was not detected on only 6 isolates over 77 . Twenty-seven isolates exhibited esterase activity and 3 different electrophoretic patterns have been revealed . No isolate was found to exhibit the 4 activities . Preliminary characterization of these activities showed high thermophily and thermostability, properties which could be used in potential biotechnological applications.

C R Acad Sci III, 1995 Apr, 318(4), 415 - 22
Thermoreduction, a hypothesis for the origin of prokaryotes; Forterre P; All thermophiles discovered so far are prokaryotes (Bacteria or Archaea) . Furthermore, reconstructions of rRNA phylogenies suggest that the progenitor of all prokaryotes was a thermophile . These data are usually interpreted as supporting the hypothesis that all present day organisms, including eukaryotes, originated from hyperthermophiles . However, this scenario is difficult to reconcile with the RNA world theory, considering the instability of RNA at very high temperatures, and it is also contradicted by the finding of sophisticated devices for thermophilic adaptation in present day hyperthermophiles . Accordingly, I propose here 2 new hypotheses to explain the correlation between the procaryotic phenotype and thermophilic life without reference to a putative hot origin of life . Firstly, eukaryotes would be unable to live in thermophilic biotopes because of the susceptibility of their mRNA to degradation at high temperature . In prokaryotes, the absence of a nuclear membrane allows these organisms to bypass the problem of mRNA heat-induced hydrolysis by coupling transcription and translation . As a corollary of this first hypothesis, I also suggest that today prokaryotes might have originated from mesophilic ancestors via reductive evolution, in the process of adaptation to thermophily.

J Pediatr Gastroenterol Nutr, 1995 Apr, 20(3), 333 - 8
Lactic acid bacteria in the treatment of acute rotavirus gastroenteritis; Majamaa H et al.; We compared different lactic acid bacteria for their effect on the immune response to rotavirus in children with acute rotavirus gastroenteritis . After initial oral rehydration, 49 children aged 6 to 35 months with rotavirus gastroenteritis randomly received either Lactobacillus casei subsp . casei strain GG (LGG), L . casei subsp . rhamnosus (Lactophilus), or a combination of Streptococcus thermophilus and L . delbruckii subsp . bulgaricus (Yalacta) twice daily for 5 days . Serum antibodies to rotavirus, total number of immunoglobulin-secreting cells (ISC), and specific antibody-secreting cells (sASC) to rotavirus were measured at the acute stage and at convalescence . The mean (SD) duration of diarrhea was 1.8 (0.8) days in children who received LGG, 2.8 (1.2) days in those receiving Lactophilus, and 2.6 (1.4) days in those receiving Yalacta (F = 3.3, p = 0.04) . The ISC response was comparable in the three study groups, but the rotavirus-specific immune responses were different . LGG therapy was associated with an enhancement of IgA sASC to rotavirus and serum IgA antibody level at convalescent stage . We conclude that certain strains of lactic acid bacteria, particularly LGG, promote serum and intestinal immune responses to rotavirus, and thus may be important in establishing immunity against rotavirus reinfections.

Inflammation, 1995 Apr, 19(2), 207 - 19
Antioxidant therapy partially blocks immune-induced lung fibrosis; Denis M; A mouse model of hypersensitivity pneumonitis was generated by challenge with a thermophilic actinomycete . Oxygen radical scavengers were administered to challenged mice: vitamin E at 1000 units daily, polyethylene glycol-superoxide dismutase (SOD) at 500 units daily, polyethylene glycol-catalase at 10,000 units daily, 1,3,dimethyl-2-thiourea (DMTU) at 2 mg daily, and the biomimetic SOD, copper(II) {diisopropyl salicylate}2 (CuDIPS) at 1 mg daily . At three weeks after actinomycete challenge, a 10-fold increase in bronchoalveolar (BAL) cell number was observed . Treatments with catalase or DMTU were without effect on the BAL cell number in challenged mice . However, infusion of vitamin E was associated with an increased BAL cell influx (15-fold increase at two and three weeks) . Similarly, treatment with PEG-SOD and CuDIPS resulted in an increase in cell number at two and three weeks . PEG-SOD or CuDIPS treatment resulted in a strong neutrophilia, whereas control challenged mice had a cellular influx mostly of macrophages and lymphocytes . Vitamin E treatment of challenged mice led to an increased T lymphocyte recruitment at two and three weeks . In vitro studies showed that actinomycete challenge was associated with an enhancement of alveolar macrophage O2- release, which was blocked by PEG-SOD, vitamin E, or DSC treatment but was unaffected by catalase or DMTU treatment . In control challenged mice, there was a 25-fold increase in the BAL albumin concentration at two weeks . PEG-SOD, vitamin E, or CuDIPS treatment all decreased the albumin concentration; the three modulators also diminished lung fibrosis at two or three weeks, as seen by a decrease in lung hydroxyproline and collagen synthesis by lung fibroblasts . Examination of sections from lungs of challenged animals showed evidence of cellular infiltrates around the bronchi and the blood vessels . Challenged mice given continuous infusions of vitamin E, SOD, or CuDIPS had lung histological scores that were significantly lower than control challenged mice or challenged mice treated with catalase or DMTU . Thus, therapies based on O2- scavenging or treatment with a general antioxidant such as vitamin E may hold some promise in the treatment of hypersensitivity pneumonitis.

J Biochem (Tokyo), 1995 Apr, 117(4), 691 - 6
Role of leucine 201 of thermostable D-amino acid aminotransferase from a thermophile, Bacillus sp . YM-1; Kishimoto K et al.; We studied the catalytic role of leucine 201 residue of the thermostable D-amino acid aminotransferase: the residue was shown crystallographically to be in the vicinity of the active site to interact with the bound pyridoxal phosphate . We replaced the leucine 201 by alanyl or tryptophanyl residues by means of site-directed mutagenesis . The L201A and L201W mutant enzymes showed anomalous kinetic behavior in the overall reaction . The reaction rates of the L201A and L201W mutant enzymes gradually decreased with an increase in the reaction time to become practically zero at a high concentration of substrates . The mutant enzymes were also inactivated in the half reaction with D-alanine, although more slowly than in the overall reaction . The absorption spectra of the mutant enzymes in the presence of D-alanine and alpha-ketoglutarate suggest that the enzyme molecules were mostly in the pyridoxamine form under the conditions employed . These phenomena were explained by assuming two (or more) enzyme species showing kinetically different catalysis for pyridoxamine form of the mutant enzymes, and the rate of conversion from one of these pyridoxamine forms to the pyridoxal form should be very low . The leucine 201 residue probably regulates the function of cofactor during the reaction of D-amino acid aminotransferase.

Environ Res, 1995 Apr, 69(1), 59 - 66
Chemotaxis of alveolar macrophages and neutrophils in response to microbial products derived from organic dust; Milanowski J et al.; The inhalation of organic dust has been implicated to cause a large number of occupational lung diseases . A common event in these diseases is an inflammatory reaction affecting airway and alveolar tissue that is characterized by the recruitment of different types of inflammatory cells . Mechanisms of chemotaxis of alveolar macrophages (AMs) and neutrophils (PMNs) in response to microbial products derived from organic dust were studied . Seven agents known to be etiological agents causing respiratory symptoms (extract and endotoxin of Enterobacter agglomerans, 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 directly attract AMs and PMNs and to stimulate 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 mechanism 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.

RNA, 1995 Apr, 1(2), 183 - 93
Enhanced self-splicing of Physarum polycephalum intron 3 by a second group I intron; Rocheleau GA et al.; The third group I intron of Physarum polycephalum is found at the same site in the large subunit rRNA as the well-characterized intron from Tetrahymena thermophila . Formation of alternative structures in the rRNA can inhibit self-splicing of the Tetrahymena intron . We report that splicing of Physarum intron 3 is also influenced by adjacent sequences . In this case, however, splicing is stimulated by the presence of a second group I intron 24 nucleotides downstream . In vitro, intron 3 self-splices 10-25-fold faster in transcripts containing both introns . In vivo, intron 3 is excised from pre-rRNA before intron 2 in the majority of transcripts, as judged by PCR amplification of processing intermediates . This is an unusual example in which self-splicing is enhanced by the juxtaposition of two group I introns . This cooperative effect may be mediated by a conformational change in the rRNA.

Int J Food Microbiol, 1995 Apr, 25(2), 179 - 90
Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus; Villani F et al.; Streptococcus thermophilus 347 isolated from yogurt produces a bacteriocin referred as thermophilin 347 . The bacteriocin was evidenced in the neutralized, filtered and catalase treated culture supernatant fluid of the producer strain . After partial purification, thermophilin 347 exhibited a bactericidal effect against Listeria monocytogenes and several closely related lactic acid bacteria . The activity of thermophilin 347 was lost after protease treatment but was maintained after heating at 100 degrees C for 1 h; after autoclaving at 121 degrees C for 15 min the activity was reduced by 50% . SDS-PAGE of partially purified thermophilin 347 was used to detect bacteriocin activity corresponding to an apparent molecular mass between 2.5 and 6.2 KDa.

Appl Environ Microbiol, 1995 Apr, 61(4), 1555 - 62
Phylogenetic diversity of the bacterial community from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii; Moyer CL et al.; The phylogenetic diversity of small-subunit rRNA genes associated with the domain Bacteria was examined (by using previously defined operational taxonomic units {C . L . Moyer, F.C . Dobbs, and D . M . Karl, Appl . Environ . Microbiol . 60:871-879, 1994}; those for Pele's Vents Bacteria are hereafter abbreviated PVB OTUs) with samples from a microbial mat at an active, deep-sea hydrothermal vent system . A cluster of phylogenetically related PVB OTUs (OTUs 2, 3, 6, and 8) was closely affiliated with Thiovulum sp . contained within the epsilon subclass of the class Proteobacteria and accounted for 60.5% of the small-subunit rRNA bacterial clone library from Pele's Vents . A second, smaller cluster of PVB OTUs (OTUs 1 and 11) was closely affiliated with Xanthomonas sp., contained within the gamma subclass of the Proteobacteria and accounted for a total of 27.1% of the bacterial clone library . The remaining five PVB OTUs each accounted for 2.1% of the clones recovered and were affiliated with the following phylogenetic groups: PVB OTU 5 was a member of the Alteromonas group; PVB OTU 12 was a member of the Colwellia assemblage; PVB OTU 4 was loosely determined to be a member of the Thiothrix group, with the endosymbiotic bacteria from Bathymodiolus thermophilus and Calyptogena magnifica as the nearest relatives; PVB OTU 10B was a member of the Myxobacterium group; and PVB OTU 9A was a member of the Paraphyletic assemblage, with the Octopus Spring microbial mat type K clone as the closest known relative.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Syst Bacteriol, 1995 Apr, 45(2), 308 - 14
Thermotoga elfii sp . nov., a novel thermophilic bacterium from an African oil-producing well; Ravot G et al.; A thermophilic, glucose-fermenting, strictly anaerobic, rod-shaped bacterium, strain SEBR 6459T (T = type strain), was isolated from an African oil-producing well . This organism was identified as a member of the genus Thermotoga on the basis of the presence of the typical outer sheath-like structure (toga) and 16S rRNA signature sequences and its ability to grow on carbohydrates (glucose, arabinose, fructose, lactose, maltose, and xylose) . Major differences in its 16S rRNA gene sequence, its lower optimum temperature for growth (66 degrees C), its sodium chloride range for growth (0 to 2.8%), its lack of lactate as an end product from glucose fermentation, and its peritrichous flagella indicate that strain SEBR 6459T is not similar to the three previously described Thermotoga species . Furthermore, this organism does not belong to any of the other genera related to the order Thermotogales that have been described . On the basis of these findings, we propose that this strain should be described as a new species, Thermotoga elfii . The type strain of T . elfii is SEBR 6459 (= DSM 9442).

