Eur J Biochem, 1991 Nov 1, 201(3), 593 - 600 Purification and characterisation of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius; Moll R et al.; A succinate dehydrogenase complex was isolated in a three-step purification from plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius . It consists of four subunits: a, 66 kDa; b, 31 kDa; c, 28 kDa and d, 12.8 kDa . In the 141-kDa native protein, the four subunits are present in an equimolar stoichiometry . The complex contains acid-non-extractable flavin, iron and acid-labile sulphide . Maximal succinate dehydrogenase activities were recorded at pH 6.5, which coincides with the internal pH of Sulfolobus cells . The temperature optimum of 81 degrees C defines the Sulfolobus succinate dehydrogenase as a thermophilic enzyme complex . The Km for succinate was found to be 1.42 mM (55 degrees C) . Similar to the mitochondrial soluble succinate dehydrogenase, this enzyme is capable of transferring electrons to artificial electron acceptors, for instance phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine and ferricyanide . In contrast to the mitochondrial succinate dehydrogenase, the archaebacterial enzyme reduces 1,4-dichloroindophenol also in the absence of phenazine methosulfate . Caldariella quinone, the physiological electron mediator in the Sulfolobus respiratory chain, was only slowly reduced under adjusted conditions . The succinate--phenazine methosulfate-(1,4-dichloroindophenol) oxidoreductase of the isolated complex was strongly inhibited by tetrachlorobenzoquinone . In plasma membranes the complex reduces molecular oxygen in a cyanide-sensitive reaction . Polyclonal Sulfolobus anti-a antibodies crossreacted with 66-67-kDa polypeptides from membranes of Thermoplasma acidophilium, Sulfolobus solfataricus and beef heart submitochondrial particles. J Protozool, 1991 Nov-Dec, 38(6), 537 - 47 Biochemical and cytological evidence for an overabundance of mucocysts in the bcd pattern mutant of Tetrahymena thermophila; Cole ES et al.; Three acidic proteins (42 kD, 43 kD and 50 kD) were present in unusually high concentrations in cortical preparations of the Tetrahymena pattern mutant broadened cortical domains (bcd) . Antisera to the 42-kD and 50-kD proteins bound to discharging mucocysts and food vacuole contents in both wild-type and mutant cells . Subsequent analysis revealed that bcd mutant cell pellicles possess five times more "docked" mucocysts than their wild-type counterparts. J Clin Microbiol, 1991 Nov, 29(11), 2438 - 45 Analysis of the role of flagella in the heat-labile Lior serotyping scheme of thermophilic Campylobacters by mutant allele exchange; Alm RA et al.; Flagellin mutations originally constructed in Campylobacter coli VC167 (serotype LIO8) by a gene replacement mutagenesis technique (P . Guerry, S . M . Logan, S . Thornton, and T . J . Trust, J . Bacteriol . 172:1853-1860, 1990) were moved from the original host into Campylobacter strains of a number of other Lior serogroups by a natural transformation procedure . This is the first report of the use of this transformation method to transfer a mutated locus among Campylobacter strains . Flagellin mutants were constructed in a number of heat-labile LIO serotypes and were serotyped and analyzed by immunoelectron microscopy with LIO typing antisera . In six cases, isogenic nonflagellated mutants were able to be serotyped in the same serogroup as their parent, and immunogold electron microscopy confirmed that antibodies in the typing antisera bound to components on the surface of both parent and mutant cells . However, in only one case, a strain belonging to serogroup LIO4, was a nonflagellated mutant untypeable, and immunogold electron microscopy showed that antibodies bound to the flagella filament of the parent but not to the cell surface . Furthermore, after introduction and expression as a flagellar filament of a LIO8 flagellin gene in this mutant, the strain could not be serotyped . These results indicate that a nonflagellar antigen is often the serodeterminant in the heat-labile Lior serotyping scheme. Vet Microbiol, 1991 Nov, 29(3-4), 225 - 35 Survival of Aujeszky's disease virus in slurry at various temperatures; Botner A; Survival of Aujeszky's disease virus in pig slurry was investigated during anaerobic storage at 5, 20, 35, 40, 45, 50 and 55 degrees C using 100-ml laboratory models simulating the conditions in slurry tanks during winter and summer seasons and during anaerobic digestion in batch reactors . The inactivation rate was found to increase with increasing temperature . Virus was inactivated at 5 and 20 degrees C in 15 weeks and 2 weeks, respectively . At 35 degrees C (mesophilic conditions) the virus was inactivated in 5 hours and at 55 degrees C (thermophilic conditions) no virus could be detected after 10 minutes. J Bacteriol, 1991 Nov, 173(22), 7070 - 6 Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability; Takemura H et al.; Acetobacter pasteurianus NCI1380, a thermophilic strain isolated from the surface culture of acetic acid fermentation, showed genetic instability to produce at high frequency spontaneous mutants which were deficient in ethanol oxidation because of the loss of alcohol dehydrogenase activity . Southern hybridization experiments with the cloned alcohol dehydrogenase-cytochrome c gene cluster as the probe showed insertion of an unknown DNA fragment into a specific position in the cytochrome c gene in most of the mutant strains . Cloning and sequencing analyses revealed that the inserted sequence was 1,665 bp in length and had a terminal inverted repeat of 15 bp . In addition, this inserted sequence was found to generate a 4-bp duplication at the inserted site upon transposition . The target site specificity was not very strict, but a TCGA sequence appeared to be preferentially used . The inserted sequence contains two long open reading frames of 461 and 222 amino acids which are overlapped and encoded by different strands . Although these open reading frames showed no homology to any protein registered in the DNA data bases, the longer open reading frame contained many basic amino acids (87 of 461), as was observed with transposases of so-called insertion sequence (IS) elements . All of these characteristics are typical of IS elements, and the sequence was named IS1380 . The copy number of IS1380 in a cell of A . pasteurianus NCI1380 was estimated to be about 100 . Several strains of acetic acid bacteria also contained IS1380 at high copy numbers . These results suggest that IS1380 is associated with the genetic loss of ethanol-oxidizing ability as well as the genetic instability of acetic acid bacteria in general. Cell, 1991 Oct 18, 67(2), 343 - 53 A conserved secondary structure for telomerase RNA; Romero DP et al.; The RNA moiety of the ribonucleoprotein enzyme telomerase contains the template for telomeric DNA synthesis . We present a secondary structure model for telomerase RNA, derived by a phylogenetic comparative analysis of telomerase RNAs from seven tetrahymenine ciliates . The telomerase RNA genes from Tetrahymena malaccensis, T . pyriformis, T . hyperangularis, T . pigmentosa, T . hegewishii, and Glaucoma chattoni were cloned, sequenced, and compared with the previously cloned RNA gene from T . thermophila and with each other . To define secondary structures of these RNAs, homologous complementary sequences were identified by the occurrence of covariation among putative base pairs . Although their primary sequences have diverged rapidly overall, a strikingly conserved secondary structure was identified for all these telomerase RNAs . Short regions of nucleotide conservation include a block of 22 totally conserved nucleotides that contains the telomeric templating region. Biochem J, 1991 Oct 1, 279 ( Pt 1), 67 - 73 Purification and characterization of endoglucanase Ss from Clostridium thermocellum; Fauth U et al.; The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate known as the cellulosome . By using a combination of ion-exchange, adsorption and hydrophobic-interaction chromatography, it was possible to isolate from extracellular broth a specific endoglucanase of interest without the use of denaturants . The endoglucanase was identified as the cellulosomal subunit Ss by the use of specific antibodies . The enzyme has an Mr of 83,000, an isoelectric point of 3.55, optimum pH of 6.6 and optimum temperature of 70 degrees C . It hydrolyses CM-cellulose and, at a higher rate, the cellodextrins, cellotetraose and cellopentaose, but does not hydrolyse a crystalline cellulose such as Avicel . Cellobiose and cellotriose are also immune to attack . It differs from endoglucanases previously isolated by others and a 76,000-Mr endoglucanase recently isolated in this laboratory. Am Rev Respir Dis, 1991 Oct, 144(4), 855 - 60 Bronchoalveolar mast cells in normal farmers and subjects with farmer's lung . Diagnostic, prognostic, and physiologic significance; Laviolette M et al.; To evaluate the specificity and significance of increased lavage mast cells in farmer's lung, we examined the lavage cell differentials of 89 farmers and 19 normal nonfarming control subjects . The farmers were divided into four groups: acute farmer's lung (n = 17), farmers with one or more prior episodes of farmer's lung who remained in daily contact with hay (n = 26) or quit farming (n = 14), and normal farmers (n = 36) . A total of 14 of the subjects with prior episodes of farmer's lung and still farming and 15 normal farmers were evaluated twice at a 2-yr interval . The lavage mast cell numbers were significantly higher in acute farmer's lung (7.5 +/- 7.3 x 10(3)/ml, mean +/- SD) and ex-farmer's lung who were still farming (1.2 +/- 1.3 x 10(3)/ml) than in normal farmers (0.1 +/- 0.1 x 10(3)/ml) (p less than 0.01) . A total of 8 of 14 exfarmer's lung patients who had quit farming and 18 of 36 normal farmers had an increased number of mast cells in lavage, but mast cell count never exceeded 0.5% of total recovered cells . In the acute farmer's lung and ex-farmer's lung-still farming groups, the mast cell count correlated with the lymphocyte count: r = 0.83 and r = 0.69 (p less than 0.001), respectively . In the two groups evaluated twice, mast cell numbers at the first study did not correlate with changes seen at the second study in chest roentgenogram and pulmonary functions . We conclude that an increase in lavage mast cells occurs commonly as a part of the immune response against thermophilic bacteria.