Thermophile

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 inhibition of F1 is a one-hit one-kill phenomenon . Isoelectric focusing gels revealed that {3H}BzADP covalently modifies both TF1 and MF1 exclusively on the beta subunit, whether or not Mg2+ is present . A single 19-residue {3H}BzADP-labeled peptide was resolved from a tryptic digest of MF1, and this peptide corresponded with the one believed to contain at least a portion of the beta subunit catalytic site domain (i.e . beta Ala-338----beta Arg-356).

J Biochem (Tokyo), 1991 Jun, 109(6), 852 - 7
Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an extreme thermophile, Thermus aquaticus YT-1; Kirino H et al.; A gene (leuB) coding for 3-isopropylmalate dehydrogenase {EC 1.1.1.85} from an extreme thermophile, Thermus aquaticus YT-1 was cloned in Escherichia coli and the nucleotide sequence was determined . It contains an open reading frame of 1,035 bp encoding 344 amino acid residues . The homology with that from T . thermophilus HB8 is 87.0% in nucleotide and 91.3% in amino acid sequences . No overlapped gene was found in the present leuB gene, in contrast to the previous prediction that Thermus leuD gene is overlapped with leuB {Croft et al . (1987) Mol . Gen . Genet . 210, 490-497} . Substitutions in the primary structure which are unique for the thermophile sequences are discussed in relation to the unusual stability of the thermophile dehydrogenase based on amino acid sequence comparison of 9 microorganisms including thermophiles and mesophiles.

Microbiol Rev, 1991 Jun, 55(2), 225 - 33
Microbiological degradation of pesticides in yard waste composting; Fogarty AM et al.; Changes in public opinion and legislation have led to the general recognition that solid waste treatment practices must be changed . Solid-waste disposal by landfill is becoming increasingly expensive and regulated and no longer represents a long-term option in view of limited land space and environmental problems . Yard waste, a significant component of municipal solid waste, has previously not been separated from the municipal solid-waste stream . The treatment of municipal solid waste including yard waste must urgently be addressed because disposal via landfill will be prohibited by legislation . Separation of yard waste from municipal solid waste will be mandated in many localities, thus stressing the importance of scrutinizing current composting practices in treating grass clippings, leaves, and other yard residues . Yard waste poses a potential environmental health problem as a result of the widespread use of pesticides in lawn and tree care and the persistence of the residues of these chemicals in plant tissue . Yard waste containing pesticides may present a problem due to the recalcitrant and toxic nature of the pesticide molecules . Current composting processes are based on various modifications of either window systems or in-vessel systems . Both types of processes are ultimately dependent on microbial bioconversions of organic material to innocuous end products . The critical stage of the composting process is the thermophilic phase . The fate and mechanism of removal of pesticides in composting processes is largely unknown and in need of comprehensive analysis.

Biochimie, 1991 Jun, 73(6), 669 - 78
Comparative analysis of ribosomal protein L5 sequences from bacteria of the genus Thermus; Jahn O et al.; The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted) . Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons . Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene . Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence . The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%) . From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine . Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25 . The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.

J Bacteriol, 1991 Jun, 173(12), 3921 - 3
Reverse gyrase in thermophilic eubacteria; Bouthier de la Tour C et al.; The presence of reverse gyrase, an unusual ATP-dependent type I topoisomerase first isolated from thermophilic archaebacteria, has been detected in four strains of Thermotogales, an order of extremely thermophilic eubacteria . This result suggests that reverse gyrase plays a key role in high-temperature-living organisms, independently of the evolutionary kingdom to which they belong.

Appl Microbiol Biotechnol, 1991 Jun, 35(3), 334 - 8
Molecular cloning in Lactobacillus helveticus by plasmid pSA3::pVA797 co-integrate formation and conjugal transfer; Thompson K et al.; A gene encoding beta-glucanase activity from Bacillus amyloliquefaciens was subcloned in both orientations into plasmid shuttle vector pSA3 . In only one orientation could a co-integrate be generated with the conjugative plasmid pVA797 . The plasmid co-integrate was conjugated into Lactobacillus helveticus strain CNRZ450, where it was stably maintained without antibiotic selection and exhibited beta-glucanase activity . This method of introducing cloned DNA into thermophilic lactobacilli will facilitate the study of heterologous gene expression in non-transformable species.