J Biol Chem, 1995 Mar 31, 270(13), 7601 - 8
Transcription in vitro of Tetrahymena class II and class III genes; Larsen LK et al.; A method for preparation of transcriptionally active nuclear extracts from the ciliated protozoan Tetrahymena thermophila is described . Cells were lysed in the presence of gum arabic, and nuclei were further purified in the presence of Ficoll 400 . Highly concentrated nuclear extracts were prepared by ultracentrifugation of nuclei in a buffer containing potassium glutamate and spermidine . These extracts supported accurate transcription initiation of T . thermophila class II and III genes . Using the histone H3-II gene as a template, we demonstrated that physiologically induced changes in transcriptional activity in vivo were reflected in the transcriptional activity of the nuclear extract in vitro . By electrophoretic mobility shift assays, five conserved sequence elements in the upstream region of the histone H3-II gene were shown specifically to bind proteins in extracts from exponentially growing as well as from starved cells, and by UV cross-linking we further characterized the specific binding of two proteins to an oligonucleotide containing a conserved CCAAT box motif . Transcription competition experiments showed that addition of this oligonucleotide decreased transcription significantly . Competition with oligonucleotides corresponding to the two proximal conserved sequence elements almost completely abolished transcription of the H3-II gene suggesting that binding of transacting factors to these elements is crucial for initiation of transcription.

Science, 1995 Mar 31, 267(5206), 1958 - 65
Architectures of class-defining and specific domains of glutamyl-tRNA synthetase; Nureki O et al.; The crystal structure of a class I aminoacyl-transfer RNA synthetase, glutamyl-tRNA synthetase (GluRS) from Thermus thermophilus, was solved and refined at 2.5 A resolution . The amino-terminal half of GluRS shows a geometrical similarity with that of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of the same subclass in class I, comprising the class I-specific Rossmann fold domain and the intervening subclass-specific alpha/beta domain . These domains were found to have two GluRS-specific, secondary-structure insertions, which then participated in the specific recognition of the D and acceptor stems of tRNA(Glu) as indicated by mutagenesis analyses based on the docking properties of GluRS and tRNA . In striking contrast to the beta-barrel structure of the GlnRS carboxyl-terminal half, the GluRS carboxyl-terminal half displayed an all-alpha-helix architecture, an alpha-helix cage, and mutagenesis analyses indicated that it had a role in the anticodon recognition.

Biochemistry, 1995 Mar 28, 34(12), 4056 - 67
A positive entropy change for guanosine binding and for the chemical step in the Tetrahymena ribozyme reaction; McConnell TS et al.; The ribozyme derived from the group I intron of Tetrahymena thermophila binds an exogenous guanosine nucleotide, which acts as the nucleophile in the sequence-specific cleavage of oligonucleotides . By examining the temperature dependence of the reaction under conditions where Km = Kd, we conclude the following: (1) Guanosine 5'-monophosphate (pG) binds to the closed ribozyme-oligonucleotide substrate complex with a positive entropy change (delta S degree' = +23 eu) and an enthalpy change (delta H degree') close to zero . This is contrary to the expectation that binding would cause increased order (negative delta S degree) and be driven by a negative delta H degree . (2) Inosine and 2-aminopurine riboside, each lacking two hydrogen-bonding moieties relative to guanosine, also bind with a positive entropy value and an unfavorable (positive) delta H degree' . From this result, we suggest that the hydrogen-bonding moieties make an enthalpic contribution to guanosine binding overcoming an intrinsic unfavorable delta H . (3) At 0 degree C, there is equally tight binding of pG in the presence and absence of oligonucleotide substrate bound to the ribozyme . Thus, energetic interactions responsible for the thermodynamic coupling between pG and oligonucleotide substrate binding seen at higher temperatures are indirect . (4) The activation barrier of the chemical step is stabilized by a positive delta S++ (+31 to 39 eu) . This stabilization is seen in four reactions using substrates with two different leaving groups in the presence and absence of pG, suggesting that the entropic contribution is inherent to the active site . The positive delta S values for the chemical step and for the binding of pG can be explained by a conformational change or release of water . Thus, although hydrogen bonding contributes to binding of nucleotides to this RNA enzyme as previously thought, it is these other events which produce a positive delta S that provide the energetic driving force for binding.

Nucleic Acids Res, 1995 Mar 25, 23(6), 988 - 94
In vitro transcription close to the melting point of DNA: analysis of Thermotoga maritima RNA polymerase-promoter complexes at 75 degrees C using chemical probes; Meier T et al.; The interaction of DNA dependent RNA polymerase of the extreme thermophile bacteria Thermotoga maritima with a promoter bearing DNA fragment was investigated in the temperature range from 20 to 85 degrees C . We show that the T . maritima RNA polymerase recognizes and utilizes the Escherichia coli T7 A1 promoter with an efficiency similar to that of the E . coli polymerase . We have investigated the interaction of both polymerases with the same promoter over a wide range of temperatures using hydroxyl radical foot-printing and osmium tetroxide probing . This study revealed that the T . maritima polymerase goes through a series of isomerisation events very similar to the E . coli polymerase, i.e . the closed, intermediate and open complexes, but the transitions themselves occur at radically different temperatures . This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence.

Nucleic Acids Res, 1995 Mar 25, 23(6), 942 - 8
Evidence for the requirement of protein synthesis and protein kinase activity in the temperature regulated stability of a Tetrahymena surface protein mRNA; McMillan PJ et al.; In Tetrahymena thermophila, the expression of the temperature-specific surface protein SerH3 is controlled primarily by a temperature-dependent change in the stability of its mRNA . The change in SerH3 mRNA stability occurs very rapidly after a shift in incubation temperature . This change in temperature could affect SerH3 mRNA stability directly by producing structural changes in the mRNA or regulatory factors acting on SerH3 mRNA . Alternatively, the temperature change could act indirectly through a signal transduction pathway leading to de novo synthesis of new regulatory factors or modifications of existing regulatory factors . To address these issues, we monitored the effect of temperature on an in vitro SerH3 mRNA decay assay and the in vivo effects of a variety of inhibitors against protein synthesis and protein kinases on SerH3 mRNA stability . The results of Northern analysis of SerH3 mRNAs in an in vitro mRNA decay assay indicate that temperature alone can not change the half-life of this mRNA . Furthermore, slot blot analysis of cytoplasmic RNAs show that protein synthesis and the action of protein kinases are not required for SerH3 mRNA turnover in cells grown at 30 degrees C . In contrast, our results indicate that the rapid decay of the SerH3 mRNA in cells grown at 30 degrees C and shifted to 40 degrees C requires a one time serine/threonine phosphorylation event which occurs at the temperature shift . In addition, the data show that a regulatory protein involved in rapid SerH3 mRNA decay must be newly and continuously synthesized following the temperature shift from 30 to 40 degrees C . These data show the complexity of temperature regulated mRNA decay and indicate that phosphorylation and protein synthesis are major factors in this process.

J Mol Biol, 1995 Mar 24, 247(2), 161 - 72
Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants; Hardt WD et al.; We have studied the interaction of 3'-end variants of a (pre-)tRNAGly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus . To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGly variants to RNase P RNAs was studied by gel retardation . A newly developed assay, based on the reduction of Pb(2+)-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants . The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNAGly variants . For E . coli RNase P RNA the following results were obtained . Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly binding by 1 to 1.5 kcal/mol . Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy . However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state . Our data suggest another kinetic step following initial substrate binding to E . coli RNase P RNA (possibly a conformational rearrangement) . For T . thermophilus RNase P RNA, product release of wild-type tRNAGly CCAAUA was not rate-limiting in the multiple turnover reaction . However, the effects of CCA mutations were similar to those attained with E . coli RNase P RNA . This supports the notion that a high-affinity binding site for the tRNA 3'-end is a ubiquitous feature of eubacterial P RNAs . Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.

J Biol Chem, 1995 Mar 17, 270(11), 6000 - 5
The phosphorylated ribosomal protein S7 in Tetrahymena is homologous with mammalian S4 and the phosphorylated residues are located in the C-terminal region . Structural characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis; Palm L et al.; A single basic ribosomal protein, protein S7, can be multiply phosphorylated in the ciliated protozoan Tetrahymena . Induction of phosphorylation is highly regulated, and the phosphorylation proceeds in a strictly sequential manner . The first site to be phosphorylated is a serine residue and the second a threonine . In this paper we report the complete primary structure of Tetrahymena thermophila ribosomal protein S7 including identification of the phosphorylated serine and threonine residues . Most of the sequence information was obtained from peptides generated by in situ digestion of S7 in two-dimensional gels using an approach that combined traditional protein chemistry with mass spectrometry . T . thermophila ribosomal protein S7 has a molecular mass of 29,459 Da and contains 259 amino acid residues . Phosphorylation takes place on Ser258 and Thr248 in the C-terminal region of the protein . Alignment of T . thermophila ribosomal protein S7 with known ribosomal proteins yielded the surprising result that T . thermophila S7 is homologous, not with mammalian ribosomal protein S6, but with mammalian ribosomal protein S4 . These findings clearly distinguish the pattern of phosphorylation of ribosomal proteins in Tetrahymena from all other eukaryotes analyzed to date.

Biochem Biophys Res Commun, 1995 Mar 8, 208(1), 55 - 62
In the thermophilic archaeon Sulfolobus solfataricus a DNA-binding protein is in vitro (Adpribosyl)ated; Faraone-Mennella MR et al.; A small protein with high affinity for homologous DNA was isolated from Sulfolobus solfataricus homogenate by mineral acid extraction . It was purified using a two-step procedure including CM-cellulose and RP-HPL chromatographies . The protein was electrophoretically homogeneous, had a molecular weight of 7.147 kDa and an amino acid composition with a high content of lysine and glutamic acid residues . The protein was able to protect DNA against thermal denaturation and DNAse I digestion in a dose-dependent manner . After incubation of the sulfolobal homogenate in the presence of 32P-NAD, followed by the purification steps, the protein was modified by ADPribose, as revealed by reaction product analysis, SDS-PAGE and autoradiography.

Chest, 1995 Mar, 107(3), 711 - 7
Hypersensitivity pneumonitis associated with home ultrasonic humidifiers; Suda T et al.; We describe five patients with hypersensitivity pneumonitis (HP) that was related to using home ultrasonic humidifiers . All patients had micronodular infiltrates on their chest radiograph, and their lung biopsy specimens revealed alveolitis with or without epithelioid cell granulomas . Challenge tests were performed on two patients with the humidifier water and three patients using the humidifier . All patients tested exhibited a positive response . Tests for precipitating antibodies against an extract of the humidifier water gave strongly positive reactions in all patients tested . Precipitins to Cephalosporium acremonium and Candida albicans were also present in all cases, whereas precipitins to thermophilic actinomycetes were not detected . Although cultures of the water grew a variety of fungal and bacterial organisms, thermophilic actinomycetes could not be detected . These findings suggest that thermophilic organisms may not be the causative antigens of HP associated with ultrasonic humidifiers . All five patients had an increase in the bronchoalveolar lavage (BAL) lymphocytes that were predominantly CD4+ lymphocytes . The T helper cell count (CD4) to suppressor T cell count (CD8) ratio was significantly higher than that observed in summer-type HP, and lower than that observed in bird fancier's lung, indicating that the phenotypes of the BAL lymphocytes may vary with the type of HP.

Mol Cell Biol, 1995 Mar, 15(3), 1144 - 53
Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila; Sheng H et al.; Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG)n double-stranded repeats with a single-stranded (TTGGGG)2 3' overhang at the end of the chromosome . We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3' telomeric repeat overhang . This protein is called TEP (telomere end-binding protein) . A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold . Changing two G's to C's in the TTGGGG repeats totally abolishes binding . However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding . A duplex structure adjacent to the 3' overhang is required for binding, although the duplex need not contain telomeric repeats . TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences . TEP has a greatly reduced affinity for RNA substrates . The copy number of TEP is at least 2 x 10(4) per cell, and it is present under different conditions of cell growth and development, although its level varies . UV cross-linking experiments show that TEP has an apparent molecular mass of approximately 65 kDa . Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.

Plant Mol Biol, 1995 Mar, 27(5), 1037 - 42
Isolation, sequence and expression in Escherichia coli of the nitrite reductase gene from the filamentous, thermophilic cyanobacterium Phormidium laminosum; Merchan F et al.; The nitrite reductase (NiR) gene (nirA) has been isolated and sequenced from the filamentous, thermophilic non-N2-fixing cyanobacterium Phormidium laminosum . Putative promoter-like and Shine-Dalgarno sequences appear at the 5' end of the 1533 bp long nir-coding region . The deduced amino acid sequence of NiR from P . laminosum corresponds to a 56 kDa polypeptide, a size identical to the molecular mass previously determined for the pure enzyme, and shows a high identity with amino acid sequences from ferredoxin-dependent NiR . This cyanobacterial NiR gene has been efficiently expressed in Escherichia coli DH5 alpha from the E . coli lac promoter and probably from the P . laminosum NiR promoter.