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1991 Oct, 229(2), 292 - 300 Organization and nucleotide sequence of the atp genes encoding the ATP synthase from alkaliphilic Bacillus firmus OF4; Ivey DM et al.; The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein . The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3 . The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits . These substitutions are in domains that have been implicated in the mechanism of proton translocation through F0-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture. Avian Dis, 1991 Oct-Dec, 35(4), 714 - 7 Isolation of Campylobacter from livers of broiler chickens with and without necrotic hepatitis lesions; Boukraa L et al.; Injured and normal livers from broiler chickens sent to slaughter plants were collected for bacterial examination . A total of 223 macroscopically abnormal livers and 50 normal livers were received . Forty-seven thermophilic Campylobacter isolates were obtained from the livers with necrotic lesions; 39 isolates were identified as Campylobacter jejuni and eight as C . coli . In normal livers, six C . jejuni isolates were obtained . C . jejuni biotype 2 was the most common isolate recovered from injured livers, and C . jejuni biotype 1 was the most frequent isolate found in normal livers . On some occasions, Campylobacter spp . could be isolated from both the liver parenchyma and bile of the same bird . Results indicated that different species and biotypes of Campylobacter can be found in the livers of broiler chickens with and without lesions of necrotic hepatitis. Biochimie, 1991 Oct, 73(10), 1281 - 5 The major pterin in Tetrahymena pyriformis is 6-(D-threo-1,2,3-trihydroxypropyl)-pterin (D-monapterin) and not 6-(L-threo-1,2-dihydroxypropyl)-pterin (ciliapterin); Klein R et al.; The major pterin in Tetrahymena pyriformis, strain W, earlier suggested to be L-threo-biopterin and named ciliapterin {1} is now identified as D-threo-neopterin (D-monapterin) . This is the first example of a natural D-monapterin . This compound was characterized by its chromatographic behavior, its fluorescence properties and by its oxidation product with alkaline permanganate . The final identification was obtained by comparison with an authentic material using an exchange ligand chromatography method with D-phenylalanine as chiral modifier and Cu (II) as metal ion . D-monapterin is also present as the major pterin in Tetrahymena pyriformis strains GL and ST, and in Tetrahymena thermophila. J Biochem (Tokyo), 1991 Oct, 110(4), 588 - 94 Temperature and solvent effects on reaction centers from Chloroflexus aurantiacus and Chromatium tepidum; Nozawa T et al.; Temperature and solvent effects on reaction center structures were examined in two thermophilic photosynthetic bacteria, Chloroflexus aurantiacus and Chromatium tepidum, in order to gain insight into the interactions among the reaction center proteins and pigment systems . Thermal stability of the reaction centers was found to be proportional to the optimum growth temperature . Circular dichroism (CD) spectra in the 250-300 nm region indicated that thermal denaturation destroyed tertiary structures (helix-to-helix interactions or amino acid residue conformation) in the native reaction center, keeping helical structures intact . Absorption and circular dichroism spectral changes showed that alcohol denatured the so-called special pair and the accessory BChl a independently . The alcohol denaturation further indicates that the coordination between BChl a and amino acid residue in the protein is one of the important interactions maintaining the pigment organization of the reaction centers. Biol Chem Hoppe Seyler, 1991 Oct, 372(10), 955 - 61 The amino-acid sequences of the Bacillus stearothermophilus ribosomal proteins S17 and S21 and their comparison to homologous proteins of other ribosomes; Herfurth E et al.; Ribosomal proteins S17 and S21 from the moderate thermophile Bacillus stearothermophilus were purified by one-step high-performance liquid chromatography from the 30S-subunit protein mixture employing a semi-preparative reversed-phase C4 column . The complete amino-acid sequences of these proteins were determined by a combination of N-terminal sequencing in picomole quantities of the protein and of appropriate peptide fragments . Proteins S17 and S21 consist of 86 and 55 amino-acid residues, corresponding to molecular masses of 10074 and 6593 Da, respectively . They are homologous to proteins S17 and S21 from the Escherichia coli ribosome, showing 50 and 55% identities in the corresponding regions, respectively . The C-terminal region of protein S21 from B . stearothermophilus has a deletion of 15 residues as compared to the E . coli S21 protein . The evolutionary relationships of the Bacillus proteins to various other members of the S17 and S21 ribosomal protein families are discussed. J Dairy Sci, 1991 Oct, 74(10), 3284 - 92 Microbiological analysis and starter culture growth in retentates; Premaratne RJ et al.; Pasteurized skim milk was concentrated by UF to 2-, 4-, and 5-fold . The retentates were evaluated for microbiological quality, heat treatments to inactivate microorganisms, and lactic acid bacterial starter culture activity . Aerobic mesophilic bacterial counts in raw milk decreased from an initial 1.4 x 10(6) to 3.9 x 10(2) cfu/ml after pasteurization . During UF, counts increased from 3.9 x 10(2) cfu/ml UF, counts increased from 3.9 x 10(2) cfu/ml in pasteurized milk to 1.4 x 10(3), 1.4 x 10(4), and 1.8 x 10(4) cfu/ml in 2-, 4- and 5-fold retentates, respectively . Psychrotrophic bacterial counts decreased from 9.9 x 10(5) cfu/ml in raw milk to 3.7 x 10(1) cfu/ml in pasteurized milk and gradually increased to 1.0 x 10(2), 2.5 x 10(2), and 1.4 x 10(3) cfu/ml in 2-, 4-, and 5-fold retentates, respectively . Thermophilic bacterial counts remained less than 10 cfu/ml in all samples . Skim milk and retentates inoculated with five starter cultures at 1% failed to decrease the pH below 4.6 in (2-, 4- and 5-fold) . The 4- and 5-fold retentates inoculated with Lactococcus lactis spp . cremoris or Lactococcus lactis spp . lactis cultures were partially coagulated with pH greater than 5.6 . In general, the pH of retentates remained higher than that of skim milk . Clotting of uninoculated samples was observed, and a spore-forming contaminant, tentatively characterized as Bacillus cereus and capable of clotting milk at a pH greater than 6, was isolated from the clotted samples.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Syst Bacteriol, 1991 Oct, 41(4), 502 - 9 Numerical analysis and DNA base compositions of some thermophilic Bacillus species; De Bartolomeo A et al.; Morphological, physiological, and biochemical characteristics and the DNA base compositions of 133 thermophilic Bacillus strains were determined . A total of 54 of these strains were received as identified species (mainly Bacillus stearothermophilus, Bacillus coagulans, Bacillus brevis, and Bacillus licheniformis) from international culture collections, and 79 newly isolated strains, which were isolated mainly from sugar diffusion juices of Italian plants, were also examined . Numerical taxonomy techniques (simple matching coefficient and unweight pair grouping using the mathematical average) and DNA G + C values showed that the strains aggregated into nine clusters . Both B . licheniformis and B . brevis were well separated from the other organisms . B . stearothermophilus and B . coagulans were confirmed as separate clusters and exhibited greater heterogeneity than previously shown . The B . stearothermophilus strains clustered into four groups, three of which have been recognized previously by other authors; the members of the fourth group had distinctive characteristics, including considerable biochemical inertness, an inability to grow at temperatures greater than 60 degrees C, and a high G + C content . Within the B . coagulans cluster the strains with characteristics very similar to those of the new species Bacillus smithii clustered together . However, the remaining strains were still clearly separated into two groups; one of these groups was considered B . coagulans sensu stricto, and the other was distinguished by morphological and biochemical criteria, such as spores which do not swell the sporangia, utilization of citrate, a higher proteolytic activity, and acidification of some carbohydrates . Our results were confirmed by comparing them with distinctive characteristics of recently described thermophilic Bacillus species. Gene, 1991 Sep 30, 106(1), 121 - 4 Native yeast telomeres are sufficient to stabilize linear DNA in Xenopus laevis oocytes; Schmid M et al.; We have constructed a linear plasmid in yeast containing the entire bovine papillomavirus genome and tested its physical stability following microinjection into stage VI oocytes of Xenopus laevis . Our results show that unmodified telomeres, in contrast to the yeast-passaged telomeres, drastically affect the stability of the injected linear plasmid . Plasmids carrying unmodified Tetrahymena thermophila telomeric sequences are rapidly degraded in oocytes . When these plasmids are passed through yeast, the telomere ends become modified by the addition of yeast telomeric sequences . These plasmids are stably maintained in X . laevis oocytes, demonstrating that yeast-modified telomeres are sufficient to prevent linear DNA degradation. FEBS Lett, 1991 Sep 23, 290(1-2), 95 - 8 Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus; Bobkova EV et al.; FPLC separation of alpha- and beta-subunits of phenylalanyl-tRNA synthetases from E . coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea . Native alpha-subunits of both enzymes were primarily alpha 2-dimers and tended to aggregate . Most E . coli enzyme beta-subunits were monomeric and only a small fraction was represented by beta 2-dimers . All thermophilic beta-subunits were beta 2-dimers . It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA(Phe) aminoacylation . For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined . The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding beta-subunits . Alpha-subunits displayed a distinctly different pH dependence of the surface charge . A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed. FEBS Lett, 1991 Sep 23, 290(1-2), 69 - 72 Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes; Yusupova G et al.; 70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNAPhe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines . A complex of N-AcPhe-tRNAPhe/(U)9/70S ribosome from Th . thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D . Trakhanov, et al., (1987) FEBS Lett . 220, 319-322). J Mol Biol, 1991 Sep 20, 221(2), 383 - 5 Preliminary X-ray diffraction analysis of a crystallizable mutant of malate dehydrogenase from the thermophile Thermus flavus; Kelly CA et al.; Malate dehydrogenase from mutant strain F428 of the thermophilic bacterium Thermus flavus has now been crystallized from polyethylene glycol 8000 in a form suitable for diffraction studies . The protein crystallizes in the orthorhombic P2(1)2(1)2(1) space group with unit cell dimensions a = 71.8 A, b = 88.6 A, c = 119.0 A . The asymmetric unit consists of one homodimer of molecular mass 67,000 Da . The X-ray diffraction extends beyond 1.7 A and a full data set to 1.9 A has been collected. J Mol Biol, 1991 Sep 20, 221(2), 375 - 7 Crystals of intact elongation factor Tu from Thermus thermophilus diffracting to high resolution; Reshetnikova LS et al.; The intact elongation factor Tu from the extreme thermophile Thermus thermophilus has been crystallized as a complex with the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate . The crystals are very stable in the X-ray beam and diffract to 1.9 A resolution . They exhibit space group C2, with a = 150.3(6) A, b = 99.6(3) A, c = 40.1(1) A, beta = 95.4(2) degrees, and contain one elongation factor Tu molecule per asymmetric unit. Gene, 1991 Sep 15, 105(2), 143 - 50 Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus; Hansen TS et al.; We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37 . The gene contains a single intron located in the 3'-part of the coding region . Two major and three minor transcription start points (tsp) were mapped 39 to 63 nucleotides upstream from the translational start codon . The uppermost tsp mapped to the first T in a putative T . thermophila RNA polymerase II initiator element, TATAA . The coding region of L37 predicts a protein of 109 amino acid (aa) residues . A substantial part of the deduced aa sequence was verified by protein sequencing . The T . thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins. Eur J Biochem, 1991 Sep 15, 200(3), 783 - 7 Characterization of two pterin derivatives isolated from Methanoculleus thermophilicum; Raemakers-Franken PC et al.; Methanoculleus thermophilicum was shown to contain two pterin derivatives . The structures of these pterin derivatives were established from amino acid analysis, 1H-NMR and fast-atom bombardment mass spectrometry data . One of the pterins was identified as tatiopterin-O, an aspartyl derivative of methanopterin with a proton at position 7 of the pterin moiety . The other pterin, which we named thermopterin, differed in the structure of the aniline group, containing two additional hydroxyl residues . The IUPAC name of thermopterin is N-{-1'-(2"-amino-4"-hydroxy-6"-pteridinyl)ethyl}-4- {2',3',4',5'-tetrahydroxypent-1'-yl(5'----1") O-alpha-ribofuranosyl-5"-phosphoric acid}-2,5-dihydroxyaniline, in which the phosphate group is esterified with alpha-hydroxyglutarylaspartic acid. Biochim Biophys Acta, 1991 Sep 2, 1075(1), 93 - 101 Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila; Saar J et al.; The membrane-bound enzyme from Tetrahymena thermophila responsible for the conversion of squalene into the quasi-hopanoid tetrahymanol was purified 297-fold to near homogeneity . Purification involved solubilization by octylthioglucoside, chromatography on DEAE-trisacryl, hydroxyapatite and FPLC ion-exchange on Mono Q . The apparent KM was found to be 18 microM . 