Biochemistry, 1991 May 28, 30(21), 5125 - 38
Stereochemistry and size of sugar head groups determine structure and phase behavior of glycolipid membranes: densitometric, calorimetric, and X-ray studies; Hinz HJ et al.; The role carbohydrate moieties play in determining the structure and energetics of glycolipid model membranes has been investigated by small- and wide-angle X-ray scattering, differential scanning densitometry (DSD), and differential scanning microcalorimetry (DSC) . The dependence of a variety of thermodynamic and structural parameters on the stereochemistry of the OH groups in the pyranose ring and on the size of the sugar head group has been studied by using an homologous series of synthetic stereochemically uniform glyceroglycolipids having glucose, galactose, mannose, maltose, or trimaltose head groups and saturated ether-linked alkyl chains with 10, 12, 14, 16, or 18 carbon atoms per chain . The combined structural and thermodynamic data indicate that stereochemical changes of a single OH group in the pyranose ring can cause dramatic alterations in the stability and in the nature of the phase transitions of the membranes . The second equally important determinant of lipid interactions in the membrane is the size of the head group . A comparison of lipids with glucose, maltose, or trimaltose head groups and identical hydrophobic moieties has shown that increasing the size of the neutral carbohydrate head group strongly favors the bilayer-forming tendency of the glycolipids . These experimental results provide a verification of the geometric model advanced by Israelachvili et al . (1980) {Israelachvili, J . N., Marcelja, S., & Horn, R . G . (1980) Q . Rev . Biophys . 13, 121-200} to explain the preferences lipids exhibit for certain structures . Generally galactose head groups confer highest stability on the multilamellar model membranes as judged on the basis of the chain-melting transition . This is an interesting aspect in view of the fact that galactose moieties are frequently observed in membranes of thermophilic organisms . Glucose head groups provide lower stability but increase the number of stable intermediate structures that the corresponding lipids can adopt . Galactolipids do not even assume a stable intermediate L alpha phase for lipids with short chain length but perform only Lc----HII transitions in the first heating . The C2 isomer, mannose, modifies the phase preference in such a manner that only L beta----HII changes can occur . Maltose and trimaltose head groups prevent the adoption of the HII phase and permit only L beta----L alpha phase changes . The DSD studies resulted in a quantitative estimate for the volume change associated with the L alpha----HII transition of 14-Glc . The value of delta v = 0.005 mL/g supports the view that the volume difference between L alpha and HII is minute.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1991 May 23, 198(1), 7 - 12
Cloning, nucleotide sequence and expression of the cytochrome c-552 gene from Hydrogenobacter thermophilus; Sanbongi Y et al.; A cytochrome c-552 gene from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, was cloned by using two oligonucleotide probes, which had been synthesized based on the known amino acid sequence of the protein . A 780-bp PstI-SphI fragment of the cloned DNA was sequenced and found to contain the entire structural gene coding for cytochrome c-552 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription . Cytochrome c-552 is synthesized in vivo as a precursor having an N-terminal signal sequence consisting of 18 amino acid residues . The cloned cytochrome c-552 gene without its own signal sequence was introduced into the pKK223-3 vector and expressed in E . coli upon induction with isopropyl beta-D-thiogalactoside . An expressed cytochrome c-552 protein had a methionine residue at the N-terminus since an initiation signal was introduced before the first amino acid residue of the mature cytochrome c-552 . The heme c was attached to apo-type cytochrome c-552 in the cytoplasm of E . coli and the holoprotein had spectral properties, similar to the authentic cytochrome c-552 from H . thermophilus.

Eur J Biochem, 1991 May 23, 198(1), 43 - 52
Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile; Canevascini G et al.; Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques . Both enzymes are slightly glycosylated flavocytochrome-b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45) . Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique . After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour . The flavin carrying component shows an atypical (for flavoprotein) three-banded spectrum indicative of the presence of a flavin derivative . Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide . Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid . With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.