J Eukaryot Microbiol, 1995 Mar-Apr, 42(2), 173 - 83
Conjugation rescue of exocytosis mutants in Tetrahymena thermophila indicates the presence of functional intermediates in the regulated secretory pathway; Sauer MK et al.; Tetrahymena thermophila possesses a regulated secretory pathway in which mucin proteins are stored in dense-core granules, called mucocysts . Exocytosis-defective mutants exist that fail to secrete mucin in response to secretagogues . Four of the mutants (SB281, SB283, SB285 and SB715) appear to be blocked at different steps of the regulated secretory pathway . SB281 and SB285 accumulate mucin proteins in heterogeneous cytoplasmic organelles which have not yet been identified; SB283 makes mucocyst-like structures but they contain no immunologically identifiable 80-kDa or 50-kDa mucin proteins; and SB715 has more than normal amounts of immature and undocked mucocysts . The organelles that accumulate in exocytosis-defective mutants could be either normal intermediates in the biosynthetic pathway or aberrant structures that form as a result of the mutations . We have used conjugation rescue to analyze steps in the biogenesis of exocytosis-competent mucocysts and to identify functional intermediates . The cytoplasmic organelles that accumulate in SB281 appear to be unidentified biosynthetic intermediates, and the defect is in a cytosolic protein essential for mucocyst maturation . The organelles which accumulate in the other mutants are likely biosynthetic, but their mutations are in proteins which are labile or not free to diffuse into the mutant conjugant.

Microbiol Res, 1995 Mar, 150(1), 93 - 8
Moulds in containers with biological wastes; Reiss J; The collection of biological wastes in separate bio-containers can lead to a favoured development of thermophilic and thermotolerant moulds, especially of mucoraceous species and aspergilli, among which the human pathogen A . fumigatus is especially frequent . The abundantly produced spores are released into the air and can evoke severe infections in persons with immune-deficiencies . In two series of experiments it was demonstrated that the following procedures can reduce the number of spores in the air in the bio-containers above the biological wastes: (1) wrapping the wastes in portions in newsprint: the number of colony-forming units (CFU) decreases for about 50-70%; (2) cleaning of the container after each emptying with diluted vinegar: the number of CFU is reduced for up to 80%; (3) placing the container at shady sites: the temperature of the air inside the bio-containers at shady sites is approximately 5-8 degrees C lower than at sunny places with the consequence that the number of CFU in the air above the biological wastes is decreased . Based on these results principles for the handling of biological wastes are set up.

Mol Biol Evol, 1995 Mar, 12(2), 285 - 90
Phylogeny of the fish parasite Ichthyophthirius and its relatives Ophryoglena and Tetrahymena (Ciliophora, Hymenostomatia) inferred from 18S ribosomal RNA sequences; Wright AD et al.; Phylogenetic relationships within the subclass Hymenostomatia were inferred from the comparisons of three new SSrRNA gene sequences from Ichthyophthirius multifiliis (1,751 bp), Ophryoglena catenula (1,748 bp), and Tetrahymena corlissi (1,753 bp) . Using maximum-parsimony and distance-matrix methods, Ichthyophthirius and Ophryoglena were consistently paired and formed a sister group to the tetrahymenines, consistent with their placement in the Ophryoglenina . Tetrahymenids formed a monophyletic group that was divided into main lineages: T . corlissi diverged from the base of the lineage that included T . thermophila.

J Biochem (Tokyo), 1995 Mar, 117(3), 521 - 6
Preparation and characterization of the hydrophilic CuA-cytochrome c domain of subunit II of cytochrome c oxidase from thermophilic bacillus PS3; Tashiro H et al.; Cytochrome c oxidase of the thermophilic bacterium, PS3, was treated with trypsin . The hydrophilic domain of 26 kDa can be easily cleaved off from the hydrophobic anchor domain at the N-terminal region of subunit II, but remains attached to the rest of the enzyme upon gel-filtration in the presence of 0.2% lauroyl sarcosinate . The separation occurred in the presence of 5 M urea in addition to 0.2% lauroyl sarcosinate . After relatively prolonged proteolysis, that induced severe activity decay, and subunit I fragmentation, the 26 kDa fragment of subunit II can be easily isolated from the rest, suggesting that this fragment with cytochrome c and CuA interacts with subunit I . The separated fragment showed absorption spectra due to CuA and cytochrome c . Reconstitution of the cytochrome oxidase activity occurred on addition of the 26 kDa fragment to the proper gel-filtration chromatographic fraction.

Proteins, 1995 Mar, 21(3), 261 - 4
Cocrystallization of lysyl-tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys; Yaremchuk AD et al.; Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNAlys or Escherichia coli tRNAlys using ammonium sulfate as precipitant . The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18-22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5 . Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 A, c = 119 A) and diffract to at least 2.7 A resolution . However the homocomplex is of space group P42(1)2 and the heterocomplex of space group I422.

Biochemistry, 1995 Feb 28, 34(8), 2545 - 52
Defining a smaller RNA substrate for elongation factor Tu; Nazarenko IA et al.; A nuclease protection assay was used to obtain equilibrium dissociation constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRNA(Phe) . Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-sized tRNAs . However, the Ala minihelix, where residue A7 is joined directly to A49, binds to EF-Tu less well than the full-sized Ala-tRNA(Ala) . Similar data were obtained for Escherichia coli EF-Tu . An in vitro selection strategy was used to isolate a substrate for EF-Tu from an RNA library where nine random nucleotides inserted between A7 and A49 in the Ala minihelix . After six rounds of enrichment, two groups of RNA were obtained that bound T . thermophilus EF-Tu as well as Ala-tRNA(Ala) . Group I molecules have the consensus sequence UNDUGACUY (N = U, C, A, G; D = U, G; Y = U, C) in the randomized region, and Group II molecules generally have 5'-terminal GUG, but are more variable in the remaining six nucleotides . The selected RNAs bind EF-Tu better than the minihelix either because they provide additional function groups for protein binding or because they have a structure more similar to the aminoacyl acceptor branch of tRNA.

FEBS Lett, 1995 Feb 27, 360(2), 187 - 90
An 8.5-kDa ribonuclease from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Fusi P et al.; Protein p3, a ribonuclease we previously isolated from the archaebacterium Sulfolobus solfataricus {P . Fusi et al . (1993) Eur . J . Biochem . 211, 305-310}, was subjected to complete amino acid sequencing . It consisted of 75 residues, with a calculated M(r) of 8582, a pI of 10.1, and had some degree of monomethylation at Lys-4 and Lys-6 . p2, a previously sequenced, 62-residue ribonuclease from the same organism, had an identical sequence for 57 consecutive residues starting from the N-terminus . p2 and p3 also showed a striking similarity to five other proteins previously isolated from Sulfolobus strains and identified as DNA-binding proteins . However, the C-terminus, 10 residue region of p3 did not show any similarity to these proteins; in contrast, it was significantly similar to stretches in three eubacterial ribonucleases from Bacillus strains . No difference between p2 and p3 has so far been detected as regards their catalytic properties . Available data suggest that these molecules have a narrow substrate specificity and probably play specific roles in RNA processing.

Biochem Biophys Res Commun, 1995 Feb 15, 207(2), 848 - 51
SECReT of the eukaryotic large ribosomal subunit; Harauz G et al.; The large ribosomal subunit of the thermophilic fungus Thermomyces lanuginosus was reconstructed in three dimensions from electron micrographs . This is the first reported reconstruction of the eukaryotic complex and demonstrates specific structural features such as an intersubunit canyon and a potential channel for the nascent peptide chain.

Biochemistry, 1995 Feb 14, 34(6), 2015 - 25
Determination of the metal ion separation and energies of the three lowest electronic states of dimanganese (II,II) complexes and enzymes: catalase and liver arginase; Khangulov SV et al.; The dimanganese (II,II) catalase from Thermus thermophilus, MnCat(II,II), arginase from rat liver, Arg(II,II), and several dimanganese(II,II) compounds, LMn2XY2, which are functional catalase mimics, all possess a pair of coupled Mn(II) ions in their catalytic sites . For each of these, we have measured by EPR spectroscopy the relative energies separating the three lowest electronic states (singlet, triplet, and quintet), described a general method for extracting the individual spectra for these states by multicomponent analysis, and determined the Mn-Mn separation . The triplet-singlet and quintet-singlet energy gaps were modeled well by fitting the temperature dependence of the EPR intensities to a Boltzmann expression for a pair of Mn(II) ions coupled by isotropic Heisenberg spin exchange (-2JS1S2) . This dependence indicates diamagnetic ground states with delta E10 (cm-1) = magnitude of 2J = 4 and 11.2 cm-1 for Arg-(II,II)(+borate) and MnCat(II,II)(phosphate), respectively . This large difference in magnitude of 2J reflects either a difference in the bridging ligands or, possibly, a weaker ligand field (larger ionization potential) for the Mn(II) ions in arginase . In n-butanol/CH2Cl2 the triplet-singlet energy gaps for {LMn2(CH3CO2)}(C1O4)2 (1), {LMn2(CH3CO2)3} (2), and {LMn2Cl3} (3), where HL = N,N,N',N'-tetrakis(2-methylenebenzimidazole)-1,3-diaminopropan+ ++-2-ol, are 23-24 cm-1 . Comparison of the Heisenberg exchange interaction constants for more than 30 dimanganese(II,II) complexes suggests a possible bridging structure of (mu-OH)(mu-carboxylate)1-2 for MnCat(II,II), while the 3-fold weaker coupling in Arg(II,II) suggests mu-aqua in place of mu-hydroxide . EPR spectra of both the triplet and quintet electronic states were extracted and found to exhibit zero-field splittings (ZFS) and resolved 55Mn hyperfine splittings indicating spin-coupled Mn2-(II,II) species . The major ZFS interaction could be attributed to the magnetic dipole-dipole interaction between the Mn(II) ions . A linear correlation is observed between the crystallographically determined Mn-Mn distance and the ZFS of the quintet state (D2) for five dimanganese pairs for which both data sets are available . Using this correlation, the Mn-Mn distance in Arg(II,II) is predicted to be 3.36-3.57 A for the native enzyme (multiple forms) and 3.59 A for MnCat(II,II)(phosphate) . Addition of the inhibitor borate to Arg(II,II) simplifies the ZFS, indicative of conversion to a single species with mean Mn-Mn separation of 3.50 A . The second metal ion in dinuclear complexes possessing a shared bridging ligand has been shown to attenuate the strength of the mu-ligand field potential, as monitored by the strength of the single ion ZFS.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1995 Feb 10, 270(6), 2827 - 32
Cloning, sequencing, and transcriptional analysis of the coenzyme F420-dependent methylene-5,6,7,8-tetrahydromethanopterin dehydrogenase gene from Methanobacterium thermoautotrophicum strain Marburg and functional expression in Escherichia coli; Mukhopadhyay B et al.; Two methylenetetrahydromethanopterin dehydrogenases have been purified from Methanobacterium thermoautotrophicum strain Marburg: one (MTD) is coenzyme F420-dependent and oxygen-stable (Mukhopadhyay, B., and Daniels, L . (1989) Can . J . Microbiol . 35, 499-507), and the other (MTH) is coenzyme F420-independent (or hydrogenase-type) and oxygen-sensitive (Zirngibl, C., Hedderich, R., and Thauer, R . K . (1990) FEBS Lett . 261, 112-116) . Based on the NH2-terminal sequence of MTD, a 36-mer oligonucleotide was designed and used to identify and clone a 6.1-kilobase pair EcoRI fragment of M . thermoautotrophicum DNA . Sequencing of this fragment revealed an 825-base pair (bp) MTD encoding gene (mtd), which was expressed in Escherichia coli yielding an enzyme that, like the native enzyme, was oxygen-stable, strictly dependent on coenzyme F420, thermostable, thermophilic, and exhibited maximum activity at an acidic pH . The amino acid sequence predicts that MTD is a hydrophobic and acidic protein with no identifiable homology to MTH (von Bunau, R., Zirngibl, C., Thauer, R . K., and Klein, A . (1991) Eur . J . Biochem . 202, 1205-1208), but comparisons with coenzyme F420 utilizing enzymes revealed a conserved region at the NH2 terminus of MTD that could correspond to the ability to interact with coenzyme F420 . The mtd transcript was approximately 900 nucleotides long and initiated 8 bp upstream of the translation initiation codon and 22 bp downstream from an archaeal promoter sequence . The mtd coding sequence was followed by several poly(dT) sequences and an inverted repeat that could be transcription termination signals.