2,3-Iminosqualene and N,N-dimethyldodecylamine-N-oxide are effective inhibitors of the cyclase with I50 values of 50 and 30 nM, respectively . The cyclase has a molecular mass of 72 kDa as judged by electrophoresis in polyacrylamide gels under denaturating conditions . The optimal enzymatic activity was obtained at pH 7.0 and 30 degrees C . The solubilized enzyme needs the presence of detergent for maintaining activity . The influence of different detergents on cyclase activity was studied . Triton X-100 proved to be a strong inactivator of the enzyme . Solubilization of the cyclase in Tween 80 and digitonin inactivates the enzyme . However, its activity can be recovered by complementation of the assay buffer with octylthioglucoside above its critical micellar concentration . We suggest that this approach might be applicable to other membrane-bound proteins. Zentralbl Hyg Umweltmed, 1991 Sep, 192(1), 14 - 24 Characterization of thermophilic campylobacters originated from a high-rate sewage treatment plant; Jacob J et al.; Campylobacter strains isolated during a one-year study from a municipal waste water treatment plant have been characterized . The study shows a considerable release of Campylobacter strains into the receiving linked river system . Campylobacters isolated here, are very similar to isolates originating from enteritic cases . In contrast to other reports we conclude that the strains released from the sewage treatment plant into the environment represent a potential risk for public health. Thorax, 1991 Sep, 46(9), 619 - 23 Influence of mode of storage and drying of fodder on thermophilic actinomycete aerocontamination in dairy farms of the Doubs region of France; Dalphin JC et al.; Airborne contamination by thermophilic actinomycetes, micromycetes and Gram negative bacteria was determined on 34 dairy farms and related to fodder drying and storage methods . Eighteen farms had a barn drying system, eight with additional heating; the remaining 16 had traditional fodder storage methods . Three air samples were obtained for each farm with a six stage Andersen sampler . The thermophilic actinomycetes were identified as Streptomyces and the dominant micromycetes as Aspergillus spp; there was no relation between the levels of these organisms . There were fewer thermophilic actinomycete colonies per Petri dish (stage 5 on the Anderson sampler) on farms with barn drying than on those with traditional storage (median (range) 7 (0-2628) and 56 (4-2628) respectively) . The three farms where no thermophilic actinomycetes were found had barn drying with heating and the four most modern farms had lower thermophilic actinomycete colony counts than the others (median (range) 3 (0-10) and 48 (0-2628)) . The level of thermophilic actinomycetes and, to a lesser degree, of micromycetes was higher where the farmer had farmer's lung . Thermophilic actinomycetes of the genus Streptomyces are probably the antigens associated with farmer's lung in the Doubs, and modern farms with barn drying and heating furnish some protection against this disease. Eur J Biochem, 1991 Sep 1, 200(2), 379 - 85 Purification and properties of a novel type of exo-1,4-beta-glucanase (avicelase II) from the cellulolytic thermophile Clostridium stercorarium; Bronnenmeier K et al.; Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium . A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II . The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9 . Determination of the N-terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I . Maximal activity of avicelase II is observed between pH 5 and 6 . In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75 degrees C . Hydrolysis of avicel occurs at a linear rate for three days at 70 degrees C . Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins . It lacks activity towards carboxymethylcellulose and barley beta-glucan . Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl-beta-D-cellobiosides . Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1 . With acid-swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose . The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non-reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose . These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non-reducing end of the cellulose chains. Eur J Biochem, 1991 Sep 1, 200(2), 295 - 300 Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu; Schirmer NK et al.; Affinity labeling in situ of the Thermus thermophilus elongation factor Tu (EF-Tu) nucleotide binding site was achieved with periodate-oxidized GDP (GDPoxi) or GTP (GTPoxi) in the absence and presence of elongation factor Ts (EF-Ts) . Lys52 and Lys137, both reacting with GDPoxi and GTPoxi, are located in the nucleotide binding region . In the absence of EF-Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTPoxi . GDPoxi reacted much more efficiently with Lys52 than with Lys137 under these conditions {Peter, M . E., Wittman-Liebold, B . & Sprinzl, M . (1988) Biochemistry 27, 9132-9138} . In the presence of EF-Ts, GDPoxi reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF-Ts with EF-Tu.GDPoxi induces a conformation resembling that of the EF-Tu.GDPoxi complex in the absence of EF-Ts . Binding of EF-Ts to EF-Tu.GDP enhances the accessibility of the Arg59-Gly60 peptide bond of EF-Tu to trypsin cleavage . Hydrolysis of this peptide bond does not interfere with the ability of EF-Ts to bind to EF-Tu . EF-Ts is protected against trypsin cleavage by interaction with EF-Tu.GDP . High concentrations of EF-Ts did not interfere significantly with aminoacyl-tRNA.EF-Tu.GTP complex formation. J Bacteriol, 1991 Sep, 173(17), 5346 - 51 Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8; Faraldo ML et al.; The gene coding for the 100 kDa monomeric protein (P100) of the S layer of Thermus thermophilus HB8 has been cloned in the Escherichia coli plasmid vector pUC9 . Recombinant plasmids were selected by colony screening with anti-P100 rabbit antiserum . The gene, named slpA (for surface layer protein A), was identified in a bacterial clone harboring a hybrid plasmid, pMF4, with a 5.8-kbp insert . This plasmid consistently expressed a protein specifically recognized by anti-P100 antiserum . Expression was apparently independent of Plac, indicating that the promoter for P100 is functional in E . coli . Most E . coli strains transformed with plasmids containing the 5.8-kbp insert cloned in pMF4 expressed two proteins with apparent masses of 52 and 50 kDa, which were strongly recognized by anti-P100 antiserum in Western immunoblots . The 52-kDa fragment could be overproduced, and the sequence of the N-terminal undecapeptide, determined by microsequencing, indicated that it could correspond to the N-terminal domain of P100 . Expression of slpA in lon mutants of E . coli led to accumulation of a protein indistinguishable from native P100, indicating that the complete gene was cloned and that the product of lon, protease La, was involved in proteolytic degradation of P100 synthesized in E . coli. Vopr Virusol, 1991 Sep-Oct, 36(5), 426 - 8 {Thermophilic eukaryotic cell cultures}; Bakhutashvili VI et al.; Thermophilic clones of lymphoblastoid cell cultures Namalwa were generated and found to be capable of life and reproduction at a temperature of 60 degrees C . The reproductive dynamics, cytology, and ultrastructure of these clones were studied. Appl Environ Microbiol, 1991 Sep, 57(9), 2602 - 9 Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria; Lovell CR et al.; The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation . The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens . Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera . A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism . No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway . DNA purified from cells extracted from horse manure was also screened with the acetogen probe . Six hybrids, indicating at least six detectable acetogen "strains," were observed. Microbiologia, 1991 Sep, 7(2), 82 - 9 Distribution of oxidizing bacterial activities and characterization of bioleaching-related microorganisms in a uranium mineral heap; de Siloniz MI et al.; The occurrence and activity of bioleaching-related microorganisms are highest at 0.25 m under the surface of a uranium mineral heap, and decrease at points deeper than 1 m inside the heap . Thiobacillus ferrooxidans, Th . acidophilus, Th . thiooxidans and Leptospirillum ferrooxidans have been found and isolated from different sites inside the heap . Other mesophilic iron- or sulphur-oxidizing bacteria and some moderate thermophile have also been isolated from deep (1 m and 4 m) sites and characterized . Several fungi and some yeasts are present in this bioleaching habitat. Mol Biol (Mosk), 1991 Sep-Oct, 25(5), 1273 - 84 {Elimination of introns from the interleukin 1 beta genome by DNA amplification and expression of interleukin 1 beta in Escherichia coli}; Chistiakov DA et al.; The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences . Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene . Synthetic oligonucleotides complementary to sequences flanking exons were used as primers . The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments . As a result the structural interleukin-1 beta gene consisting of three exons was assembled . DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers . The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene . We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells . It was used for expression of the present gene . The interleukin 1 beta synthesized in E . coli had biological activity. Plasmid, 1991 Sep, 26(2), 94 - 107 Plasmids in Bacillus stearothermophilus coding for bacteriocinogeny and temperature resistance; Stahl SR; Obligately thermophilic strains of Bacillus stearothermophilus were screened for the presence of plasmids by agarose gel electrophoresis . All strains in our collection contained large plasmids (20 x 10(6)-80 x 10(6)) and were divided into four groups with respect to their plasmid pattern and production of bacteriocins . The major plasmid species were designated pSE407 (38.7 x 10(6)), pSE409 (29.0 x 10(6)), pSE411 (21.5 x 10(6)), and pSE410 (23.5 x 10(6)) . Their physical endonuclease maps were constructed, and by Southern blots and hybridizations it was shown that these plasmids were related . From curing experiments and electrotransformations (electroporations) we conclude that pSE407, pSE410, and pSE411 code for temperature resistance . In addition pSE410 codes for bacteriocin production and resistance . Plasmid pSE409 probably also codes for bacteriocin production and resistance. Genetics, 1991 Sep, 129(1), 57 - 67 Tel-1 transposon-like elements of Tetrahymena thermophila are associated with micronuclear genome rearrangements; Wyman C et al.; The micronuclear genome of Tetrahymena thermophila contains Tel-1 elements that structurally resemble transposons . Here we present molecular evidence that Tel-1 transposon-like elements are mobile . The arrangements of Tel-1 elements in the micronuclear genomes of several T . thermophila strains and cell lines were assayed by Southern blotting . The molecular evidence for Tel-1 transposition is most striking in strains that have undergone unusual laboratory-induced meioses . The genetic history of the strains exhibiting evidence of Tel-1 transposition is consistent with periods of genome restructuring in response to genomic "shock" that B . McClintock has suggested could result in transposon activation. Biochem J, 1991 Sep 1, 278 ( Pt 2), 595 - 9 The role of histidine-118 of inorganic pyrophosphatase from thermophilic bacterium PS-3; Hirano N et al.; Treatment of the inorganic pyrophosphatase from thermophilic bacterium PS-3 with diethyl pyrocarbonate resulted in the almost complete loss of its activity, which followed pseudo-first-order kinetics . The presence of Mg2+ prevented the inactivation . Enzyme inactivated with diethyl pyrocarbonate was re-activated by hydroxylamine . The inactivation parallelled the amount of modified histidine residue, and a plot of the activity remaining against the amount of modified histidine residue suggested that the modification of one of two histidine residues totally inactivated the enzyme . The site involved was found to be located in a single lysyl endopeptidase-digest peptide derived from the ethoxy{14C}carbonylated enzyme . Amino acid analysis and sequence analysis of the peptide revealed that it comprised residues 96-119 of the inorganic pyrophosphatase from thermophilic bacterium PS-3 . These results, when compared with those reported for the Escherichia coli and yeast enzymes, imply that His-118 of the inorganic pyrophosphatase from thermophilic bacterium PS-3 is located near the Mg(2+)-binding site and thus affects the binding of Mg2+. Biochem Biophys Res Commun, 1991 Aug 30, 179(1), 565 - 71 Gene structure of heat shock proteins 61KDa and 12KDa (thermophilic chaperonins) of thermophilic bacterium PS3; Tamada H et al.; Heat shock proteins 60 (hsp60) and 10 (hsp10) are essential for the formation and restoration of many supramolecular structures . For reconstitution of these structures, we isolated stable hsps of 61kDa and 12kDa, which are similar to hsp60 and hsp10, respectively, from the supernatant fraction of thermophilic bacterium PS3 by ATP-Agarose chromatography . Using synthetic DNA of the deduced sequence, the 1.6kbp double stranded DNA encoding both proteins was obtained by the polymerase chain reaction (PCR) . The complete sequence of the resulting reading frames showed high homology to those of the genes encoding GroEL (hsp60) and GroES (hsp10) of E . coli, and hsp60s and hsp10s of several other species . The genes for the 12K and 61K were present in the same operon . 