Biochemistry, 1991 May 21, 30(20), 4844 - 54
Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom; Herschlag D et al.; The L-21 ScaI ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a guanosine-dependent endonuclease reaction that is analogous to the first step in self-splicing of this intervening sequence . We now describe pre-steady-state kinetic experiments, with sulfur substituting for the pro-RP (nonbridging) phosphoryl oxygen atom at the site of cleavage, that test aspects of a kinetic model proposed for the ribozyme reaction (Herschlag, D., & Cech, T . R . (1990) Biochemistry 29, 10159-10171) . Thio substitution does not affect the reaction with subsaturating oligonucleotide substrate and saturating guanosine ((kcat/Km)S), consistent with the previous finding that binding of the oligonucleotide substrate limits this rate constant . In contrast, there is a significant decrease in the rate of single-turnover reactions of ribozyme-bound (i.e., saturating) oligonucleotide substrate upon thio substitution, with decreases of 2.3-fold for the reaction with guanosine ((kcat/Km)G) and 7-fold for hydrolysis {i.e., with solvent replacing guanosine; kc(-G)} . These "thio effects" are consistent with rate-limiting chemistry, as shown by comparison with model reactions . Nonenzymatic nucleophilic substitution reactions of the phosphate diester, methyl 2,4-dinitrophenyl phosphate monoanion, are slowed 4-11-fold by thio substitution for reactions with hydroxide ion, formate ion, fluoride ion, pyridine, and nicotinamide . In addition, we have confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA (Burgers, P . M . J., & Eckstein, F . (1979) Biochemistry 18, 592-596) . Considering the strong preference of Mg2+ for binding to oxygen rather than sulfur, the modest thio effect on the chemical step of the ribozyme-catalyzed reaction and the absence of a thio effect on the equilibrium constant for binding of the oligonucleotide substrate suggest that the pro-RP oxygen atom is not coordinated to Mg2+ in the E.S complex or in the transition state . General implications of thio effects in enzymatic reactions of phosphate diesters are discussed.

J Mol Biol, 1991 May 20, 219(2), 335 - 58
Manganese superoxide dismutase from Thermus thermophilus . A structural model refined at 1.8 A resolution; Ludwig ML et al.; The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods . The R-factor {formula: see text} for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell . The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet . Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet . Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions . The tetramer possesses 222 symmetry but is held together by only two types of interfaces . The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents . The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain . Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry . One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170 . Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem Biophys Res Commun, 1991 May 15, 176(3), 1313 - 8
Molecular cloning of phosphofructokinase 1 gene from a thermophilic bacterium, Thermus thermophilus; Xu J et al.; Phosphofructokinase 1 (PFK1) from Thermus thermophilus differs from other bacterial PFKs in that it is regulated by effector-induced reversible tetramer-dimer conversion rather than conformational change alone . We have cloned its gene and the deduced amino acid sequence was compared with that of other PFKs . While almost all amino acid residues involved in substrate binding sites, which are assigned from crystal structures of other PFKs, are well conserved, some possibly important changes are found at subunit interfaces.

Nucleic Acids Res, 1991 May 11, 19(9), 2321 - 3
BsiY I, a novel thermophilic restriction endonuclease that recognizes 5' CCNNNNNNNGG 3' and the discovery of a wrongly sequenced site in pACYC177; Mok YK et al.; A new type II restriction endonuclease designated BsiY I has been purified from a thermophilic soil Bacillus stearothermophilus strain . This enzyme recognizes and cleaves the highly degenerate sequence 5' CCNNNNN!NNGG 3' . During the identification of the recognition sequence of BsiY I, we discovered that there should be five G nucleotides instead of four at position 1227-1230 of the plasmid pACYC177.

Eur J Biochem, 1991 May 8, 197(3), 699 - 705
Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases; Lauraeus M et al.; The expression of two different aa3-type cytochrome oxidases is demonstrated in Bacillus subtilis . One of them (denoted caa3-605), was predicted by DNA-sequencing of Bacillus cytochrome oxidase genes, but has not been found previously . It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3 . The other oxidase (denoted aa3-600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al . {de Vrij, W., Azzi, A . & Konings, W . N . (1983) Eur . J . Biochem . 131, 97-103} . It shows no immunological cross-reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem-A chromophores . It catalyses oxidation of quinols, which is proposed to be its physiological function.