Mol Cell Biochem, 1995 Feb 9, 143(1), 21 - 34
Ribosomal proteins of Thermomyces lanuginosus--characterisation by two-dimensional gel electrophoresis and differential disassembly; Wu J et al.; One- and two-dimensional gel electrophoresis were employed to characterise the proteins derived from the ribosomes of the thermophilic fungus Thermomyces lanuginosus . Approximately 32 (29 basic and 3 acidic) and 45 (43 basic and 2 acidic) protein spots were resolved from Th . lanuginosus small and large ribosomal subunits, respectively . The molecular weight of the small subunit proteins ranged from 9,800-36,000 Da with a number average molecular weight of 20,300 Da . The molecular weight range for the large subunit proteins was 12,000-48,500 Da with a number average molecular weight of 25,900 Da . Most proteins appeared to be present in unimolar amounts . These data are comparable with but not identical to those from other eukaryotic ribosomes . The sensitivities of the ribosomal proteins to increasing concentrations of NH4Cl were also evaluated by two-dimensional gel electrophoresis . Most ribosomal proteins were gradually released over a wide range of salt concentrations but some were preferentially enriched in one or two salt conditions.

Biochemistry, 1995 Feb 7, 34(5), 1646 - 60
Structure-function in Escherichia coli iron superoxide dismutase: comparisons with the manganese enzyme from Thermus thermophilus; Lah MS et al.; The crystal structure of dimeric Fe(III) superoxide dismutase (SOD) from Escherichia coli (3006 protein atoms, 2 irons, and 281 solvents) has been refined to an R of 0.184 using all observed data between 40.0 and 1.85 A (34,879 reflections) . Features of this structure are compared with the refined structure of MnSOD from Thermus thermophilus . The coordination geometry at the Fe site is distorted trigonal bipyramidal, with axial ligands His26 and solvent (proposed to be OH-), and in-plane ligands His73, Asp156, and His160 . Reduction of crystals to the Fe(II) state does not result in significant changes in metal-ligand geometry (R = 0.188 for data between 40.0 and 1.80 A) . The arrangement of iron ligands in Fe(II) and Fe(III)SOD closely matches the Mn coordination found in MnSOD from T . thermophilus {Ludwig, M.L., Metzger, A.L., Pattridge, K.A., & Stallings, W.C . (1991) J . Mol . Biol . 219, 335-358} . Structures of the Fe(III) azide (40.0-1.8 A, R = 0.186) and Mn(III) azide (20.0-1.8 A, R = 0.179) complexes, reported here, reveal azide bound as a sixth ligand with distorted octahedral geometry at the metal; the in-plane ligand-Fe-ligand and ligand-Mn-ligand angles change by 20-30 degrees to coordinate azide as a sixth ligand . However, the positions of the distal azide nitrogens are different in the FeSOD and MnSOD complexes . The geometries of the Fe(III), Fe(II), and Fe(III)-azide species suggest a reaction mechanism for superoxide dismutation in which the metal alternates between five- and six-coordination . A reaction scheme in which the ligated solvent acts as a proton acceptor in the first half-reaction {formation of Fe(II) and oxygen} is consistent with the pH dependence of the kinetic parameters and spectroscopic properties of Fe superoxide dismutase.

Eur J Biochem, 1995 Feb 1, 227(3), 848 - 56
The thermosome of Thermoplasma acidophilum and its relationship to the eukaryotic chaperonin TRiC; Waldmann T et al.; A high molecular-mass protein complex from the archaebacterium Thermoplasma acidophilum, referred to here as the 'thermosome', is built from two subunits (M(r) 58 and 60) . The thermosome has been purified to homogeneity . The molecular mass of the native complex was determined to be 1061 +/- 30 Da by scanning transmission electron microscopy . It shows a weak ATPase activity and is able to bind denatured polypeptides . Averages obtained from electron micrographs of negatively stained molecules in the end-on and side-on orientations, respectively, were compared with those of the t-complex polypeptide 1 ring complex (TRiC), isolated from bovine testes . Both molecules consist of two stacked pseudo eightfold symmetric rings which build up a cylindrical particle with a large cavity in the center . Sequence alignments of peptides generated from both subunits of the thermosome and different subunits of TRiC reveal a high partial similarity to each other and to the archaebacterial chaperonin thermophilic factor 55 from Sulfolobus shibatae as well as to eukaryotic TCP1 proteins . These striking structural similarities confirm the proposition that all these molecules belong to a single protein family which is structurally and functionally related to the GroEL class of molecular chaperones.

Carcinogenesis, 1995 Feb, 16(2), 245 - 52
Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation; Baricault L et al.; Epidemiological and in vivo and in vitro experimental studies have suggested that fermented milks may interfere with the emergence and/or the development of colon cancer . The results, however, remain inconclusive . This prompted us to develop a new approach based on the use of HT-29, a cultured human colon cancer cell line, to study at the cellular level the effect of fermented milks on colon cancer cell growth and differentiation characteristics . Undifferentiated HT-29 cells have been grown in the continuous presence of milks fermented by one of the following bacterial populations: Lactobacillus helveticus, Bifidobacterium, L.acidophilus or a mix of Streptococcus thermophilus and L . bulgaricus . Penicillin G was added to the cell culture medium, resulting in a complete blockade of bacterial growth without significant effect on bacterial viability . One out of the four bacteria species studied, namely L.acidophilus, was without effect on both cell growth and differentiation . The three other bacterial strains induced a significant, although variable, reduction in the growth rate of HT-29 cells, which resulted in a 10-50% decrease in the cell number at steady-state (i.e . at cell confluency) . The most efficient strains in lowering the HT-29 growth rate were L . helveticus and Bifidobacterium . Concomitantly, the specific activities of dipeptidyl peptidase IV (DPP IV), a sensitive and specific marker of HT-29 cell differentiation, and that of three other brush border enzymes (sucrase, aminopeptidase N and alkaline phosphatase) were significantly increased, thus suggesting that these cells may have entered a differentiation process . Altogether, these results indicate that the use of cultured colon cancer cells may be a useful tool to further study the effect of fermented milks on colon cancer and that bacterial strains may exert a different and specific effect on cancer cell growth and differentiation when used in fermented milk products.

Zoolog Sci, 1995 Feb, 12(1), 133 - 5
The mutant gene product of a Tetrahymena cell-division-arrest mutant cdaA is localized in the accessory structure of specialized basal body close to the division furrow; Numata O et al.; A division arrest mutant, cdaA, of Tetrahymena thermophila is known to have a temperature sensitive-defect in the determination of the division plane, and its gene product had been shown to be a protein designated as p85 (Mr = 85,000; pI = 4.7) . Here the localization of p85 was shown to be the accessary structure of specialized basal body close to the division furrow by immunoelectron microscopy using anti-p85 antiserum.

Biol Chem Hoppe Seyler, 1995 Feb, 376(2), 127 - 30
Studies on S14 protein from Thermus thermophilus possessing zinc finger-like motifs; Tsiboli P et al.; The amino acid sequence of the ribosomal protein S14 of Thermus thermophilus has been determined both by automated sequence analysis of the intact protein as well as by DNA sequence analysis of the gene . The carboxy-terminal region was verified by both amino acid sequence analysis of the carboxy-terminal peptide produced after Glu-C digestion and by DNA sequence analysis . The protein contains 60 amino acid residues with a calculated molecular weight of 7008 . The most extensive homology is observed in the carboxy-terminal regions of all S14 proteins compared . Interestingly, the carboxy-terminal region of most S14 proteins of all species studied so far, form zinc-finger domains in the variety of C2-C2 form.

Biochem Mol Biol Int, 1995 Feb, 35(2), 397 - 407
Refolding of glutamate dehydrogenase from Bacillus acidocaldarius after guanidinium chloride-induced unfolding; Consalvi V et al.; The hexameric NAD-dependent glutamate dehydrogenase from the thermophilic eubacterium Bacillus acidocaldarius is the first glutamate dehydrogenase which spontaneously refolds in vitro . After 24 h unfolding in 6 M guanidinium chloride at 20 degrees C, refolding was achieved by dilution of the denaturant . The yield of reconstitution obtained in the presence of 1,4 dithio-DL-threitol in the unfolding/refolding mixture was 75% . The unfolding/refolding equilibria have been studied by fluorescence, circular dichroism and catalytic activity, which was 50% inhibited at 0.08 M guanidinium chloride, a value 30-fold lower than the transition midpoint detected by physical changes . Refolding was attempted in the presence of various additives, at different temperatures and varying enzyme and residual guanidinium chloride concentration . The refolded enzyme, after removal of inactive aggregated species, completely resembles the native enzyme in term of its physicochemical and kinetic properties as well as thermophilicity.

J Biochem (Tokyo), 1995 Feb, 117(2), 408 - 13
The crystal structures of mutated 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 and their relationship to the thermostability of the enzyme; Moriyama H et al.; The structures of two mutant forms (G240A and L246E/V249M) of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 were studied by X-ray crystallography . In the case of G240A, the replacement of glycine by alanine at residue 240 was expected to decrease the thermostability as a result of abnormal contacts between the methyl group of alanine and the peptide chain . However, the normal van der Waals' contacts were achieved owing to a shift in a bundle of beta-strands that yielded a vacant space for the alanine residue . The extended hydrogen bonds within the beta-sheet are the major reason for the decreased thermostability of G240A . The mutations in L246E/V249M are located in an alpha-helix region which is involved in subunit-subunit contact via hydrophobic interaction . Loosening of the subunit-subunit contact owing to ionic repulsion was the major cause of the lower heat stability of L246E/V249M.

Appl Environ Microbiol, 1995 Feb, 61(2), 729 - 33
Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp . strain JW/SL-YS485; Shao W et al.; Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp . strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth . Both enzymes released acetic acid from chemically acetylated larch xylan . Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II) . The isoelectric points were 4.2 and 4.3, respectively . As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively . Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively . At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.

Eur J Epidemiol, 1995 Feb, 11(1), 83 - 6
Diffusion of thermophilic Campylobacter in the Pesaro-Urbino area (Italy) from 1985 to 1992; Baffone W et al.; The results of research on the spreading of campylobacter in the Pesaro-Urbino area carried out from 1985 to 1992 are presented . Materials of different origin were examined: 822 samples of human faeces, 533 animal rectal swabs, 192 samples of domestic sewage, 48 of river water, 96 of sea water and 632 of various types of food . Two hundred and nine strains of campylobacter were isolated (9%), most of which were Campylobacter jejuni (80%), with particular frequency in food products (chicken carcass 45.7%, ground meat and sausage 18.1%) and in river water (31.3%) . In contrast, the samples of sea water and dairy cheese products were always negative . It may be concluded that the spreading of campylobacter in the Pesaro-Urbino area is mainly associated with food products of animal origin . Therefore, better controls in the processing of these products may be necessary.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 778 - 82
Regulation of bacterial sugar-H+ symport by phosphoenolpyruvate-dependent enzyme I/HPr-mediated phosphorylation; Poolman B et al.; The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein(s) domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . C-terminal truncation mutants were constructed and expressed in Escherichia coli and their properties were analyzed . Remarkably, the entire IIA domain (160 amino acids) could be deleted without significant effect on lactose-H+ symport and galactoside equilibrium exchange . Analysis of the LacS mutants in S . thermophilus cells suggested that transport is affected by PTS-mediated phosphorylation of the IIA domain . For further studies, membrane vesicles of S . thermophilus were fused with cytochrome c oxidase-containing liposomes, and, when appropriate, phosphoenolpyruvate (PEP) plus purified enzyme I and heat-stable protein HPr were incorporated into the hybrid membranes . Generation of a protonmotive force (delta p) in the hybrid membranes resulted in accumulation of lactose, whereas uptake of the PTS sugar sucrose was not observed . With PEP and the energy-coupling proteins enzyme I and HPr of the PTS on the inside, high rates of sucrose uptake were observed, whereas delta p-driven lactose uptake by wild-type LacS was inhibited . This inhibition was not observed with LacS(delta 160) and LacS(H552R), indicating that PEP-dependent enzyme I/HPr-mediated phosphorylation of the IIA domain (possibly the conserved His-552 residue) modulates lactose-H+ symport activity.