61K was also partially similar to the F1 alpha subunit of thermophilic ATP synthase, which is highly reconstitutable to form the alpha beta complex. Biochim Biophys Acta, 1991 Aug 30, 1079(2), 161 - 8 Magnetization of manganese superoxide dismutase from Thermus thermophilus; Peterson J et al.; The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data . Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein . The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0 . The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027 . These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds . In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present . The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S . and Nakazawa, K . (1978) J . Biochem . 83, 1165-1171) . The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed. Nucleic Acids Res, 1991 Aug 25, 19(16), 4429 - 36 A self-splicing group I intron in the nuclear pre-rRNA of the green alga, Ankistrodesmus stipitatus; Davila-Aponte JA et al.; The nuclear small subunit ribosomal RNA gene of the unicellular green alga Ankistrodesmus stipitatus contains a group I intron, the first of its kind to be found in the nucleus of a member of the plant kingdom . The intron RNA closely resembles the group I intron found in the large subunit rRNA precursor of Tetrahymena thermophila, differing by only eight nucleotides of 48 in the catalytic core and having the same peripheral secondary structure elements . The Ankistrodesmus RNA self-splices in vitro, yielding the typical group I intron splicing intermediates and products . Unlike the Tetrahymena intron, however, splicing is accelerated by high concentrations of monovalent cations and is rate-limited by the exon ligation step . This system provides an opportunity to understand how limited changes in intron sequence and structure alter the properties of an RNA catalytic center. Nucleic Acids Res, 1991 Aug 25, 19(16), 4443 - 9 Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo; Itaya M et al.; A DNA fragment encoding Ribonuclease H (EC 3 . 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant . The primary amino acid sequence showed 56% similarity to that of E . coli RNase HI but little or no homology to E . coli RNase HII . Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts . However, T . thermophilus RNase H has one more cysteine than its E . coli homologue . Stability of the RNase H in extracts of T . thermophilus to elevated temperatures was the same for the protein expressed in E . coli . T . thermophilus RNase H should, therefore, be a useful tool for editing RNA-DNA hybrid molecules at higher temperatures and may also be stable enough to be used in a cyclical process . It was suggested that regulation of expression of the RNase H may be different from that of E . coli . RNase HI. FEBS Lett, 1991 Aug 19, 288(1-2), 98 - 100 Molecular cloning, nucleotide sequence and expression of the tufB gene encoding elongation factor Tu from Thermus thermophilus HB8; Satoh M et al.; The tufB gene encoding elongation factor Tu (EF-Tu) of Thermus thermophilus HB8 was cloned and expressed . Compared with the known tufA gene of T . thermophilus, nucleotide differences were found at 10 positions out of 1221 nucleotides, and amino acid substitutions were found at 4 positions out of 406 amino acids . The tufB product was 70.9% homologous to the corresponding sequence of the tufB product of E . coli . The G+C content of the third base of the codon in the tufB gene was 84.8% and G was especially preferred in this position. FEBS Lett, 1991 Aug 19, 288(1-2), 154 - 8 Haem O2 can replace haem A in the active site of cytochrome c oxidase from thermophilic bacterium PS3; Sone N et al.; Thermophilic bacterium PS3 cultured under slightly air-limited conditions showed a mitochondrion-like cytochrome pattern similar to that in vigorously aerated cells, but an o-type cytochrome replaced cytochrome a3 as the CO-binding centre . Cytochrome cao-type oxidase was purified from the cell membranes by almost the same procedure as used for cytochrome caa3 . The turnover number of cytochrome cao was higher than that of cytochrome caa3, but the Km's of the two enzymes for cytochrome c and O2 were almost the same . Gel electrophoresis in the presence of sodium dodecyl sulfate gave bands of four subunits at the identical positions both for cytochrome cao and cytochrome caa3 . Cytochrome cao contained a novel kind of haem in addition to haems C and A . This novel haem is likely to be haem O, very recently found as the chromophore of the cytochrome bo complex in Escherichia coli . These data suggest that cytochrome cao is an alternative form of cytochrome c oxidase (cytochrome caa3), in which the cytochrome a3 centre of the enzyme is replaced with cytochrome o. Anal Biochem, 1991 Aug 15, 197(1), 83 - 90 A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions; Dedon PC et al.; Using the single cell eukaryote Tetrahymena thermophila, a simple method was developed for studying protein-DNA associations by cross-linking proteins to DNA with formaldehyde and immunoprecipitating the solubilized chromatin fragments with a specific antiserum . The protocol uses crude antiserum and involves only three steps: cross-linking, shearing to solubilize the chromatin, and immunoprecipitation . Methods for optimizing certain critical parameters, such as fixation time and NaCl concentration, are described . The method is likely to be generally useful for a variety of nuclear antigens. Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 6921 - 5 Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better; Herschlag D; Kinetic and thermodynamic factors that determine specificity of RNA cleavage by ribozymes are illustrated with examples from recent work with a ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA . The conclusions also apply to other ribozymes, to antisense oligonucleotide experiments, and to RNA and DNA cleavage agents that can recognize a single-stranded or double-stranded region of variable length . At first, adding bases to a ribozyme's recognition sequence is expected to increase cleavage of the target RNA relative to cleavage of other RNAs . However, adding more bases ultimately reduces this discrimination, as cleavage occurs essentially every time the target RNA or a mismatched RNA binds the ribozyme . This occurs despite the weaker binding of the mismatched RNA because dissociation becomes too slow (binding is too strong) to allow the ribozyme to "choose" between cleavage of the target RNA and a mismatched RNA . In summary, more (base pairing) isn't always better, because maximal discrimination requires equilibrium binding prior to cleavage . The maximum discrimination that can be obtained is expected to be greater with an A + U-rich recognition sequence than with a G + C-rich recognition sequence . This is because the weaker A.U base pairs (relative to G-C base pairs) allow recognition to be spread over a larger number of bases while preventing binding that is too strong . Finally, creating an A-rich ribozyme rather than a U-rich ribozyme avoids the loss in discrimination expected with U-rich ribozymes from the formation of U.G wobble pairs in addition to the "targeted" Watson-Crick U.A pair. Biochemistry, 1991 Aug 6, 30(31), 7661 - 6 Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase; Myers TW et al.; A recombinant DNA polymerase derived from the thermophilic eubacterium Thermus thermophilus (Tth pol) was found to possess very efficient reverse transcriptase (RT) activity in the presence of MnCl2 . Many of the problems typically associated with the high degree of secondary structure present in RNA are minimized by using a thermostable DNA polymerase for reverse transcription, and predominantly full-length products can be obtained . The cDNA can also be amplified in the polymerase chain reaction (PCR) with the same enzyme . The Tth pol was observed to be greater than 100-fold more efficient in a coupled RT/PCR than the analogous DNA polymerase from Thermus aquaticus (Taq pol) . The sensitivity of the reactions performed by Tth pol allowed for the detection of ethidium bromide stained products starting with as little as 100 copies of synthetic cRNA . Similar results were also obtained with RNA from a Philadelphia-chromosome positive cell line . Detection of IL-1 alpha mRNA was possible starting with 80 pg of total cellular RNA . The ability of Tth pol to perform both reverse transcription and DNA amplification will undoubtedly prove useful in the detection, quantitation, and cloning of cellular and viral RNA. FEBS Lett, 1991 Aug 5, 287(1-2), 5 - 9 Identities of four low-molecular-mass subunits of the photosystem I complex from Anabaena variabilis ATCC 29413 . Evidence for the presence of the psaI gene product in a cyanobacterial complex; Ikeuchi M et al.; Photosystem I (PSI) complex of Anabaena variabilis ATCC 29413 consists of at least 11 subunits, 9 of which are resolved by high resolution gel electrophoresis . N-terminal amino acid sequences of the four subunits with molecular masses of 6.8, 5.2, 4.8 and 3.5 kDa were determined . Based on the sequence homology, the 3.5 kDa subunit was revealed to correspond to PSI-I (the gene product of psaI), which had so far been detected only in higher plant PSI complexes . The 6.8 kDa protein and 4.8 kDa protein were identified as gene products of psaK and psaJ, respectively . The 5.2 kDa protein was homologous to a 4.8 kDa subunit of PSI of the thermophilic cyanobacterium Synechococcus vulcanus, suggesting that this protein is a component of PSI in cyanobacteria. J Biol Chem, 1991 Aug 5, 266(22), 14294 - 9 Role of threonine 190 in modulating the catalytic function of malate dehydrogenase from a thermophile Thermus flavus; Nishiyama M et al.; Random mutagenesis of malate dehydrogenase from a thermophilic bacterium, Thermus flavus AT-62, had revealed that a Thr190----Ile replacement near the essential catalytic residue His187 caused marked modulation of the catalytic properties . For further exploration of a role of the residue at this position, this residue was substituted with each of the other amino acids by site-directed mutagenesis . Most of the mutations except for substitution with Ser caused increases in Km for oxaloacetate and increases Ki for oxaloacetate of 2-110 times . Substitution with His or Pro was characterized by the complete loss of substrate inhibition, along with a marked increase in Km for oxaloacetate . Kinetic analyses of the native and altered malate dehydrogenases at various pHs revealed that both Km and Ki for oxaloacetate decreased proportionally to the decrease in pH from 8.40 to 5.75, whereas kcat was nearly constant within the pH range . Apparent shifts of the optimum pH values toward acidity observed with most of the altered malate dehydrogenases were attributed to the increase in Ki, which facilitated the