Proc Natl Acad Sci U S A, 1991 May 1, 88(9), 4015 - 9
Switching substrate preference of thermophilic xylose isomerase from D-xylose to D-glucose by redesigning the substrate binding pocket; Meng M et al.; The substrate specificity of thermophilic xylose isomerase from Clostridium thermosulfurogenes was examined by using predictions from the known crystal structure of the Arthrobacter enzyme and site-directed mutagenesis of the thermophile xylA gene . The orientation of glucose as a substrate in the active site of the thermophilic enzyme was modeled to position the C-6 end of hexose toward His-101 in the substrate-binding pocket . The locations of Met-87, Thr-89, Val-134, and Glu-180, which contact the C-6-OH group of the substrate in the sorbitol-bound xylose isomerase from Arthrobacter {Collyer, C.A., Henrick, K . & Blow, D . M . (1990) J . Mol . Biol . 212, 211-235}, are equivalent to those of Trp-139, Thr-141, Val-186, and Glu-232 in the thermophilic enzyme . Replacement of Trp-139 with Phe reduced the Km and enhanced the kcat of the mutant thermophilic enzyme toward glucose, whereas this substitution reversed the effect toward xylose . Replacement of Val-186 with Thr also enhanced the catalytic efficiency of the enzyme toward glucose . Double mutants with replacements Trp-139----Phe/Val-186----Thr and Trp-139----Phe/Val-186----Ser had a higher catalytic efficiency (kcat/Km) for glucose than the wild-type enzyme of 5- and 2-fold, respectively . They also exhibited 1.5- and 3-fold higher catalytic efficiency for D-glucose than for D-xylose, respectively . These results provide evidence that alteration in substrate specificity of factitious thermophilic xylose isomerases can be achieved by designing reduced steric constraints and enhanced hydrogen-bonding capacity for glucose in the substrate-binding pocket of the active site.

J Bacteriol, 1991 May, 173(10), 3078 - 83
Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases; Dekker K et al.; The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe . The complete nucleotide sequence of the gene was determined . T . thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized . The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000 . The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases . Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T . thermophilus . Moreover, the enzyme from E . coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min . The characteristics of the enzyme made in E . coli are the same as those of enzyme made in T . thermophilus . Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved . Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T . thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.

Res Microbiol, 1991 May, 142(4), 389 - 96
Characterization of gram-positive broad host-range plasmids carrying a thermophilic replicon; De Rossi E et al.; The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (CmR and ApR) from the Escherichia coli plasmid pJH101 . The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed CmR in B . subtilis and CmR and ApR in E . coli . Different shuttle vectors for Gram+ bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194 . The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B . subtilis at a copy number of 60 . By electroporation, these plasmids were introduced and stably maintained in B . subtilis, B . amyloliquefaciens, S . aureus, S . carnosus and Lactobacillus reuteri.

Biol Chem Hoppe Seyler, 1991 May, 372(5), 363 - 72
Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, XI . Engineering thermostability and activity of lactate dehydrogenases from bacilli; Zulli F et al.; An extensive comparative structural analysis of lactate dehydrogenase (LDH) sequences from thermophilic, mesophilic and psychrophilic bacilli revealed characteristic primary structural differences . These specific amino-acid substitutions were found in the entire LDH molecule . However, in certain regions of the LDH an accumulation of these exchanges could be detected . These regions seem to be particularly important for the temperature adaptation of the enzyme . The influence of one of such regions at the N-terminus on stability and activity of LDHs was analysed by the construction of hybrid mutants between LDH sequences from thermophilic, mesophilic and psychrophilic bacilli and also by site-directed mutagenesis experiments at five different positions . The substitutions of Thr-29 or Ser-39 to Ala residues in the LDH from the mesophilic B . megaterium increased the thermostability of the enzyme drastically (15 degrees C) . An increase of 20 degrees C could be observed when both amino-acid substitutions were introduced . These amino-acid substitutions resulted in an increase of Km for pyruvate and led to a three-fold reduction of the activity (kcat/Km) at 40 degrees C compared with the wild type enzyme . The influence of these amino-acid substitutions was also investigated in the LDHs from thermophilic and psychrophilic bacilli . The high heat resistance of the LDH from the thermophilic B . stearothermophilus was not altered by the Ala to Thr and Ser substitutions at positions 29 and 39, respectively . This indicates a cooperatively stabilized conformation of this LDH . However, in this mutant of the B . stearothermophilus LDH the activity (kcat/Km) was increased two-fold.