Biochim Biophys Acta, 1995 Jan 19, 1246(2), 151 - 9
ADP-ribosylation reactions in Sulfolobus solfataricus, a thermoacidophilic archaeon; Faraone-Mennella MR et al.; An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C . The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent . It proved to be highly thermostable, with about 30% decrease of 14C incorporation from {14C}NAD on incubation at 80 degrees C for up to 24 h . The main reaction product was found to be mono-ADP-ribose . Testing both {adenine-14C(U)}NAD and {adenine-14C(U)}ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity . The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

Genes Dev, 1995 Jan 15, 9(2), 248 - 55
Transient DNA breaks associated with programmed genomic deletion events in conjugating cells of Tetrahymena thermophila; Saveliev SV et al.; Thousands of programmed genomic deletion events occur during macronuclear development in Tetrahymena thermophila . Two of the deleted segments, called M and R, have been particularly well-characterized . Using ligation-mediated PCR, we have detected DNA strand breaks that correlate temporally and structurally with the deletion events in the M and R regions . The ends appear at positions that correspond precisely to boundaries of deleted sequences, as defined by observed chromosomal junctions found after deletion is complete . They occur exclusively during the known DNA rearrangement period in macronuclear development . The breaks are staggered by 4 bp in the complementary strands . Several alternative breaks were found at the end of one deleted region, consistent with multiple alternative chromosomal junctions detected previously . The free 5' ends generated at the breaks are phosphorylated . A purine residue always occurs at the free 3' ends, with an adenosine appearing in 11 of 12 cases . Patterns found in the detected break sites suggest rules that define the ends of the deleted segments within a transposon-like deletion mechanism.

Biochem J, 1995 Jan 15, 305 ( Pt 2), 539 - 48
Catalytic-rate improvement of a thermostable malate dehydrogenase by a subtle alteration in cofactor binding; Alldread RM et al.; The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2'- and 3'-hydroxy groups of the adenine-ribose of the cofactor . This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position . The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid . The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation . When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions . The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme . This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.

J Biotechnol, 1995 Jan 15, 38(2), 183 - 92
Aerobic thermophilic treatment of sewage sludge at pilot plant scale . 2 . Technical solutions and process design; Ponti C et al.; The performance of the ATS process depends essentially on the oxygen transfer efficiency . Improvement of the mass transfer capacity of a bioreactor allowed to reduce the incubation time necessary to attain sludge stabilization . It is important to use equipment with a high aeration efficiency such as an injector aeration system . The ratio between the total oxygen consumption and the organic matter degradation (delta COD) ranged between 0.4 and 0.8 in the pilot plant, whereas 1.23 was found in completely mixed bioreactors (Bomio, 1990) . No significant improvement of the bacterial degradation efficiency was attained with a specific power input exceeding 6-8 kW m-3 . A mean residence time of less than 1 d allowed organic matter removals up to 40% with specific power consumption of 10 kWh kg-1 COD oxidized . The sludge hygienization is one of the objectives and benefits of the thermophilic treatment: not only temperature but also the total solids content were important factors affecting inactivation of pathogens . The inactivation rate was promoted by the increase of temperature, while the residual colony forming units decreased with reducing the total solids content of sewage sludge . It is concluded that continuous operation mode would not affect the quality of the hygienization but could display the high degradation potential of the aerobic system.

J Biotechnol, 1995 Jan 15, 38(2), 173 - 82
Aerobic thermophilic treatment of sewage sludge at pilot plant scale . 1 . Operating conditions; Ponti C et al.; The aerobic thermophilic treatment process of sewage sludge was studied at different bioreactor scales in a pilot plant installation . Since, for a satisfactory sludge disinfection, the Swiss legislation requires minimal incubation times of all volume elements, the bioreactors were operated in repetitive batch mode (draw and fill) . Different retention times and frequencies of the volume changes were applied in order to prove the capability of the particular operation modes in assuring high degradative potential . The main enzymatic activity involved during the aerobic treatment was proteolysis: the RQ values ranged between 0.8 and 0.9 depending on the applied operating conditions . Although not in a linear manner, the efficiency of the microflora decreased as the bioreactor scale increased, when this increase corresponded with a reduction of the specific power input . The sludge oxidation rates can be tuned by some process operating conditions such as the volume change frequency, the changed volume quantities and the retention times . It was possible to improve the microbial degradative efficiency by an increased frequency of the changes, while the mean retention time influenced in particular the ultimate product quality, described as residual organic matter content of the sludge . The microflora present was also satisfactorily active at mean hydraulic retention times of less than 10 h . The organic matter concentration of the inlet sewage sludge plays an important role: it influences the aerobic degradation process positively.

Proc Natl Acad Sci U S A, 1995 Jan 3, 92(1), 175 - 9
Supramolecular structure of the photosystem II complex from green plants and cyanobacteria; Boekema EJ et al.; Photosystem II (PSII) complexes, isolated from spinach and the thermophilic cyanobacterium Synechococcus elongatus, were characterized by electron microscopy and single-particle image-averaging analyses . Oxygen-evolving core complexes from spinach and Synechococcus having molecular masses of about 450 kDa and dimensions of approximately 17.2 x 9.7 nm showed twofold symmetry indicative of a dimeric organization . Confirmation of this came from image analysis of oxygen-evolving monomeric cores of PSII isolated from spinach and Synechococcus having a mass of approximately 240 kDa . Washing with Tris at pH 8.0 and analysis of side-view projections indicated the possible position of the 33-kDa extrinsic manganese-stabilizing protein . A larger complex was isolated that contained the light-harvesting complex II (LHC-II) and other chlorophyll a/b-binding proteins, CP29, CP26, and CP24 . This LHC-II-PSII complex had a mass of about 700 kDa, and electron microscopy revealed it also to be a dimer having dimensions of about 26.8 and 12.3 nm . From comparison with the dimeric core complex, it was deduced that the latter is located in the center of the larger particle, with additional peripheral regions accommodating the chlorophyll a/b-binding proteins . It is suggested that two LHC-II trimers are present in each dimeric LHC-II-PSII complex and that each trimer is linked to the reaction center core complex by CP24, CP26, and CP29 . The results also suggest that PSII may exist as a dimer in vivo.

Essays Biochem, 1995, 29, 193 - 207
Protein stability at high temperatures; Cowan DA; The enzymology of hyperthermophilic micro-organisms is a growing field . As increasing numbers of novel high-temperature organisms are isolated and made available through culture collections, and, as biomass becomes more readily available, more laboratories will undoubtedly expand their research interests into this area . The prospect of totally novel enzyme systems and of new approaches to the investigation of fundamental molecular properties will continue to stimulate interest in this field . Studies of thermostable enzymes have already provided valuable data on the relationships between protein stability and activity . The subtle molecular mechanisms which have evolved to stabilize these proteins provide the clues needed for the intelligent design of stabilized mesophilic enzymes, an important target where a combination of high activity at 'low' temperatures and resistance to denaturation is required . The current role of hyperthermophilic enzymes in biotechnology is relatively minor, despite these enzymes having a high 'profile' . While early over-enthusiastic predictions that these enzymes would revolutionize biotechnology should be disregarded, it can reasonably be assumed that, where functional and economic criteria are suitable, thermophilic enzymes will be readily incorporated into current and future biotechnology.

Crit Rev Microbiol, 1995, 21(3), 165 - 74
Is thermophily a transferrable property in bacteria?
Lindsay JA.
Bacteria exhibit unique diversity in their ability to grow at different temperatures . Indeed, eubacteria and archaebacteria are the only organisms able to grow above 65 degrees C . The temperature range for a species is generally considered to be a stable character; however, mutants may be isolated that have a Tmin or Tmax below or above the parent organism . Some bacteria may also be coaxed to grow at different temperature by training cultures, through an incremental increase or decrease of temperature . Genetic approaches, for example, the transformation of mesophilic Bacillus to thermophily using DNA from closely related thermophiles, has been very controversial . A major problem has been the lack of stability of the high-temperature phenotype upon subculture, which has not allowed extensive genetic and biochemical characterization of the transformants . The mechanism whereby the thermophilic phenotype is carried is unknown, although it is possible that the adapter genes are plasmid encoded . Studies using phenotypically stable transformants indicated that the thermostability of some cellular components was significantly increased, both in the vegetative cell and spore state . Enzyme thermostability, for example, appeared to be associated with an increased use of hydrophobic amino acids; however, the biochemical mechanisms for these alterations remain unknown . Thermophily is still a challenging problem with some interesting molecular biology.

Nucleic Acids Symp Ser, 1995, (34), 205 - 6
Similarities and differences in tRNA identity between Escherichia coli and Saccharomyces cerevisiae: evolutionary conservation and divergence; Nameki N et al.; Identity elements, which allow correct recognition of tRNAs by their cognate aminoacyl-tRNA synthetase, have been well elucidated in Escherichia coli to begin to see a pattern for tRNA recognition . We examined the identity elements of several tRNA species from Saccharomyces cerevisiae and Thermus thermophilus using in vitro transcripts . Comparison of identity elements among different organisms indicates not only conservation but also evolutionary divergence of tRNA recognition.

Chin J Biotechnol, 1995, 11(3), 171 - 6
High-concentration ethanol production from cooked corn starch by using medium-temperature cooking process; Chi Z et al.; An improved process for high-concentration ethanol production from cooked corn starch by Saccharomyces sp . W4 has been well developed . Simultaneous cooking and liquefaction of corn starch were performed by using thermophilic alpha-amylase at 95 degrees to 105 degrees C . The mash was then saccharified at 60 degrees C by using high-efficiency glucoamylase . Saccharomyces sp . W4 could produce 18.3% (v/v) ethanol at 30 degrees C within 60 hrs, with 1.2% reducing sugar and 4.1% total sugar remaining in the fermented mash . It was found that ammonium sulfate could accelerate the fermentation rate . When 0.9 g of ammonium sulfate was added to 280 mL of the fermentation media, 18.9% (v/v) ethanol could be reached in the mash after 50 hrs of fermentation, leaving 0.27% reducing sugar and 3.1% total sugar in the fermented media.

Microbios, 1995, 84(339), 127 - 30
Effect of a detergent Triton X-100 on growth and alpha-glucosidase production by the thermophilic fungus Malbranchea sulfurea; Gupta AK et al.; A detergent Triton X-100 was found to affect maltose-induced synthesis of extracellular, mycelial and intracellular alpha-glucosidase in the thermophilic fungus Malbranchea sulfurea . The extracellular fraction of the total alpha-glucosidase yield was found to be 90.7% and 40.4% in the presence and absence of the detergent, respectively . Data suggest that supplementation of the detergent in the medium resulted in the partial solubilization of the cell-bound alpha-glucosidase and caused its release in the growth medium.

Microbios, 1995, 83(337), 217 - 28
Mycobacterium paratuberculosis and inflammatory bowel disease: frequency distribution in serial colonoscopic biopsies using the polymerase chain reaction; Murray A et al.; An association between Mycobacterium paratuberculosis and Crohn's disease is suspected but the evidence remains controversial . Using a one-step DNA extraction procedure with the thermophilic protease PRETAQ and amplification by the polymerase chain reaction, M . paratuberculosis DNA was detected in 22% of patients with Crohn's disease, and in 13% of patients with ulcerative colitis . M . paratuberculosis DNA was not found in any biopsy tissue from control non-inflammatory bowel disease patients . The biopsy tissues in which M . paratuberculosis was detected all came from regions which were inflamed when viewed microscopically . Overall, 7.7% of biopsies from such inflamed areas were positive . This low frequency of detection could be explained on the basis of extremely low abundance of the organism in relation to the area of mucosa sampled, or be consistent with a non-aetiological role for M . paratuberculosis in inflammatory bowel disease.

Annu Rev Microbiol, 1995, 49, 1 - 28
The road to Yellowstone--and beyond; Brock TD; This memoir describes the professional life and times of Thomas D . Brock, with an emphasis on those aspects of his career relating to research in microbial ecology, and how this work led to field research in Yellowstone . The first discovery of extremely thermophilic bacteria is described, followed by a discussion of some of the consequences of this discovery for biotechnology and microbiology . Also covered briefly in this memoir are Brock's activities in textbook writing, publishing, computers, and the history of science.