release from the substrate inhibition at a lower pH . Replacement of Thr310, a possible counterpart with which Thr190 forms a hydrogen bond, by Ile caused changes in the catalytic properties similar to those of the Thr190-substituted enzymes . These results suggest that not only the loss of the hydrogen bond between Thr190 and Thr310 but also properties of the residues introduced at position 190 cause modulation of the catalytic properties, probably through dislocation of the loop structure that contains the catalytic residue His187. J Mol Biol, 1991 Aug 5, 220(3), 549 - 50 Crystals of protein S6 from the 30 S ribosomal subunit of Thermus thermophilus; Sedelnikova SE et al.; Crystals of protein S6 from the small ribosomal subunit of an extreme thermophile, Thermus thermophilus, have been obtained by the hanging-drop/vapor diffusion technique using methane pentanediol as a precipitant in the presence of potassium fluoride . The crystals belong to the space group C222 with cell parameters a = 106.7, b = 52.8, c = 41.0 A . They diffract to 2.0 A resolution. EMBO J, 1991 Aug, 10(8), 1979 - 87 Maturation of dense core granules in wild type and mutant Tetrahymena thermophila; Turkewitz AP et al.; Tetrahymena thermophila, a ciliated protozoan, has a well-developed pathway of regulated secretion from dense core granules called mucocysts . Since exocytosis-defective mutants are available, steps in the biogenesis of dense core granules and their fusion with the plasma membrane may be resolved genetically . To describe the steps in biochemical terms, we have generated antisera against mucocyst content proteins . One antiserum is directed against a calcium binding protein, p40, that is released on stimulation of exocytosis . p40 is shown to associate with an insoluble matrix in mature mucocysts . In addition, the antiserum recognizes a larger protein, p60, that is soluble, is not found in mature mucocysts and is not released on stimulation . Pulse-chase experiments support a precursor-product relationship between p60 and p40 . Using these proteins as markers, two mutant Tetrahymena strains defective in exocytosis have been shown to accumulate the putative precursor p60 in organelles that can be distinguished from one another and from wild type mucocysts on the basis of density . The kinetics of appearance of insoluble p40 and the mutant phenotypes suggest a model of mucocyst maturation in which sorting precedes matrix condensation. J Gen Microbiol, 1991 Aug, 137 ( Pt 8), 1931 - 7 Survival of a plasmid-bearing strain of Bacillus subtilis introduced into compost; Amner W et al.; Survival of Bacillus subtilis strain 168 containing plasmid pAB224, which carries a gene for tetracycline resistance, was studied in mushroom compost under mesophilic and thermophilic conditions . Stable populations of B . subtilis were maintained as spores in both sterile and fresh mushroom compost incubated at 37 degrees C . At 65 degrees C, the introduced B . subtilis populations declined during incubation but spores were still detectable after 28 d . Survival at the higher temperature was greater in fresh than in sterile compost . There was no apparent loss of plasmid pAB224 or plasmid-determined phenotype from the introduced B . subtilis population at either incubation temperature . The frequency of tetracycline resistance in the indigenous Bacillus population was very low (10(-5), but some tetracycline-resistant isolates contained plasmid DNA . Four plasmid DNA profiles were found associated with five Bacillus phenotypes, and some evidence for homology with pAB224 was found . However, pAB224 was found to be a suitable marker for release studies because it was easily recovered, readily distinguished from indigenous plasmids on agarose gels, and was maintained in compost-grown B . subtilis 168 in the absence of any selective pressure. Wei Sheng Wu Xue Bao, 1991 Aug, 31(4), 261 - 6 {Studies on the classification of thermophilic actinomycetes . IV . Determination of thermophilic Streptomyces hygroscopicus group}; Lu YY et al.; The thermophilic streptomyces hygroscopicus is one of the pathogens of farmer's lung disease . The identified 60 strains of thermophilic streptomyces hygroscopicus coming from the isolates from the haystack, moldy hay and sputa collected from Jiangsu, Hubei province and Shanghai in China . These strains are grown around 50 degrees C and have moist patches on the surface of colonies . It is proved that protease can be extracted from cultured H9-4 strain . This protease is provided with antigen, the farmer's lung disease of rabbit can be induced in animals experiments . On clinical diagnosis the farmer's lung disease can be detected by sera test . From the identification of the 60 thermophilic streptomyces hygroscopicus strains, we found the morphological cultural, physiological characteristics and cell wall composition of H9-4 and T562 well different from description hitherto . So two of them are identified as new species . H9-4 is named as Streptomyces thermoendus sp . nov . and T562 is named as Streptomyces thermobicorno-hygroscopicus sp . nov. Protein Seq Data Anal, 1991 Aug, 4(2), 93 - 6 Amino acid sequence of 10-kDa protein in photosystem I reaction-center complex from a thermophilic cyanobacterium, Synechococcus elongatus Naegeli; Kotani N et al.; Four small subunits (14, 13, 10, and 8 kDa) of the photosystem I reaction-center complex were isolated from a thermophilic cyanobacterium Synechococcus elongatus . The complete amino acid sequence of the 10-kDa subunit was determined to consist of 80 amino acid residues giving a molecular mass of 8855.3, excluding iron and sulfur atoms, and containing two special sequences of cysteine residues, Cys-X-X-Cys-X-X-Cys-X-X-X-Cys-Pro, at residues 10-21 and 47-58, which indicates that the subunit is an apoprotein carrying two iron-sulfur centers, FA and FB, assigned as {4Fe-4S} clusters . The amino acid sequence indicated an 87.5% identity compared with those deduced from the nucleotide sequences of chloroplast gene psa C from several plants. Protein Seq Data Anal, 1991 Aug, 4(2), 81 - 6 Amino acid sequence of the 14-kDa protein in the photosystem I reaction center complex from Synechococcus elongatus Naegeli; Kotani N et al.; One of the small components (14 kDa) of the photosystem I reaction center complex was isolated from a thermophilic alga, Synechococcus elongatus . The amino acid sequence was determined . The protein consists of 137 amino acid residues, corresponding to the molecular mass of 15,319 . Alignment of this sequence with the ferredoxin-binding proteins of photosystem I from other cyanobacteria and higher plants suggests the possible biologically important residues and the residues responsible for thermostability in the sequence. J Mol Evol, 1991 Aug, 33(2), 142 - 51 Phylogenetic depth of Thermotoga maritima inferred from analysis of the fus gene: amino acid sequence of elongation factor G and organization of the Thermotoga str operon; Tiboni O et al.; The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacterium Thermotoga maritima was identified and sequenced . The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf) . The rpsG, fus, and tuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in the str operon of Escherichia coli . The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii) . Unrooted phylogenetic dendrograms, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies between Thermotoga and the other bacterial lineages. Int J Food Microbiol, 1991 Aug, 13(4), 273 - 83 Changes in chemical composition and sensory qualities of peanut milk fermented with lactic acid bacteria; Lee C et al.; The effects of fermentation of aqueous extracts of peanuts (peanut milk) with Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus salivarius ssp . thermophilus, separately and in combination, on selected chemical and sensory qualities were investigated . Changes in pH, titratable acidity and viable cell populations indicated that there was a synergistic interaction between L . delbrueckii ssp . bulgaricus and S . salivarius ssp . thermophilus during fermentation . Analysis of headspace volatiles revealed that hexanal, which is one of the compounds responsible for undesirable green/beany flavor in peanut milk, completely disappeared as a result of fermentation . S . salivarius ssp . thermophilus was more effective than L . delbrueckii ssp . bulgaricus in reducing the hexanal content . The acetaldehyde content of peanut milk increased during fermentation . Changes in concentrations of these volatile compounds were correlated with sensory evaluation scores which showed that a significant (P less than or equal to 0.05) decrease in green/beany flavor and a significant increase in creamy flavor occurred as a result of fermentation. Eur J Biochem, 1991 Aug 1, 199(3), 529 - 37 Properties of the elongation factor 1 alpha in the thermoacidophilic archaebacterium Sulfolobus solfataricus; Masullo M et al.; The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S . The purified protein binds {3H}GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP . These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu) . Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1 . The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii . The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared . aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of {3H}GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual {3H}GDP-binding ability was still 90% and 54% of the control, respectively . The affinity of aEF-1 alpha for GDP and GTP was also evaluated . At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E . coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E . coli and T . thermophilus or EF-1 alpha from A . salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS) Plant Mol Biol, 1991 Aug, 17(2), 209 - 19 Nucleotide and derived amino acid sequence of the cyanogenic beta-glucosidase (linamarase) from white clover (Trifolium repens L.); Oxtoby E et al.; The nucleotide sequence and derived amino acid sequence of two different beta-glucosidase cDNA clones were determined . One clone (TRE104) was identified as the cyanogenic beta-glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme . The biological function of the other beta-glycosidase (TRE361) is not known . Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome . Both TRE104 and TRE361 fragments co-segregate with cyanogenic beta-glucosidase activity . Extensive homology was found between the white clover beta-glucosidase sequences and a group of prokaryote and mammalian beta-glycosidases . This group of sequences has no homology with a separate set of beta-glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum. Biochem J, 1991 Aug 1, 277 ( Pt 3), 887 - 90 Thermostable cellobiohydrolase from the thermophilic eubacterium Thermotoga sp . strain FjSS3-B.1 . Purification and properties; Ruttersmith LD et al.; Exo-1,4-beta-cellobiohydrolase (EC 3.2.1.91) was isolated from the culture supernatant of Thermotoga sp . strain FjSS3-B.1, an extremely thermophilic eubacterium that grows optimally at 80 degrees C . The enzyme was purified to homogeneity as determined by SDS/PAGE and has an Mr of 36,000 . The enzyme is the most thermostable cellulase reported to date, with a half-life at 108 degrees C of 70 min in buffer . In a 40 min assay, maximal activity was recorded at 105 degrees C . Cellobiohydrolase from strain FjSS3-B.1 is active against amorphous cellulose and CM-cellulose but only effects limited hydrolysis of filter paper or Sigmacell 20 . The only product identified by h.p.l.c . is the disaccharide cellobiose . The enzyme has a pH optimum around neutral and is stabilized by the presence of 0.8 M-NaCl. J Bacteriol, 1991 Aug, 173(16), 5047 - 53 Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase; Lauer G et al.; We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus . A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli . The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes . We have engineered the expression of the T . thermophilus gene in Escherichia coli, and we show that E . coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step. J Clin Microbiol, 1991 Aug, 29(8), 1620 - 4 Characterization of an immunoreactive species-specific 19-kilodalton outer membrane