Appl Environ Microbiol, 1991 May, 57(5), 1333 - 9
Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp . thermophilus; Slos P et al.; A promoter probe vector, pTG244, was constructed with the aim of isolating transcription initiation signals from Streptococcus thermophilus (Streptococcus salivarius subsp . thermophilus) . pTG244 is based on the Escherichia coli-streptococcus shuttle vector pTG222, into which the promoterless chloramphenicol acetyltransferase gene of Bacillus pumilus (cat-86) was cloned . Random Sau3A fragments from the S . thermophilus A054 chromosomal DNA were cloned upstream of the cat-86 gene by using E . coli as the host . The pool of recombinant plasmids were introduced into S . thermophilus and Lactococcus lactis subsp . lactis in order to search for promoter activity in these hosts . For S . thermophilus, it was necessary to first select erythromycin-resistant transformants and then to screen for chloramphenicol resistance among these . Direct selection of chloramphenicol-resistant clones was, however, possible in L . lactis subsp . lactis . Six fragments exhibiting promoter activity were characterized in S . thermophilus by measuring the levels of cat-86 transcription and/or chloramphenicol acetyltransferase specific activity . Three of the promoter-carrying fragments were sequenced . The 5' ends of their corresponding mRNAs were determined by S1 mapping and shown to correspond to a purine residue in all cases . Upstream from these potential transcription start points, sequences homologous to the E . coli sigma 70 and the Bacillus subtilis vegetative sigma 43 (or sigma A) consensus promoters were identified.

Biochimie, 1991 May, 73(5), 573 - 81
The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci; Robitaille D et al.; The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography . The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85) . However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase . Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis . Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact . The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months . Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein . Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius . Rabbit polyclonal antibodies directed against each form reacted with both proteins . The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions . A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1991 May, 173(10), 3138 - 48
Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria); Edmonds CG et al.; Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (Thermoplasma acidophilum) to 105 degrees C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermotoga maritima (80 degrees C) . Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree . 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya . A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known . From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.

Biochim Biophys Acta, 1991 Apr 29, 1077(3), 291 - 8
Characterisation of arginase from the extreme thermophile 'Bacillus caldovelox'; Patchett ML et al.; A thermostable arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile 'Bacillus caldovelox' (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor . The purified enzyme is a hexamer with a subunit Mr of 31,000 +/- 2000 and contains greater than or equal to 1 Mn atom per subunit . Maximum activation on incubation with Mn2+ is 29% . Activity is optimal at pH 9 and at 60 degrees C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM . Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60 degrees C (t1/2 (half life) = 4.5 min) and individually 0.1 mM Mn2+ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce greater than 1000-fold increase and a t1/2 = 105 min at 95 degrees C . Aspartic acid and other species that bind Mn2+ can replace BSA, and it is suggested that arginase can be inactivated by free Mn2+ . A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5 . Reassociation occurs at pH 9 and is unusual in that it does not require Mn2+.

Biochim Biophys Acta, 1991 Apr 29, 1077(3), 281 - 4
Modification of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding; Kaneko S et al.; The effect of N-bromosuccinimide (NBS) on the activity of the inorganic pyrophosphatase (PPiase) from thermophilic bacterium PS-3 was studied . The enzyme was almost completely inactivated on chemical modification with NBS, depending upon the concentration of NBS . The presence of a complex of Mg2+ and a substrate analogue, imidodiphosphate (PNP), provided extensive protection against the inactivation, whereas Mg2+ or PNP alone showed no protective effect . Amino acid analysis of the NBS-modified enzyme after hydrolysis with 6 M HCl indicated no change in the amino acid composition . However, the magnetic circular dichroism (MCD) bands around 293 nm due to the tryptophan residue and the optical density at 280 nm, decreased concomitantly with modification by NBS . These results strongly suggested that the tryptophan residue at position 143, which is the only tryptophan residue per subunit in the thermophilic PPiase (Ichiba, T., Takenaka, O., Samejima, T . and Hachimori, A . (1990) J . Biochem . 108, 572-578), might be involved in the active site or be located in the vicinity of the active site . The circular dichroism (CD) spectrum in the far ultraviolet region showed no significant alteration during the modification, indicating that the polypeptide chain backbone of the enzyme remained unaltered . However, the modification considerably altered the CD bands in, the near ultraviolet region, indicating that a conformational change occurred in the vicinity of the active site in the enzyme molecule.