Appl Environ Microbiol, 1995 Jan, 61(1), 290 - 6
Conservation of the genes for dissimilatory sulfite reductase from Desulfovibrio vulgaris and Archaeoglobus fulgidus allows their detection by PCR; Karkhoff-Schweizer RR et al.; The structural genes for dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris Hilden-borough were cloned as a 7.2-kbp SacII DNA fragment . Nucleotide sequencing indicated the presence of a third gene, encoding a protein of only 78 amino acids, immediately downstream from the genes for the alpha and beta subunits (dsvA and dsvB) . We designated this protein DsvD and the gene encoding it the dsvD gene . The alpha- and beta-subunit sequences are highly homologous to those of the dissimilatory sulfite reductase from Archaeoglobus fulgidus, a thermophilic archaeal sulfate reducer, which grows optimally at 83 degrees C . A gene with significant homology to dsvD was also found immediately downstream from the dsrAB genes of A . fulgidus . The remarkable conservation of gene arrangement and sequence across domain (bacterial versus archaeal) and physical (mesophilic versus thermophilic) boundaries indicates an essential role for DsvD in dissimilatory sulfite reduction and allowed the construction of conserved deoxyoligonucleotide primers for detection of the dissimilatory sulfite reductase genes in the environment.

J Appl Bacteriol, 1995 Jan, 78(1), 34 - 8
The effect of sewage sludge treatment processes on oocysts of Cryptosporidium parvum; Whitmore TN et al.; The effect of common sewage sludge treatment processes on oocysts of the coccidian protozoan Cryptosporidium was evaluated in laboratory simulations . The ability of primary sewage sedimentation to remove Cryptosporidium oocysts was found to be poor . Thermophilic (55 degrees C) aerobic digestion and sludge pasteurization at the same temperature were found to be effective treatments to inactivate Cryptosporidium oocysts . Approximately 10% of the oocyst population were found to be viable after 18 d exposure to mesophilic (35 degrees C) anaerobically digesting sludge . The viability of Cryptosporidium oocysts decreased within the range 20-40% in sludge-treated soil mesocosms over 30 d . The survival results obtained, however, indicated that oocysts would survive well beyond this period.

Plant Mol Biol, 1995 Jan, 27(1), 199 - 204
Cloning and sequence analysis of a signal peptidase I from the thermophilic cyanobacterium Phormidium laminosum; Packer JC et al.; Type I signal peptidases are a widespread family of enzymes which remove the presequences from proteins translocated across cell membranes, including thylakoid and cytoplasmic membranes of cyanobacteria and thylakoid membranes of chloroplasts . We have cloned and sequenced a signal peptidase gene from the thermophilic cyanobacterium Phormidium laminosum which is believed to encode an enzyme common to both membrane systems . The deduced amino acid sequence is 203 residues long and although the overall similarity among signal peptidases is rather low there are a number of identifiable conserved regions present . The P . laminosum enzyme is predicted to have a single transmembrane domain, in contrast to other Gram-negative bacterial sequences, but similar to other type I signal peptidases.

Plant Mol Biol, 1995 Jan, 27(1), 179 - 90
Characterization of plastocyanin from the cyanobacterium Phormidium laminosum: copper-inducible expression and SecA-dependent targeting in Escherichia coli; Varley JP et al.; Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gene cloned and sequenced . The derived amino acid sequence indicates that the plastocyanin protein is initially synthesized with an N-terminal leader sequence of 34 amino acids to direct it across the thylakoid membrane . The leader sequence consists of a positively charged N-terminal region, a hydrophobic region and a cleavage site, which are characteristic both of higher-plant chloroplast thylakoid transfer domains and of bacterial leader peptides . The petE gene and flanking regions have been cloned in Escherichia coli, and the plastocyanin protein is expressed and directed to the periplasmic space, with concomitant processing to the mature form . Targeting to the periplasm and processing of the plastocyanin protein in E . coli appears to be dependent on components of the Sec apparatus, since the unprocessed precursor accumulates in the cytoplasm of a secA mutant . Expression of plastocyanin in E . coli is copper-inducible and apparently controlled at the level of transcription, leading to the conclusion that copper-regulated promoters exist in the regions flanking the gene and are recognized in a heterologous system . Possible implications for gene expression and protein targeting in the cyanobacterium are discussed.

Int J Syst Bacteriol, 1995 Jan, 45(1), 9 - 16
Bacillus thermoamylovorans sp . nov., a moderately thermophilic and amylolytic bacterium; Combet-Blanc Y et al.; A moderately thermophilic, facultatively anaerobic, amylolytic bacterium was isolated from palm wine, a tropical alcoholic beverage that was sampled in Senegal . The cells were gram positive, catalase positive, non-spore forming, rod shaped, and slightly motile with peritrichous flagella . The strain which we examined did not possess cytochrome and produced L-(+)-lactate, acetate, ethanol, and formate but not hydrogen during carbohydrate fermentation . Growth occurred at pH values ranging from 5.4 to 8.5, and optimum growth occurred at around pH 7.0 . The optimum temperature for growth was around 50 degrees C, and the upper temperature limit for growth was 58 degrees C . The guanine-plus-cytosine content of the DNA was 38.8 +/- 0.2 mol% . A sequence analysis of the 16S rRNA gene revealed that the new organism is closely related phylogenetically to members of genus Bacillus . Despite the lack of spores, we propose that on the basis of phylogenetic characteristics, the new isolate should be classified as a new Bacillus species, Bacillus thermoamylovorans . The type strain is strain DKP (= Collection of Institut Pasteur CNCM I-1378).

Int J Syst Bacteriol, 1995 Jan, 45(1), 67 - 77
Thermocrispum gen . nov., a new genus of the order Actinomycetales, and description of Thermocrispum municipale sp . nov . and Thermocrispum agreste sp . nov; Korn-Wendisch F et al.; Ten strains of thermophilic actinomycetes were isolated from waste and mushroom composts, as well as from the air of compost plants and a refuse incineration plant in Germany . These organisms produce white aerial mycelia and form hyphae with so-called pseudosporangia that fragment into rod-like structures . The organisms have type III cell walls (meso-diaminopimelic acid and whole-cell sugar type C), the phospholipid type is type PII, and mycolic acids are not present . The major menaquinone is MK-9(H4), and the fatty acids are mainly iso- and anteiso-branched fatty acids, hydroxy fatty acids, and 10-methyl-branched fatty acids . The guanine-plus-cytosine content of the DNA is 69 to 73 mol% . The chemotaxonomic markers (especially whole-cell sugar type C) and 16S ribosomal DNA sequence data indicated that these organisms represent a new genus of the order Actinomycetales, for which the name Thermocrispum is proposed . On the basis of phylogenetic and phenotypic data, this new genus is closely related to members of the family Pseudonocardiaceae and related taxa and contains two species: Thermocrispum municipale sp . nov . and Thermocrispum agreste sp . nov . The type species of the genus is T . municipale, with type strain MKD 35 (= DSM 44069), and the type strain of T . agreste is CHB 77 (= DSM 44070).

Genes Dev, 1995 Jan 1, 9(1), 59 - 71
An unusual sequence arrangement in the telomeres of the germ-line micronucleus in Tetrahymena thermophila; Kirk KE et al.; The ciliated protozoan Tetrahymena thermophila contains two nuclei that differ dramatically in function, chromosome size and number, chromatin structure, and mode of division . It is possible that the telomeres of the two nuclei have different functions . Although macronuclear telomeric DNA has been well characterized and consists of tandem G4T2/C4A2 repeats that are synthesized by the enzyme telomerase, micronuclear telomeres have not been isolated previously . Here, we report the identification and cloning of micronuclear telomeres and demonstrate that although they contain the same terminal tandem G4T2 repeats as macronuclear telomeres, they are strikingly different in three respects . First, the tracts of G/C-rich telomeric repeats are approximately seven times longer in the micronucleus than in the macronucleus (approximately 2.0-3.4 vs . approximately 0.3-0.5 kb, respectively) from the same cell population . Second, the immediate telomere-associated sequences (TASs) from six different micronuclear chromosome ends have an unusually high G/C content and degree of homology to one another, unlike macronuclear TASs . The TAS from at least one micronuclear chromosome is unique to micronuclear telomeres and is not present in the macronucleus . Finally, and unexpectedly, all micronuclear telomere clones contain an inner homogeneous tract of a variant G4T3 repeat adjacent to the distal tract of G4T2 repeats . The native micronuclear telomeric DNA is composed of approximately 30% G4T3 repeats, corresponding to 0.6-1.0 kb per average telomere, positioned centromere-proximally to most or all of the G4T2 repeats . Neither the G4T3 sequence nor any other variant repeat is found in macronuclear telomeres . Furthermore, such a homogeneous tract of a variant repeat has not been found in the telomeres of any eukaryote.

Biochem J, 1995 Jan 1, 305 ( Pt 1), 17 - 20
The effect of low temperatures on enzyme activity; More N et al.; The stability of two enzymes from extreme thermophiles (glutamate dehydrogenase from Thermococcales strain AN1 and beta-glucosidase from Caldocellum saccharolyticum expressed in Escherichia coli) has been exploited to allow measurement of activity over a 175 degrees C temperature range, from +90 degrees C to -85 degrees C for the glutamate dehydrogenase and from +90 degrees C to -70 degrees C for the beta-glucosidase . The Arrhenius plots of these enzymes, and those for two mesophilic enzymes (glutamate dehydrogenase from bovine liver and beta-galactosidase from Escherichia coli), exhibit no downward deflection corresponding to the glass transition, found by biophysical measurements of several non-enzymic mesophilic proteins at about -65 degrees C and reflecting a sharp decrease in protein flexibility as the overall motion of groups of atoms ceases.

J Bacteriol, 1995 Jan, 177(2), 482 - 5
Purification and characterization of a maltase from the extremely thermophilic crenarchaeote Sulfolobus solfataricus; Rolfsmeier M et al.; A soluble maltase (alpha-glucosidase) with an apparent subunit mass of 80 kDa was purified to homogeneity from Sulfolobus solfataricus . The enzyme liberates glucose from maltose and malto-oligomers . Maximal activity was observed at 105 degrees C, with half-lives of 11 h (85 degrees C), 3.0 h (95 degrees C), and 2.75 h (100 degrees C) . The enzyme was generally resistant to proteolysis and denaturants including aliphatic alcohols . n-Propanol treatment at 85 degrees C increased both Km and Vmax for maltose hydrolysis.

J Biochem (Tokyo), 1995 Jan, 117(1), 113 - 9
ATP-hydrolyzing excitation state of the reconstituted alpha 3 beta 3 complex of ATP synthase from the thermophilic bacterium PS3: structural characteristics shown by time-resolved small-angle X-ray scattering with synchrotron radiation; Sato M et al.; The ATP-hydrolyzing excitation state of the alpha 3 beta 3 complex of the ATP synthase from the thermophilic bacterium PS3 was investigated using time-resolved small-angle X-ray scattering with synchrotron radiation . The results showed the presence of the alpha 3 beta 3 complex at a steady state during ATP hydrolysis when the alpha 3 beta 3 hexamer reacted with Mg-ATP . The radius of gyration of the complex in the steady state was significantly larger than that of the Mg-AMP-PNP-hexamer complex, indicating a conformational change to an expanded structure during catalysis . This alpha 3 beta 3 complex dissociated into alpha 1 beta 1 heterodimers with apparent first-order reaction kinetics after all the ATPs were converted to ADPs . In contrast, when the alpha 3 beta 3 complex reacted with Mg-ADP, the complex dissociated into dimers with apparent first-order reaction kinetics without showing the steady state of the complex . The dimers, however, re-associated into the hexamer when Mg-ATP was added . The results were well-explained by a computer simulation based on non-linear chemical dynamics, in which a reaction mechanism that incorporates the dynamic structure of the hexamer in the steady state was considered.

Protein Sci, 1995 Jan, 4(1), 84 - 92
Modeling substrate binding in Thermus thermophilus isopropylmalate dehydrogenase; Zhang T et al.; The Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) and Escherichia coli isocitrate dehydrogenase (ICDH) are two functionally and evolutionarily related enzymes with distinct substrate specificities . To understand the determinants of substrate specificities of the two proteins, the substrate and coenzyme in IPMDH were docked into their respective binding sites based on the published structure for apo IPMDH and its sequence and structural homology to ICDH . This modeling study suggests that (1) the substrate and coenzyme (NAD) binding modes of IPMDH are significantly different from those of ICDH, (2) the interactions between the substrates and coenzymes help explain the differences in substrate specificities of IPMDH and ICDH, and (3) binding of the substrate and coenzyme should induce a conformational change in the structure of IPMDH.