protein from Helicobacter pylori by using a monoclonal antibody; Drouet EB et al.; Immunoblotting experiments on hyperimmune rabbit serum and sera from patients with Helicobacter pylori gastritis showed a consistent antibody response to a 19-kDa outer membrane protein antigen . A monoclonal antibody, designated HP 40, which reacted by Western immunoblotting with this protein was produced . It was shared by all H . pylori strains tested (D 273, NCTC 11637, and 24 wild strains) but not by the thermophilic Campylobacter species, Campylobacter fetus, Helicobacter mustellae, or Helicobacter fennelliae . Immunogold staining suggested that the 19-kDa antigen was exposed on the outer surface of the bacteria . Its functional role and effectiveness as a serological diagnostic tool are under study. Eur Cytokine Netw, 1991 Aug-Sep, 2(4), 299 - 303 Interferon induction by Lactobacillus bulgaricus and Streptococcus thermophilus in mice; Pereyra BS et al.; This study investigates the effect of intraperitoneal injection of L . bulgaricus and S . thermophilus on interferon production by Swiss mice . The serum from mice given 5 x 10(7) L . bulgaricus in 0.5 ml saline showed a maximal production of 300 U/ml of alpha/beta interferon activity six hours after injection . Cellular integrity appears to be necessary for stimulation; heat-treated bacteria had little effect, while irradiated-bacteria had a greater effect . TNF was also produced, the sera of mice with high IFN also contained 300 U/ml TNF . Streptococcus thermophilus produced no detectable increase in serum IFN, but the 2'-5' A synthetase activity of peritoneal cells was elevated suggesting that small amounts of interferon were produced . Injection of Streptococcus thermophilus plus Lactobacillus bulgaricus did not change the serum interferon response to L . bulgaricus . These observations suggest that non-pathogenic bacteria such as those used in food processing, can stimulate IFN production in mice . There is some evidence that the bacterial cell walls might be responsible for at least part of this effect. Curr Opin Biotechnol, 1991 Aug, 2(4), 551 - 60 Analysis and modulation of protein stability; Fontana A; Numerous site-directed mutagenesis experiments have provided new insights into the stabilizing role of the individual forces and interactions within a globular protein molecule . Some useful guidelines and procedures are now available for producing genetically more stable proteins . Examples are the introduction of disulfide bonds, ion-binding sites, salt bridges, hydrophobic residues or hydrogen bonds, and the improvement of hydrophobic packing or alpha-helix propensity . Moreover, it is now clearly recognized that thermophilic (and, in general, extremophilic) bacteria produce highly stable proteins and enzymes of practical interest. J Biol Chem, 1991 Jul 25, 266(21), 13834 - 41 The extremely thermophilic eubacterium, Thermotoga maritima, contains a novel iron-hydrogenase whose cellular activity is dependent upon tungsten; Juszczak A et al.; Thermotoga maritima is the most thermophilic eubacterium currently known and grows up to 90 degrees C by a fermentative metabolism in which H2, CO2, and organic acids are end products . It was shown that the production of H2 is catalyzed by a single hydrogenase located in the cytoplasm . The addition of tungsten to the growth medium was found to increase both the cellular concentration of the hydrogenase and its in vitro catalytic activity by up to 10-fold, but the purified enzyme did not contain tungsten . It is a homotetramer of Mr 280,000 and contains approximately 20 atoms of Fe and 18 atoms of acid-labile sulfide/monomer . Other transition metals, including nickel (and also selenium), were present in only trace amounts (less than 0.1 atoms/monomer) . The hydrogenase was unstable at both 4 and 23 degrees C, even under anaerobic conditions, but no activity was lost in anaerobic buffer containing glycerol and dithiothreitol . Under these conditions the enzyme was also quite thermostable (t50% approximately 1 h at 90 degrees C) but extremely sensitive to irreversible inactivation by O2 (t50% approximately 10 s in air) . The optimum pH ranges for H2 evolution and H2 oxidation were 8.6-9.5 and greater than or equal to 10.4, respectively, and the optimum temperature for catalytic activity was above 95 degrees C . In contrast to mesophilic Fe hydrogenases, the T . maritima enzyme had very low H2 evolution activity, did not use T . maritima ferredoxin as an electron donor for H2 evolution, was inhibited by acetylene but not by nitrite, and exhibited EPR signals typical of {2Fe-2S}1+ clusters . Moreover, the oxidized enzyme did not exhibit the rhombic EPR signal that is characteristic of the catalytic iron-sulfur cluster of mesophilic Fe hydrogenases . These data suggest that T . maritima hydrogenase has a different FeS site and/or mechanism for catalyzing H2 production . The potential role of tungsten in regulating the activity of this enzyme is discussed. J Mol Biol, 1991 Jul 20, 220(2), 531 - 8 Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus; Varley PG et al.; 3-Phosphoglycerate kinases from yeast and the extreme thermophilic bacterium Thermus thermophilus HB8 have been used as models for investigating the relationship between stability, dynamics and activity . It was found that while at a given temperature the thermophilic protein is more stable, its conformational dynamics as measured by the ability of acrylamide to quench the fluorescence of a buried tryptophan as well as its specific activity, are both lower than for the mesophilic protein . As the temperature is increased, the thermodynamic stability of the thermophilic protein approaches that of the mesophilic protein at its working temperature . Its conformational dynamics and specific activity however were both shown to increase, until at the physiologically operational temperature, they become similar to those of the mesophilic enzyme at its operational temperature . These results confirm the proposal that a direct relationship and balance holds between thermodynamic stability, dynamics and specific activity in globular proteins . They demonstrate also the constraining effect of increased stability upon conformational dynamics and enzyme activity. Biochem J, 1991 Jul 15, 277 ( Pt 2), 413 - 7 An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga; Simpson HD et al.; Endo-1,4-beta-xylanase (EC 3.2.1.8) was isolated from the culture supernatant of Thermotoga sp . strain FjSS3-B.1, an extremely thermophilic anaerobic eubacterium which grows optimally at 80 degrees C . Activity was purified 165-fold by anion-exchange and hydroxyapatite chromatography . The enzyme has an Mr of 31,000 as determined by SDS/PAGE and 35,000 by analytical gel filtration . The optima for activity and stability for purified xylanase were between pH 5.0 and 5.5 . At pH 5.5, which is the optimum pH for thermostability, t1/2 (95 degrees C) is 90 min . The thermostability was improved by immobilization of the xylanase on to porous glass beads; t1/2 (105 degrees C) is 10 min . Several additives, such as sorbitol and xylan, were also found to increase the thermostability . At 130 degrees C, the half-life of immobilized xylanase in the presence of 90% sorbitol was 1.3 min . At 130 degrees C in molten sorbitol half of the enzyme denatured rapidly, but the remainder appeared to have a half-life of about 60 min. Eur J Biochem, 1991 Jul 15, 199(2), 395 - 400 S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus . Purification, molecular properties and studies on the covalently bound pyruvate; Cacciapuoti G et al.; S-Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87 degrees C, has been purified to homogeneity . The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min-1 (mg protein)-1 and the overall yield 8% . The enzyme is thermophilic with an optimum at 75 degrees C, is thermostable, and does not require divalent cations or putrescine for activity . It has a molecular mass of 32 kDa, and appears to be a monomeric protein . S-Adenosylmethionine decarboxylase from S . solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time-dependent process by NaCNBH3, in the presence of both the substrate and the product . Incubation with decarboxylated S-adenosyl{Me-3H}methionine and NaCNBH3 resulted in the labeling of the protein at the active site. Biochim Biophys Acta, 1991 Jul 12, 1078(3), 377 - 82 Distribution and purification of aspartate racemase in lactic acid bacteria; Okada H et al.; The distribution of aspartate racemase (EC 5.1.1.13) in various kinds of bacteria demonstrated that the enzyme occurs in lactic acid bacteria, such as Streptococcus species and Lactobacillus species . The enzyme from Streptococcus thermophilus IAM10064 was more thermostable than that from Streptococcus lactis IAM1198 which contained the enzyme most abundantly among the lactic acid bacteria we examined here . We purified the enzyme about 3400-fold to homogeneity from cell-free extract of S . thermophilus, which is composed of two identical subunits with a molecular weight of 28,000 as a homodimer . The enzyme utilizes specifically aspartate as a substrate, but not alanine and glutamate . Maximal reaction velocity was observed at 37 degrees C and around pH 8.0 . The sequence of the NH2-terminal amino acids of the enzyme was determined to be Met-Glu-Asn-Phe-Phe-Ser-Ile-Leu-Gly-XXX-Met-Gly-Thr-Met-Ala-Thr-Glu-Ser- Phe-. J Biol Chem, 1991 Jul 5, 266(19), 12321 - 8 DNA topoisomerase III from extremely thermophilic archaebacteria . ATP-independent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature; Slesarev AI et al.; A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus . In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase III is an ATP-independent relaxing topoisomerase . It is a monomer with Mr 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography . Dam topoisomerase III, like other bacterial type I topoisomerases, absolutely requires Mg2+ for activity and is specific for single-stranded DNA . At 60-80 degrees C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA . At 95 degrees C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA . The peculiarities of DNA topoisomerization at high temperatures are discussed. J Bacteriol, 1991 Jul, 173(14), 4464 - 73 Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes; Mollet B et al.; By complementing appropriate gal lesions in Escherichia coli K802, we were able to isolate the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes of Lactobacillus helveticus . Tn10 transposon mutagenesis, together with in vivo complementation analysis and in vitro enzyme activity measurements, allowed us to map these two genes . The DNA sequences of the genes and the flanking regions were determined . These revealed that the two genes are organized in the order galK-galT in an operonlike structure . In an in vitro transcription-translation assay, the galK and galT gene products were identified as 44- and 53-kDa proteins, respectively, data which corresponded well with the DNA sequencing data . The deduced amino acid sequence of the galK gene product showed significant homologies to other prokaryotic and eukaryotic galactokinase sequences, whereas galactose-1-phosphate uridyl transferase did not show any sequence similarities to other known proteins . This observation, together with a comparison of known gal operon structures, suggested that the L . helveticus operon developed independently to a translational expression unit having a different gene order than that in E . coli, Streptococcus lividans, or Saccharomyces cerevisiae . DNA sequencing of the flanking regions revealed an open reading frame downstream of the galKT operon . It was tentatively identified as galM (mutarotase) on the basis of the significant amino acid sequence homology with the corresponding Streptococcus thermophilus gene. J Bacteriol, 1991 Jul, 173(13), 4155 - 62 Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum; Morag E et al.; In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome . A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage . The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography . The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations . The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements . The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate . Cellobiose dramatically inhibited the cellulolytic activities . In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C . Cysteine further enhanced the stabilizing effect of calcium . This is the first report of a defined cellobiohydrolase in C . thermocellum . Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex. J Bacteriol, 1991 Jul, 173(13), 3943 - 8 Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes; Ohshima T et al.; Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes . We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield . The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration . The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min . The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively . Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism . The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively . The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate. Am Ind Hyg Assoc J, 1991 Jul, 52(7), 271 - 9 Airborne dust, ammonia, microorganisms, and antigens in pig confinement houses and the respiratory health of exposed farm workers; Crook B et al.; This study investigated the environmental conditions on pig farms and the respiratory health of pig farmers and their immunological response to airborne contaminants . Airborne concentrations of dust and ammonia were measured in 20 pig houses; viable microorganisms, endotoxins, and aeroallergens were measured in 6 of these houses, chosen to represent the range in dustiness . The 29 farmers employed on the farms completed a questionnaire and underwent lung function tests; 24 of them provided blood samples for the measurement of specific IgE and IgG antibody to extracts of pig squames and urine, feed components, and bacterial isolates . Mean airborne dust and ammonia concentrations in the pig houses ranged from 1.66 to 21.04 mg/m3 and from 1.50 to 13.23 ppm, respectively . Factors affecting these concentrations include time of year, feed systems used, and levels of ventilation . There was no direct relationship between airborne dust and ammonia concentrations . Airborne microorganisms ranged from 10(5) to more than 10(7) colony-forming units (cfu)/m3; most were bacteria, with few fungi or thermophilic actinomycetes isolated . Gram-positive bacterial genera (Staphylococcus, Micrococcus, and Bacillus spp.) predominated . Concentrations of endotoxin in collected airborne dust were low . Work-related respiratory symptoms, typically chest tightness/wheeze and nasal and eye irritation, were reported by 23 of the 29 workers . Three farmers had specific IgE to pig squames or urine and eight to feed components but none to the microbial extracts . Specific IgG to pig squames or urine and to feed components was demonstrated in 14 and 9 workers, respectively . Specific IgE responses occurred mainly in subjects with chest tightness or wheeze, although specific IgG responses were not related to symptoms.(ABSTRACT TRUNCATED AT 250 WORDS) Plasmid, 1991 Jul, 26(1), 30 - 9 The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions; Oskam L et al.; Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species . It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913 . Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline . The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism . This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913 . A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region . The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis. Fundam Appl Toxicol, 1991 Jul, 17(1), 136 - 49 Evaluation of seven in vitro alternatives for ocular safety testing; Bruner LH et al.; Seven in vitro assays were evaluated to determine if any were useful as screening procedures in ocular safety assessment . Seventeen test materials (chemicals, household cleaners, hand soaps, dishwashing liquids, shampoos, and liquid laundry detergents) were tested in each assay . In vivo ocular irritation scores for the materials were obtained from existing rabbit low volume eye test (LVET) data . The seven assays evaluated included the silicon microphysiometer (SM), luminescent bacteria toxicity test (LBT), neutral red assay (NR), total protein assay (TP), Tetrahymena thermophila motility assay (TTMA), bovine eye/chorioallantoic membrane assay (BE/CAM), and the EYTEX system (ETS) . For the seventeen materials used in this study there was a significant correlation between the in vivo irritant potential and in vitro data for all the tests except the EYTEX System (SM, r = -0.87; LBT, r = -0.91; NR, r = -0.85; TTMA, r = 0.78; TP, r = -0.86; ETS, r = 0.29) . The irritation classifications provided by the BE/CAM also did not correspond with the actual in vivo irritancy potential of the test materials . The result of this study suggested it may be possible to classify materials into broad irritancy categories with some of the assays . This would allow their use as screens prior to limited in vivo confirmation in the ocular safety assessment process. Equine Vet J, 1991 Jul, 23(4), 247 - 52 Prevalence of serum precipitating antibodies in horses to fungal and thermophilic actinomycete antigens: effects of environmental challenge; Madelin TM et al.; Sera from 54 two- to three-year-old Thoroughbred horses from an English racing stable were examined for precipitins to antigen extracts prepared from 18 species of moulds (fungi and thermophilic actinomycetes) isolated from the same stable . Twenty-seven horses exhibited serum precipitins to one or more antigens; sixteen of the mould antigens elicited positive reactions in sera from one or more horses . Significantly more precipitins occurred in sera of those horses stabled in a barn than among those stabled in individual boxes . This indicated a possible association between type of housing, level of exposure to airborne moulds and presence of serum precipitins . None of the horses had overt respiratory disease . This study agrees with reports of the presence of serum precipitating antibodies to mould antigens in clinically healthy horses and confirms that serological tests, therefore, are of little value in the diagnosis of chronic obstructive pulmonary disease (COPD) or 'heaves'. J Mol Evol, 1991 Jul, 33(1), 83 - 91 The main regulatory region of mammalian mitochondrial DNA: structure-function model and evolutionary pattern; Saccone C et al.; The evolution of the main regulatory region (D-loop) of the mammalian mitochondrial genome was analyzed by comparing the sequences of eight mammalian species: human, common chimpanzee, pygmy chimpanzee, dolphin, cow, rat, mouse, and rabbit . The best alignment of the sequences was obtained by optimization of the sequence similarities common to all these species . The two peripheral left and right D-loop domains, which contain the main regulatory elements so far discovered, evolved rapidly in a species-specific manner generating heterogeneity in both length and base composition . They are prone to the insertion and deletion of elements and to the generation of short repeats by replication slippage . However, the preservation of some sequence blocks and similar cloverleaf-like structures in these regions, indicates a basic similarity in the regulatory mechanisms of the mitochondrial genome in all mammalian species . We found, particularly in the right domain, significant similarities to the telomeric sequences of the mitochondrial (mt) and nuclear DNA of Tetrahymena thermophila . These sequences may be interpreted as relics of telomeres present in ancestral linear forms of mtDNA or may simply represent efficient templates of RNA primase-like enzymes . Due to their peculiar evolution, the two peripheral domains cannot be used to estimate in a quantitative way the genetic distances between mammalian species . On the other hand the central domain, highly conserved during evolution, behaves as a good molecular clock . Reliable estimates of the times of divergence between closely and distantly related species were obtained from the central domain using a Markov model and assuming nonhomogeneous evolution of nucleotide sites. Appl Environ Microbiol, 1991 Jul, 57(7), 2085 - 90 Potential for thermophilic (50 degrees C) anaerobic dechlorination of pentachlorophenol in different ecosystems; Larsen S et al.; Thermophilic (50 degrees C) anaerobic biodegradation of pentachlorophenol (PCP) was investigated by using different inocula from natural ecosystems and anaerobic digesters . The inocula tested were three freshwater sediments, four anaerobic sewage sludge samples from digesters treating sludge from wastewater plants with various industrial inputs, and digested manure from an anaerobic reactor . Only one digested-sludge sample and the manure sample were from thermophilic environments . The initial PCP concentration was 7.5 or 37.5 microM . After 8 months, PCP had disappeared from the sediment samples and various, less chlorinated intermediates were present . Additions of extra PCP were degraded within 4 weeks, and a maximal observed dechlorination rate of 1.61 mumol/liter/day in the vials with addition of 7.5 microM PCP and 7.50 mumol/liter/day in the vials with addition of 37.5 microM PCP were measured for a freshwater sediment . In contrast, only 2.8 to 17.5% of the initial PCP added had disappeared from the sludge samples after 8 months of incubation . The complex pattern of intermediates formed indicated that the dechlorination of PCP proceeded via different pathways, involving at least two different populations in the dechlorination processes. EMBO J, 1991 Jul, 10(7), 1711 - 22 A novel ATPase complex selectively accumulated upon heat shock is a major cellular component of thermophilic archaebacteria; Phipps BM et al.; We have discovered a large cylindrical protein complex which is an abundant component of the cytoplasm of extremely thermophilic archaebacteria . Structural analysis by image processing of electron micrographs suggests that the complex is composed of two stacked rings of eight subunits each; the rings enclose a central channel . The complex purified from the hyperthermophile Pyrodictium occultum is composed of equal quantities of two polypeptides of Mr 56,000 and 59,000 . It exhibits an extremely thermostable ATPase activity with a temperature optimum of 100 degrees C . The basal level of the ATPase complex in the cell is high, and it becomes highly enriched as a result of heat shock (shift from 102 degrees C to 108 degrees C) or balanced growth at temperatures near the physiological upper limit . Immunoblotting results indicate that a related protein is present in most thermophilic archaebacteria and in Escherichia coli . This protein complex may play an important role in the adaptation of thermophilic archaebacteria to life at high temperature. J Protozool, 1991 Jul-Aug, 38(4), 306 - 11 PCR amplification of Tetrahymena rDNA segments starting with individual cells; Orias E et al.; To facilitate studies of rDNA molecular genetics in Tetrahymena thermophila, we attempted the detection of polymorphisms in the nontranscribed spacers (NTSs) using polymerase chain reaction (PCR), starting with minute amounts of DNA . The targeted polymorphic regions are 85% adenine-thymine (AT) . We found conditions of efficient and specific in vitro amplification of targeted segments in the replication domain of the 5'NTS and in the subtelomeric segment of the 3'NTS . The identity of the amplified segments was confirmed by restriction enzyme digestion and DNA sequence analysis . Digestion of the template DNA at restriction sites upstream and downstream of the targeted region increased the efficiency of amplification, presumably because the targeted segments are in a palindromic molecule . Starting from total cell DNA corresponding to as little as 0.03 picogram (equivalent to the DNA content of 0.003 cells or about 30 rDNA molecules), we observed the amplified band after agarose gel electrophoresis and ethidium bromide staining . The yield indicated more than 10-billion-fold amplification . Amplification of the subtelomeric fragment yielded homogeneous product of minimum possible length even though the telomeric-specific primer can bind, at least initially, at a multiplicity of GGGGTT repeats . Amplified 5'NTS product also was detected in an ethidium-bromide-stained gel when PCR was started with a single cell. Biochimie, 1991 Jul-Aug, 73(7-8), 1037 - 43 Overproduction of the Thermus thermophilus elongation factor Tu in Escherichia coli; Ahmadian MR et al.; The elongation factor Tu (EF-Tu) encoded by the tufl gene of the extreme thermophilic bacterium Thermus thermophilus HB8 was expressed under control of the tac promoter from the recombinant plasmid pEFTu-10 in Escherichia coli . Thermophilic EF-Tu-GDP, which amounts to as much as 35% of the cellular protein content, was separated from the E coli EF-Tu-GDP by thermal denaturation at 60 degrees C . The overproduced E coli-born T thermophilus EF-Tu was characterized by: i) recognition through T thermophilus anti-EF-Tu antibodies; ii) analysis of the peptides obtained by cyanogen bromide cleavage; iii) thermostability; iv) guanine nucleotide binding activity in the absence and the presence of elongation factor Ts; and v) ternary complex formation with phenylalanyl-tRNAPhe and GTP. Biochimie, 1991 Jul-Aug, 73(7-8), 887 - 97 Thermus thermophilus ribosomes for crystallographic studies; Yusupov MM et al.; Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and threonyl- and seryl-tRNA synthetases from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, have been obtained at our institute . X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 A and 60 A, respectively . Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy . Structural studies of crystals of 2 ribosomal proteins, L1 and S6, elongation factor G and threonyl- and seryl-tRNA synthetases are also in progress . At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies. Agric Biol Chem, 1991 Jul, 55(7), 1739 - 44 Purification and some properties of a thermostable metal proteinase produced by Thermomicrobium sp . KN-22 strain; Murao S et al.; An extreme thermophile that produces a heat-stable proteinase was isolated from hot-spring water and classified as Thermomicrobium sp . KN-22 (growth temperature, 50-83 degrees C; and optimum growth temperature, 70 degrees C) . The proteinase was purified from the culture broth of this strain by fractionation with ammonium sulfate, chromatography on columns of DEAE-cellulose and CM-Sepharose CL-6B, and HPLC on TSKgel CM-5PW . The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and a single peak after HPLC (yield 8.8%) . The enzyme had maximum activity at pH 8.5 and at 75 degrees C and it was stable up to 60 degrees C . The molecular weight of the enzyme was 35,000 by SDS-PAGE . Since the enzymatic activity was completely inhibited by EDTA, o-phenanthroline, and phosphoramidon, it appears that the enzyme is a metal proteinase. J Biol Chem, 1991 Jun 25, 266(18), 11455 - 60 Small-angle X-ray scattering studies of Mg.AT(D)P-induced hexamer to dimer dissociation in the reconstituted alpha 3 beta 3 complex of ATP synthase from thermophilic bacterium PS3; Harada M et al.; The alpha 3 beta 3 complex of ATP synthase obtained from a thermophilic bacterium PS3 was isolated and found to show the ATPase activity (Kagawa, Y., Ohta, S., and Otawara-Hamamoto, Y . (1989) FEBS Lett . 249, 67-69) . The structure and the nucleotide binding effects of the alpha 3 beta 3 complex were investigated by means of small-angle x-ray scattering and high performance liquid chromatography . The scattering profile from the alpha 3 beta 3 complex was explained with a model in which the complex is made of an ellipsoid of revolution with the axes of 121.8, 121.8, and 72.0 A having an elliptical hollow cavity with the axes of 35.4, 35.4, and 72.0 A . By the addition of Mg.AT(D)P, significant changes in the scattering profile were observed, in which the radius of gyration decreased from 44 to 35 A . This change was found by gel filtration to be caused by the dissociation reaction from the alpha 3 beta 3 hexamer to the alpha beta dimer . The dissociation of the alpha 3 beta 3 complex was not induced by unhydrolyzable ATP analogue, nor by Pi, Mg2+, and Pi + Mg2+ . The structure of the dimer was well explained by the triaxial ellipsoidal model with the axes of 105.2, 39.4, and 108.2 A . The dissociation into the dimer is considered to be related to the ATPase activity because the AT(D)P-induced dissociation is observed only in the presence of Mg2+ ions. Biochim Biophys Acta, 1991 Jun 19, 1084(1), 101 - 4 Definition of total biosynthesis pathway of taurolipids in Tetrahymena cells; Kaya K et al.; Taurine-combined fatty acids were found in the lipotaurine fraction of cells of Tetrahymena thermophila . Taurine and fatty acid moieties of the compounds were identified by nuclear magnetic resonance and gas chromatography-mass spectrometry, respectively . The molar ratio of fatty acid methyl esters and taurine in the hydrolysate of the lipotaurine fraction by methanolic hydrochloric acid hydrolysis, was 1.06:1.00 . From the results, the structures of six taurine-combined fatty acids including lipotaurine in the fraction were identified . These structures suggest that the compounds are precursors of lipotaurine as an intermediate of taurolipids biosynthesis, and lipotaurine is biosynthesized via 2-(octadecanoylamino)ethanesulfonic acid and 2-(7-hydroxy-13-octadecenoylamino)ethanesulfonic acid . From the results of the present study and our previous studies, the total biosynthesis pathway of taurolipids is defined. FEBS Lett, 1991 Jun 17, 284(1), 39 - 41 A small basic ribosomal protein from the extreme thermophilic archaebacterium Sulfolobus solfataricus that has no equivalent in Escherichia coli; Ramirez C et al.; The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene has been confirmed by partial amino acid sequencing . The protein shows no sequence similarity to any of the ribosomal proteins from eubacteria (Escherichia coli) or to those that have been reported from eukaryotes. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5312 - 6 Monomeric and trimeric forms of photosystem I reaction center of Mastigocladus laminosus: crystallization and preliminary characterization; Almog O et al.; Photosystem I (PSI) reaction centers (RCs) of the thermophilic cyanobacterium Mastigocladus laminosus were purified and characterized . The PSI RC was obtained in two forms, monomeric and trimeric . The two forms contained the same number of pigments per P700 and displayed similar photochemical activities . The two forms had nearly identical polypeptide subunit compositions; the only observed difference was an additional subunit of about 12 kDa observed in the trimeric form . The purified preparations of both the monomeric and the trimeric forms were used for crystallization and preliminary crystallographic analysis . The trimeric PSI RC preparations produced several three-dimensional crystal forms, one of which, the "hexagonal needle" form (THN), had a hexagonal unit cell with dimensions of 300 x 300 x 160 A, containing four PSI RC trimers . The monomeric preparations also produced single crystals of several forms under various crystallization conditions . One of these crystal forms, the "hexagonal plate" (MHP), diffracted to a resolution of about 5.5 A . It had a hexagonal unit cell with dimensions of 192 x 192 x 163 A, containing six PSI RC monomers . Comparison of the PSI RCs in the crystals with those in the precrystallization preparations demonstrated that neither the monomeric nor the trimeric form of PSI RC was altered by the crystallization process . Both forms retained their original polypeptide subunit composition and their pigment content. FEMS Microbiol Lett, 1991 Jun 15, 65(2), 123 - 8 A species-specific DNA probe obtained from Streptococcus salivarius subsp . thermophilus detects strain restriction polymorphism; Colmin C et al.; Genomic polymorphism in Streptococcus salivarius subsp . thermophilus was revealed by DNA restriction pattern analysis . A 4.2-kb variable DNA fragment was cloned from strain NST7 and hybridised with the DNA of 25 strains allowing an easy detection of intraspecific RFLP . Strong and weak hybridisation signals were observed and the latter were specifically revealed by a 2.1-kb fragment of the probe . Probe specificity was demonstrated by the absence of homology with DNA of strains belonging to 10 other species, with the exception of S . salivarius subsp . salivarius, confirming a close relationship between S . salivarius and S . thermophilus. Biochim Biophys Acta, 1991 Jun 13, 1089(2), 234 - 40 Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus; Yohda M et al.; The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli . The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids . The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S . thermophilus . The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence . In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein . No significantly homologous proteins were found in a protein sequence data bank . Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low . Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18 . The amount of aspartate racemase increases with the growth of E . coli and almost no degradation of the enzyme was observed . The maximum amount of the produced enzyme reached approx . 20% of the total protein of E . coli. J Mol Biol, 1991 Jun 5, 219(3), 399 - 402 Crystallization and preliminary diffraction studies of 5 S rRNA from the thermophilic bacterium Thermus flavus; Lorenz S et al.; Crystals of purified 5 S rRNA from Thermus flavus have been obtained . The crystals diffract up to 8 A resolution, using synchrotron radiation, and have the monoclinic space-group C2 . The unit cell has the dimensions a = 190 A, b = 110 A, c = 138 A and beta = 117 degrees . The cell volume suggests the presence of four 5 S rRNA molecules per asymmetric unit. J Biol Chem, 1991 Jun 5, 266(16), 10570 - 7 Isoleucyl-tRNA synthetase of Methanobacterium thermoautotrophicum Marburg . Cloning of the gene, nucleotide sequence, and localization of a base change conferring resistance to pseudomonic acid; Jenal U et al.; The ileS gene encoding the isoleucyl-tRNA synthetase of the thermophilic archaebacterium Methanobacterium thermoautotrophicum Marburg was isolated and sequenced . ileS was closely flanked by an unknown open reading frame and by purL and thus is arranged differently from the organizations observed in several eubacteria or in Saccharomyces cerevisiae . The deduced amino acid sequence of isoleucyl-tRNA synthetase was compared with primary sequences of isoleucyl-, valyl-, leucyl-, and methionyl-tRNA synthetases from eubacteria and yeast . The archaebacterial enzyme fitted well into this group of enzymes . It contained the two short consensus sequences observed in class I aminoacyl-tRNA synthetases as well as regions of homology with enzymes of the isoleucine family . Comparison between the isoleucyl-tRNA synthetases of M . thermoautotrophicum yielded 36% amino acid identity with the yeast enzyme and 32% identity with the corresponding enzyme from Escherichia coli . The ileS gene of the pseudomonic acid-resistant M . thermoautotrophicum mutant MBT10 was also sequenced . The mutant enzyme had undergone a glycine to aspartic acid transition at position 590, in a conserved region comprising the KMSKS consensus sequence . The inhibition constants of pseudomonic acid, KiIle and KiATP, for the mutant enzyme were 10-fold higher than those determined for the wild-type enzyme . Both the mutant and the wild-type ileS gene were expressed in E . coli, and their products displayed the expected difference in sensitivity toward pseudomonic acid. J Biol Chem, 1991 Jun 5, 266(16), 10368 - 76 Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1 . The binding change motif is independent of the F1 gamma delta epsilon subunits; Aloise P et al.; Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+) . Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+ . Titration of near stoichiometric {MgBzADP}/{F1} ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association . Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes . With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme . Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites . Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme . As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H., Grubmeyer, C., and Coleman, P.S . (1987) J . Biol . Chem . 262, 13765-13772), this supports the fact that the photocovalent inhibi