FEBS Lett, 1991 Apr 22, 282(1), 132 - 4
Expression and purification of plasmid-encoded Thermoplasma acidophilum citrate synthase from Escherichia coli; Sutherland KJ et al.; The citrate synthase gene from the thermophilic archaebacterium Thermoplasma acidophilum was expressed in Escherichia coli, yielding an active product of the expected molecular weight . Manipulation of the citrate synthase gene in a series of pUC19 constructs showed that the presumed Thermoplasma ribosome binding site is recognized by the E . coli ribosome . A rapid purification of the expression product to homogeneity was achieved, based on the thermostability of Thermoplasma citrate synthase.

FEBS Lett, 1991 Apr 22, 282(1), 122 - 6
Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritima; Schumann J et al.; The hyperthermophilic eubacterium Thermotoga maritima uses starch as a substrate, without releasing amylase activity into the culture medium . The enzyme is associated with the 'toga' . Its expression level is too low to allow the isolation of the pure enzyme . Using cycloheptaamylose and acarbose affinity chromatography and common chromatographic procedures, two enzyme fractions are obtained . They differ in specificity, pH-optimum, temperature dependence and stability . Substrate specificity and Ca2+ dependence indicate alpha-, beta- and gluco-amylase activity . Compared with alpha-amylase from Bacillus licheniformis (Tmax = 75 degrees C), the amylases from Thermotoga maritima show exceedingly high thermal stability with an upper temperature limit at 95 degrees C . Significant turnover occurs only between 70 and 100 degrees C, i.e . in the range of viability of the microorganism.

FEBS Lett, 1991 Apr 9, 281(1-2), 173 - 6
Bacteriochlorophyll c formation via the C5 pathway of 5-aminolevulinic acid synthesis in Chloroflexus aurantiacus; Oh-hama T et al.; Biosynthesis of 5-aminolevulinic acid (ALA) in Chloroflexus aurantiacus, a thermophilic bacterium forming bacteriochlorophyll c, is shown to proceed via the C5 pathway by demonstrating (1) the specific labeling of its chlorin ring with {1 - 13C}glutamate and (2) the enzyme activity to produce ALA from glutamate in a cell-free extract . From the phylogenetic distribution it is suggested that ALA synthetase distributed in some aerobic eubacteria could be monophyletic in origin.

J Biol Chem, 1991 Apr 5, 266(10), 6195 - 200
Early assembly proteins of the large ribosomal subunit of the thermophilic archaebacterium Sulfolobus . Identification and binding to heterologous rRNA species; Altamura S et al.; Studies of ribosome structure in thermophilic archaebacteria may provide valuable information on (i) the mechanisms involved in the stabilization of nucleic acid-protein complexes at high temperatures and (ii) the degree of evolutionary conservation of the ribosomal components in the primary kingdoms of cell descent . In this work we investigate certain aspects of RNA/protein interaction within the large ribosomal subunits of the extremely thermophilic archaebacterium Sulfolobus solfataricus . The ribosomal proteins involved in the early reactions leading to in vitro particle assembly have been identified; it is shown that they can interact with the RNA in a temperature-independent fashion, forming a thermally stable "core" particle that can subsequently be converted into complete 50 S ribosomes . Among the protein components of the core particle, those capable of independently binding to 23 and 5 S RNA species have also been identified . Finally, we show that the early assembly proteins of Sulfolobus large ribosomal subunits are able to interact cooperatively with 23 S RNAs from other archaebacteria or from eubacteria, thereby suggesting that RNA/protein recognition sites are largely conserved within prokaryotic ribosomes . By contrast, no specific binding of the archaebacterial proteins to eukaryotic RNA could be demonstrated.