Protein Eng, 1995 Jan, 8(1), 39 - 43
Thermal stability of chimeric isopropylmalate dehydrogenase genes constructed from a thermophile and a mesophile; Numata K et al.; Chimeric isopropylmalate dehydrogenases were constructed by connecting the genes isolated from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis . These genes were expressed in Escherichia coli . The enzymes were purified and analysed . Enzymes of T.thermophilus and B.subtilis and chimeric enzymes showed similar enzymological characteristics except for thermal stability . The stability of each enzyme was approximately proportional to the content of the amino acid sequence from the T.thermophilus enzyme . The results suggested that amino acid residues contributing the thermal stability distribute themselves, in general, evenly at least in the N-terminal half of the amino acid sequence of T.thermophilus isopropylmalate dehydrogenase.

Protein Eng, 1995 Jan, 8(1), 31 - 7
Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability; Ammendola S et al.; The alcohol dehydrogenase gene from the thermophilic archaeum Sulfolobus solfataricus has been subcloned and expressed in Escherichia coli under the control of the T7 inducible promoter . The recombinant protein shows properties analogous to those of the native enzyme, including thermostability, despite the fact that E.coli does not post-translationally modify two lysine residues which are N-epsilon-methylated in the native enzyme . We constructed a 3-D model of the S.solfataricus alcohol dehydrogenase using the known structure of its isozyme from horse liver as a template . Our analysis of the structural zinc binding site suggested that this site is present and functional in the S.solfataricus enzyme and that a glutamate ligand can contribute to thermostability by influencing electrostatic interactions around the metal centre . To investigate this hypothesis, we constructed, expressed and characterized a mutant where the glutamate is replaced by a cysteine, thus restoring the zinc binding site of mesophilic alcohol dehydrogenases . The mutant shows the same activity but a reduced thermostability with respect to the wild-type recombinant protein, as suggested by our model.

Appl Microbiol Biotechnol, 1995 Jan, 42(5), 807 - 11
Studies on the biosorption of uranium by Talaromyces emersonii CBS 814.70 biomass; Bengtsson L et al.; Residual biomass, produced by the thermophilic fungus, Talaromyces emersonii CBS 814.70, following growth on glucose-containing media, was examined for its ability to take up uranium from aqueous solution . It was found that the biomass had a relatively high observed biosorption capacity for the uranium (280 mg/g dry weight biomass) . The calculated maximum biosorption capacity obtained by fitting the data to a Langmuir model was calculated to be 323 mg uranium/g dry weight biomass . Pretreatment of the biomass with either dilute HCl or NaOH brought about a significant decrease in biosorptive capacity for uranium . Studies on the effects of variation in temperature on the biosorptive capacity demonstrated no significant change in binding between 20 degrees C and 60 degrees C . However, a significant decrease in biosorptive capacity was observed at 5 degrees C . Binding of uranium to the biomass at all temperatures reached equilibrium within 2 min . While the routine binding assays were performed at pH 5.0, adjustment of the pH to 3.0 gave rise to a significant decrease in biosorption capacity by the biomass . The biosorptive capacity of the biomass for uranium was increased when extraction from solution in sea-water was examined.

Plasmid, 1995 Jan, 33(1), 7 - 14
Native promoter-plasmid vector system for heterologous cholesterol oxidase synthesis in Streptococcus thermophilus; Somkuti GA et al.; The cholesterol oxidase gene (choA) of a streptomycete was used as a model for studying heterologous gene expression in Streptococcus thermophilus, an essential bacterium in dairy food fermentations . The vectors pER82 and pER82P were developed from the 2.2-kb indigenous plasmid (pER8) of S . thermophilus ST108, and sP1, a 51-bp synthetic promoter patterned after a chromosomal sequence of S . thermophilus . The presence of sP1 promoter in pER82PbCOb with the choA insert aligned with the cat gene was essential for the intracellular production of cholesterol oxidase . The pER82PbCOb was apparently stable in S . thermophilus with no detectable evidence of deletion mutational events.

Antonie Van Leeuwenhoek, 1995, 67(1), 91 - 102
Methanogenesis in thermophilic biogas reactors; Ahring BK; Methanogenesis in thermophilic biogas reactors fed with different wastes is examined . The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined . Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors . The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor . The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor . The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present . Methanogens that reacted with the probe against Methanobacterium thermoautotrophicum were the most numerous in this reactor . For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN . The majority of acetate utilizing methanogens in the reactor were Methanosarcina spp . single cells, which is a difficult form of the organism to cultivate in vitro . No reactions were observed with antibody probes raised against Methanothrix soehngenii or Methanothrix CALS-1 in any of the thermophilic biogas reactors examined . Studies using 2-14C-labeled acetate showed that at high concentrations (more than approx . 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane . When the concentration of acetate was less than approx . 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane . In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche of Methanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems . Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56 degrees C . Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55 degrees C could be obtained at 61 degrees C after a long adaptation period . However, propionate degradation was inhibited by increasing the temperature.

Antonie Van Leeuwenhoek, 1995, 67(1), 3 - 28
Anaerobic digestion and wastewater treatment systems; Lettinga G; Upflow Anaerobic Sludge Bed (UASB) wastewater (pre-)treatment systems represent a proven sustainable technology for a wide range of very different industrial effluents, including those containing toxic/inhibitory compounds . The process is also feasible for treatment of domestic wastewater with temperatures as low as 14-16 degrees C and likely even lower . Compared to conventional aerobic treatment systems the anaerobic treatment process merely offers advantages . This especially is true for the rate of start-up . The available insight in anaerobic sludge immobilization (i.e . granulation) and growth of granular anaerobic sludge in many respects suffices for practice . In anaerobic treatment the immobilization of balanced microbial communities is essential, because the concentration of intermediates then can be kept sufficiently low . So far ignored factors like the death and decay rate of organisms are of eminent importance for the quality of immobilized anaerobic sludge . Taking these factors into account, it can be shown that there does not exist any need for 'phase separation' when treating non- or slightly acidified wastewaters . Phase separation even is detrimental in case the acidogenic organisms are not removed from the effluent of the acidogenic reactor, because they deteriorate the settleability of granular sludge and also negatively affect the formation and growth of granular sludge . The growing insight in the role of factors like nutrients and trace elements, the effect of metabolic intermediates and end products opens excellent prospects for process control, e.g . for the anaerobic treatment of wastewaters containing mainly methanol . Anaerobic wastewater treatment can also profitably be applied in the thermophilic and psychrophilic temperature range . Moreover, thermophilic anaerobic sludge can be used under mesophilic conditions . The Expanded Granular Sludge Bed (EGSB) system particularly offers big practical potentials, e.g . for very low strength wastewaters (COD << 1 g/l) and at temperatures as low as 10 degrees C . In EGSB-systems virtually all the retained sludge is employed, while compared to UASB-systems also a substantially bigger fraction of the immobilized organisms (inside the granules) participates in the process, because an extraordinary high substrate affinity prevails in these systems . It looks necessary to reconsider theories for mass transfer in immobilized anaerobic biomass . Instead of phasing the digestion process, staging of the anaerobic reactors should be applied . In this way mixing up of the sludge can be significantly reduced and a plug flow is promoted . A staged process will provide a higher treatment efficiency and a higher process stability . This especially applies for thermophilic systems.

Antonie Van Leeuwenhoek, 1995, 67(4), 339 - 44
Thermophilic actinomycetes in cane sugar mills: an aeromicrobiologic and seroepidemiologic study; Khan ZU et al.; Aerial prevalence of clinically important thermophilic actinomycetes and occurrence of precipitating antibodies against them in sera of 153 exposed workers have been reported . The study was carried out in two cane sugar mills namely, the Upper Doab Sugar Mills and the Ramala Sugar Mills, located in north-west India . In both the sugar mills, T . sacchari was the predominant species, it accounted for 55.1% and 50.3% of the total population of thermophilic actinomycetes, followed by T . vulgaris (19.7% and 23.7%), T . thalpophilus (21.1% and 17.1%), Saccharomonospora viridis (3.4% and 5.0%) and Saccharopolyspora rectivirgula (Faenia rectivirgula) (0.7% and 3.9%), respectively . Precipitating antibodies against thermophilic actinomycetes were demonstrable in 34 (22.2%) workers; T . sacchari alone accounted for 20 of the positive precipitin reactions, followed by S . rectivirgula in 10 . The mean absorbance values for IgG antibody activity against T . sacchari as well as S . rectivirgula were found to be elevated significantly in the symptomatic workers than in the asymptomatic workers (p < 0.05) or unexposed controls (p < 0.001) . However, the difference in IgG antibody activity was insignificant between precipitin-positive symptomatic workers and precipitin-positive asymptomatic workers . The results indicate that clinically important thermophilic actinomycetes are widely prevalent in cane sugar mills, and T . sacchari and S . rectivirgula are the major species involved in the sensitization of the bagasse workers in India.

Acta Vet Scand, 1995, 36(1), 79 - 85
Survival of salmonellas and Ascaris suum eggs in a thermophilic biogas plant; Plym-Forshell L; In a continuous biogas plant, receiving manure from 200 dairy cows and 400 calves and young stock, survival of salmonellas and Ascaris suum eggs was studied . The bacteria and parasite eggs were kept in filter sacs in the manure that had a temperature of 55 degrees C . No viable salmonellas or Ascaris suum eggs could be found after 24h in the digester . Survival of salmonellas and Ascaris suum eggs was also studied in the manure pit where the manure was stored after digestion . The temperature in the manure pit varied between 22-27 degrees C . Salmonellas survived 35 but not 42 days . On day 56, when the experiments had to be stopped, 60% of the Ascaris eggs were viable.

J Eukaryot Microbiol, 1995 Jan-Feb, 42(1), 32 - 43
Circular rDNA replicons persist in Tetrahymena thermophila transformants synthesizing GGGGTC telomeric repeats; Romero DP et al.; Site-directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo . The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach . The telomerase RNA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection . Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo . However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons . The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation.

Gene, 1994 Dec 30, 151(1-2), 231 - 5
Sequence of the macronuclear DNA encoding large subunit ribosomal protein 29 (L29) in Euplotes crassus and cycloheximide sensitivity; Jahn CL et al.; As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines . All of the cell lines are highly sensitive to Chx . Progeny cell lines show a wider range of sensitivities than the parental lines . Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila {Yao and Yao, Proc . Natl . Acad . Sci . USA 88 (1991) 9493-9497}, we isolated and sequenced the entire E . crassus macronuclear DNA carrying RPL29 . The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T . thermophila and Saccharomyces cerevisiae to mouse . In E . crassus, the codon that has been mutated to confer Chx resistance in both S . cerevisiae and T . thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms . Thus, E . crassus RPL29 is not a convenient source of a selectable marker . Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.

Gene, 1994 Dec 30, 151(1-2), 177 - 80
Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases; Jonsson ZO et al.; By dideoxynucleotide sequencing of a genomic clone, we have determined the complete nucleotide sequence of the gene encoding NAD(+)-dependent DNA ligase (EC 6.5.1.2) of the thermophilic bacterium Thermus scotoductus . The gene encodes a 674-amino-acid thermostable enzyme highly similar to other bacterial DNA ligases and to parts of the deduced gene product of Escherichia coli ORF f562, 5' to the spoR gene encoding 5' guanosyl kinase.

Biochem Biophys Res Commun, 1994 Dec 30, 205(3), 1869 - 74
Does the eukaryotic large ribosomal subunit have a channel passing through it?
Harauz G, Beniac DR, Wu J, Zuzan H.
The large ribosomal subunit of the thermophilic fungus Thermomyces lanuginosus was treated with 2.96 M NH4Cl to remove specific complements of ribosomal proteins, and the core particles thereby derived were imaged by bright field transmission electron microscopy, and recurring views computed by single particle electron image analysis . A new characteristic projection was elucidated which showed a large depression or channel passing through the subunit . Such a channel has been perceived in the prokaryotic large ribosomal subunit under certain conditions and has been postulated to be the exit pathway for the nascent polypeptide chain, but its existence has not hitherto been demonstrated in eukaryotes.

Biochim Biophys Acta, 1994 Dec 30, 1188(3), 302 - 10
Over-expression of membrane-bound cytochrome c-551 from thermophilic Bacillus PS3 in Bacillus stearothermophilus K1041; Noguchi S et al.; Cytochrome c-551 is a lipoprotein of about 10500 Da, found in thermophilic Bacillus PS3 grown under air-limited conditions . An expression vector was constructed from a structural gene of PS3 cytochrome c-551, synthetic oligonucleotide as a promoter for Bacillus stearothermophilus and a shuttle vector for Escherichia coli and B . stearothermophilus . The transformed cells of B . stearothermophilus K1041 expressed cytochrome c-551 as much as 5 nmol/mg membrane protein . The effects of over-expression on the host cells are analyzed; a slightly slower growth rate and an increased synthesis of cytochrome oxidase (about twofold) occurred . Over-expressed (4-10-fold) cytochrome c-551 were purified and its properties were examined to know whether the protein is processed as in PS3 cells grown under air-limited conditions . The molecular mass determination and treatment with Rhizopus lipase suggested that the same processes, cleavage of signal peptidase, blocking of the N-terminal group and acylation of glycerol residue by two fatty acids, took place in the over-expression system . Fatty acylation seems useful for the cytochrome c to be effectively oxidized.

Biochemistry, 1994 Dec 27, 33(51), 15433 - 6
Protein coordination to manganese determines the high catalytic rate of dimanganese catalases . Comparison to functional catalase mimics; Shank M et al.; Catalysis of hydrogen peroxide dismutation by the dimanganese catalase from Thermus thermophilus has been measured and found to obey Michaelis-Menton kinetics with no evidence for substrate inhibition at concentrations up to 0.45 M H2O2 . Comparison among three dimanganese catalases (Thermus thermophilus, Thermoleophilium album, and Lactobacillus plantarum) reveals that their apparent second-order rate constants, Kcat/Km, differ by at most a factor of 5, even though the individual kinetic constants differ by as much as a factor of 20 . This similarity suggests that all three enzymes may have the same rate-determining step . For T . thermophilus catalase we find that kcat/Km approximately kbi, the bimolecular rate constant at limiting substrate concentrations . Thus, the rate of the rate-determining step is unaltered over the entire range of substrate concentrations, unlike T . album and L . plantarum catalases where substrate inhibition has been reported . Comparison to structurally characterized dimanganese complexes and dimetalloproteins (arginase, hemerythrin), which are functional, albeit kinetically slow, catalase mimics, reveals that high catalase activity correlates with a greater number of stronger sigma-ligand donors like anionic carboxylatos vs neutral histidines that stabilize the oxidized Mn2(III,III) state over reduced Mn2(II,II) . A critical feature for enzymatic functionality in vivo is suppression of one-electron chemistry leading to formation of the mixed-valence forms, Mn2(III,IV) and Mn2(II,III), which are kinetically inactive or precursors to inactive species, respectively . Evidence is presented from model compounds suggesting that the mu-carboxylato bridge between Mn ions in catalase may play the key role in suppressing formation of these detrimental oxidation states through destabilization of these one-electron redox processes.

Nucleic Acids Res, 1994 Dec 25, 22(25), 5702 - 8
Detection of circular excised DNA deletion elements in Tetrahymena thermophila during development; Yao MC et al.; Extensive programmed DNA deletion occurs in ciliates during development . In this study we examine the excised forms of two previously characterized deletion elements, the R- and M-element, in Tetrahymena . Using divergently oriented primers in polymerase chain reactions we have detected the junctions formed by joining the two ends of these elements, providing evidence for the presence of circular excised forms . These circular forms were detected in developing macronuclear DNA from 12-24 h after mating began, but not in micronuclear or whole cell DNA of vegetative cells . They are present at very low abundance, detectable after PCR only through hybridization with specific probes . Sequence analysis shows that the circle junctions occur at or very near the known ends of the elements . There is sequence microheterogeneity in these junctions, which does not support a simple reciprocal exchange model for DNA deletion . A model involving staggered cuts and variable mismatch repair is proposed to explain these results . This model also explains the sequence microheterogeneity previously detected among the junction sequences retained in the macronuclear chromosome.

Nucleic Acids Res, 1994 Dec 25, 22(25), 5695 - 701
The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila; Saveliev SV et al.; Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila . In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR . However, the circles are not normal products of the reaction pathway . The circular forms occur at very low levels in conjugating cells, but are stable . Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions . The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region . The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1) . Some new chromosomal junctions were detected in the present study . The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly . The species is only rarely converted to the stable circles we detect . The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5).

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12837 - 41
Heterogeneity of genomes: measures and values; Karlin S et al.; Genomic homogeneity is investigated for a broad base of DNA sequences in terms of dinucleotide relative abundance distances (abbreviated delta-distances) and of oligonucleotide compositional extremes . It is shown that delta-distances between different genomic sequences in the same species are low, only about 2 or 3 times the distance found in random DNA, and are generally smaller than the between-species delta-distances . Extremes in short oligonucleotides include underrepresentation of TpA and overrepresentation of GpC in most temperate bacteriophage sequences; underrepresentation of CTAG in most eubacterial genomes; underrepresentation of GATC in most bacteriophage; CpG suppression in vertebrates, in all animal mitochondrial genomes, and in many thermophilic bacterial sequences; and overrepresentation of GpG/CpC in all animal mitochondrial sets and chloroplast genomes . Interpretations center on DNA structures (dinucleotide stacking energies, DNA curvature and superhelicity, nucleosome organization), context-dependent mutational events, methylation effects, and processes of replication and repair.

Eur J Biochem, 1994 Dec 15, 226(3), 811 - 8
Thermodynamics of the formylmethanofuran dehydrogenase reaction in Methanobacterium thermoautotrophicum; Bertram PA et al.; Purified formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum, which is a thermophilic methanogenic Archaeon growing on H2 and CO2, was shown to catalyze the reversible reduction of CO2 to N-formylmethanofuran with 1,1',2,2'-tetramethylviologen (E'0 = -550 mV) as electron donor . The rate of CO2 reduction was approximately 25 times higher than the rate of N-formylmethanofuran dehydrogenation . From determinations of equilibrium concentrations at 60 degrees C and pH 7.0 a midpoint potential (E'0) for the CO2 + methanofuran/formylmethanofuran couple of approximately -530 mV was estimated . The initial step of methanogenesis from CO2 thus has a midpoint potential considerably more negative than that of the H+/H2 couple (E'0 = -460 mV at 60 degrees C) . Evidence is described indicating that the as-yet unidentified physiological electron donor of the formylmethanofuran dehydrogenase is present in the soluble cell fraction.

Structure, 1994 Dec 15, 2(12), 1157 - 67
The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum; Russell RJ et al.; BACKGROUND: The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity . Different members of the thermophilic Archaea tolerate temperatures in the range 55-110 degrees C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15-45 degrees C) may provide important information on the structural basis of protein thermostability . We have chosen citrate synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies . RESULTS: We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%) . CONCLUSIONS: The three-dimensional structural comparison of thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.

Nucleic Acids Res, 1994 Dec 11, 22(24), 5391 - 8
High frequency vector-mediated transformation and gene replacement in Tetrahymena; Gaertig J et al.; Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells . Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus . We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments . Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus . Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold . The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid . However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used . This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG . Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene . This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid . Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena . As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.

FEBS Lett, 1994 Dec 5, 355(3), 233 - 6
Pyridine dinucleotide biosynthesis in archaebacteria: presence of NMN adenylyltransferase in Sulfolobus solfataricus; Raffaelli N et al.; The enzyme NMN adenylyltransferase, leading to NAD synthesis, has been observed for the first time in soluble extracts from the extreme acidothermophilic archaeon Sulfolobus solfataricus . Comparison of its molecular and kinetic properties with those of the enzyme isolated from prokaryotes and eukaryotes revealed significant differences, knowledge of which may contribute to the understanding of metabolic evolutionary mechanisms . The thermophilic enzyme shows a molecular mass of about 66,000 and an isoelectric point of 5.4 . The Km values for ATP, NMN and nicotinic acid mononucleotide are 0.08 microM, 1.4 microM and 17 microM, respectively . The enzyme shows a remarkable degree of thermophilicity, with an activation energy of 95 kJ/mol.

J Immunol Methods, 1994 Dec 2, 176(2), 235 - 43
In situ transcription with Tth DNA polymerase and fluorescent nucleotides; Chang H; We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST) . They are simple alternatives to the tedious steps of in situ hybridization with labeled probes . We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods . We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST) . Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight . They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried . The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip . The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip . Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2 . After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried . The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy . Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer . FIST appears to be useful for the detection of specific mRNAs in single cells.

J Immunol Methods, 1994 Dec 2, 176(2), 153 - 62
Amplified electrochemical immunoassay for thyrotropin using thermophilic beta-NADH oxidase; Athey D et al.; The use of the highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase) from the thermophilic organism Thermus aquaticus in combination with alcohol dehydrogenase in an amperometric amplified immunoassay for thyrotropin (TSH) is described . NADH oxidase catalyses the oxidation of reduced nicotinamide adenine dinucleotide (NADH) with concomitant two electron reduction of di-oxygen to hydrogen peroxide . Hydrogen peroxide can be detected by oxidation at a platinum electrode poised at +650mV vs . Ag/AgCl . The enzyme amplification system described has advantages over existing amplification techniques in terms of sensitivity, specificity and operational pH dependence . The electrochemical enzyme-amplified assay for TSH was compared with a spectrophotometric enzyme-amplified system and with a non-amplified electrochemical immunoenzymometric TSH assay . The dynamic range of the electrochemical enzyme-amplified TSH immunoassay was 0.2-100 mIU/l, which was four times that of the enzyme-amplified spectrophotometric assay while the detection limits of both techniques were comparable.

J Bacteriol, 1994 Dec, 176(24), 7744 - 7
A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance; Aagaard C et al.; Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes . Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius . Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees . Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol . Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA) . The single-site mutation also conferred resistance to carbomycin . The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.

J Bacteriol, 1994 Dec, 176(24), 7703 - 10
Phylogenetic depth of S10 and spc operons: cloning and sequencing of a ribosomal protein gene cluster from the extremely thermophilic bacterium Thermotoga maritima; Sanangelantoni AM et al.; A segment of Thermotoga maritima DNA spanning 6,613 bp downstream from the gene tuf for elongation factor Tu was sequenced by use of a chromosome walking strategy . The sequenced region comprised a string of 14 tightly linked open reading frames (ORFs) starting 50 bp downstream from tuf . The first 11 ORFs were identified as homologs of ribosomal protein genes rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, and rps17 (which in Escherichia coli constitute the S10 operon, in that order); the last three ORFs were homologous to genes rpl14, rpl24, and rpl5 (which in E . coli constitute the three promoter-proximal genes of the spectinomycin operon) . The 14-gene string was preceded by putative -35 and -10 promoter sequences situated 5' to gene rps10, within the 50-bp spacing between genes tuf and rps10; the same region exhibited a potential transcription termination signal for the upstream gene cluster (having tuf as the last gene) but displayed also the potential for formation of a hairpin loop hindering the terminator; this suggests that transcription of rps10 and downstream genes may start farther upstream . The similar organization of the sequenced rp genes in the deepest-branching bacterial phyla (T . maritima) and among Archaea has been interpreted as indicating that the S10-spc gene arrangement existed in the (last) common ancestor . The phylogenetic depth of the Thermotoga lineage was probed by use of r proteins as marker molecules: in all except one case (S3), Proteobacteria or the gram-positive bacteria, and not the genus Thermotoga, were the deepest-branching lineage; in only two cases, however, was the inferred branching order substantiated by bootstrap analysis.

J Bacteriol, 1994 Dec, 176(24), 7413 - 22
Physical and genetic map of Streptococcus thermophilus A054; Roussel Y et al.; The three restriction endonucleases SfiI, BssHII, and SmaI were found to generate fragments with suitable size distributions for mapping the genome of Streptococcus thermophilus A054 . A total of 5, 8, and 24 fragments were produced with SfiI, BssHII, and SmaI, respectively . An average genome size of 1,824 kb was determined by summing the total fragment sizes obtained by digestions with these three enzymes . Partial and multiple digestions of genomic DNA in conjunction with Southern hybridization were used to map SfiI, BssHII, and SmaI fragments . All restriction fragments were arranged in a unique circular chromosome . Southern hybridization analysis with specific probes allowed 23 genetic markers to be located on the restriction map . Among them, six rrn loci were precisely located . The area of the chromosome containing the ribosomal operons was further detailed by mapping some of the ApaI and SgrAI sites . Comparison of macrorestriction patterns from three clones derived from strain A054 revealed two variable regions in the chromosome . One was associated with the tandem rrnD and rrnE loci, and the other was mapped in the region of the lactose operon.

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