Scand J Work Environ Health, 1991 Apr, 17(2), 117 - 22
Feeding and bedding materials as sources of microbial exposure on dairy farms; Kotimaa MH et al.; Hay, grain, silage, and bedding are the sources of mold dust in agriculture . The aim of the present study was to evaluate the effect of different farming methods on exposure to airborne microbes . The study material comprised 50 silage, 54 hay, 47 grain, and 70 bedding samples taken on 18 farms in the beginning, middle, and end of the indoor feeding season . The modified wind-tunnel technique and six-stage impactors were used to determine the number of mesophilic bacteria, xerophilic fungi, mesophilic fungi, thermotolerant fungi, and thermophilic actinomycetes liberated from each material . Baled hay and straw liberated the largest amounts of microbes . Hay, except when dried in storage, liberated great numbers of fungal spores . The proportion of respirable airborne microbe-bearing particles was greatest in the highest concentrations . Theoretically, choosing the best possible alternative work methods could diminish exposure to microbes to one-tenth of the present level.

Biotechnol Appl Biochem, 1991 Apr, 13(2), 238 - 45
Transfer and expression of a Streptomyces cholesterol oxidase gene in Streptococcus thermophilus; Somkuti GA et al.; The recombinant plasmid pNCO937 (8.1 kbp) containing a Streptomyces sp . cholesterol oxidase gene was introduced into Streptococcus thermophilus by electrotransformation . Transformation frequency was 7.2 x 10(5) colony forming units/micrograms of DNA . The presence of the cholesterol oxidase gene in S . thermophilus was confirmed with Southern blot analysis using a biotinylated probe . Thin-layer chromatographic analysis showed the expression of the Streptomyces cholesterol oxidase gene resulting in the oxidation of cholesterol to 4-cholesten-3-one . S . thermophilus may be a suitable host for the expression of other genes regulating prokaryotic cholesterol metabolism.

J Bacteriol, 1991 Apr, 173(8), 2681 - 90
Processing and termination of 23S rRNA-5S rRNA-tRNA(Gly) primary transcripts in Thermus thermophilus HB8; Hartmann RK et al.; The two 23S rRNA-5S rRNA-tRNAGly operons from the extreme thermophilic eubacterium Thermus thermophilus HB8 were used to characterized the in vivo processing and termination of 23S rRNA-5S rRNA-tRNAGly primary transcripts in this organism by nuclease S1 mapping . A processing site in the pre-23S rRNA 3'-flanking region is located approximately 25 nucleotides upstream of 5S rRNA and precedes a putative 23S-5S rRNA spacer antitermination box A . Cleavage at this site and 5S rRNA 5' end formation were shown to be inseparable events . Termination of transcription at the uridine cluster following the termination-associated hairpin was shown to be efficient but leaky . Subsequent to the operon, a functional promoter was detected whose -35 box coincided with the uridine-rich termination region . The promoter directed synthesis of a beta-galactosidase fusion protein in Escherichia coli.

J Bacteriol, 1991 Apr, 173(8), 2481 - 7
Purification and properties of methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila; Jablonski PE et al.; Methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila TM-1 was purified 16-fold from a cell extract to apparent homogeneity as determined by native polyacrylamide gel electrophoresis . Ninety-four percent of the methylreductase activity was recovered in the soluble fraction of cell extracts . The estimated native molecular weight of the enzyme was between 132,000 (standard deviation {SD}, 1,200) and 141,000 (SD, 1,200) . Denaturing polyacrylamide gel electrophoresis revealed three protein bands corresponding to molecular weights of 69,000 (SD, 1,200), 42,000 (SD, 1,200), and 33,000 (SD, 1,200) and indicated a subunit configuration of alpha 1 beta 1 gamma 1 . As isolated, the enzyme was inactive but could be reductively reactivated with titanium (III) citrate or reduced ferredoxin . ATP stimulated enzyme reactivation and was postulated to be involved in a conformational change of the inactive enzyme from an unready state to a ready state that could be reductively reactivated . The temperature and pH optima for enzyme activity were 60 degrees C and between 6.5 and 7.0, respectively . The active enzyme contained 1 mol of coenzyme F430 per mol of enzyme (Mr, 144,000) . The Kms for 2-(methylthio)ethane-sulfonate and 7-mercaptoheptanoylthreonine phosphate were 3.3 mM and 59 microM, respectively.

J Dairy Sci, 1991 Apr, 74(4), 1145 - 50
Inhibition of Clostridium tyrobutyricum by bacteriocin-like substances produced by lactic acid bacteria; Thuault D et al.; Lactic acid bacteria were selected for their inhibitory activity against Clostridium tyrobutyricum under conditions that eliminate the effects of lactic acid and hydrogen peroxide . Four strains were isolated belonging to the species Lactococcus lactis ssp . lactis . The sensitivity of the inhibitory substances to pronase and trypsine indicates that they are proteins or peptides different from nisin . Their resistance to phospholipase D indicates that they are also different from lactostrepcin . The inhibitory substances are produced during the exponential phase of growth . Their activity is bactericidal and directed toward some strains of Clostridium tyrobutyricum, Lactobacillus helveticus, and Streptococcus thermophilus, but strains used as dairy starters, Lactobacillus lactis, Streptococcus thermophilus, and Propionibacterium shermanii, are not all affected by the inhibition.

Mol Gen Mikrobiol Virusol, 1991 Apr, (4), 3 - 7
{Cloning the gene of beta-galactosidase from the industrial strain of Streptococcus lactis 111 in E . coli cells and conjugated transfer of this gene to Streptococcus thermophilus cells}; Molotov SV et al.; The ability of the industrial strains of Streptococcus lactis to synthesize the enzyme beta-galactosidase was studied . Five strains among sixteen were found to produce high levels of the enzyme . The beta-galactosidase gene in the most active strain Streptococcus lactis 111 was shown to be located on the 50 kb conjugative plasmid . The plasmid was transferred by conjugation into Streptococcus thermophilus cells and subsequently the gene for beta-galactosidase was studied in transconjugants . The beta-galactosidase gene from Streptococcus lactis 111 was subcloned in Escherichia coli cells on the plasmid pBR322 . The gene was localized on the 4.8 kb BgIII fragment of DNA . Following the restriction of DNA by the Sau3A the gene was subcloned on the birepliconed plasmid vector pCB20 capable of replication in the Gram-negative as well as Gram-positive microorganisms . The recombinant derivatives of pCB20 were isolated that carry the beta-galactosidase gene on the DNA fragments of different size.

Zentralbl Hyg Umweltmed, 1991 Apr, 191(4), 423 - 37
{Acanthamoeba, Naegleria and invertebrates in wet areas of physiotherapy equipment in hospitals}; Michel R et al.; In the course of investigations on the occurrence of primary free-living amoebae in different aquatic habitats, especially Naegleria and Acanthamoeba, we now investigated the moist areas in physiotherapeutic departments of 10 hospitals . As a result 61% of the swabs taken in those areas were positive with one or several species of amoebae cultivated on NN-agar according to Page . Among many other species observed during this study we were able to isolate 47 strains of Acanthamoeba and only two strains of Naegleria . The Naegleria-strains did not appear to be thermophilic . Therefore they are part of the N . gruberi-complex, which is considered to include only non pathogenic strains . Six from 47 strains of Acanthamoebae isolated revealed pathogenic characters as demonstrated in the MIT (Mice inoculation test) . As a remarkable result four of these 6 strains with proven pathogenicity were not thermophilic at + 40 degrees C . Eventually all strains even not being thermophilic should in future be tested on pathogenicity . Additionally, nematodes and rotifers were isolated from several samples.

Antonie Van Leeuwenhoek, 1991 Apr, 59(3), 167 - 75
Occurrence and significance of precipitating antibodies against thermophilic actinomycetes in the sera of dairy herd workers, Nangali, Delhi; Gangwar M et al.; The study was prompted by the lack of information on the role of thermophilic actinomycetes in hypersensitivity pneumonitis in India . It reports the occurrence of precipitating antibodies against clinically important thermophilic actinomycetes in the sera of a population sample of dairy herd workers, Nangali, Delhi . Of 112 workers investigated, 28 (25%) showed precipitins against Faenia rectivirgula, 4 (3.2%) against Saccharomonospora viridis, 2 against Thermoactinomyces thalpophilus and one each against T . vulgaris and T . sacchari . The results of enzyme-linked immunosorbent assay (ELISA) indicated that IgG antibody activity against F . rectivirgula was significantly higher in the symptomatic group than in the asymptomatic group (p less than 0.05) of workers and the controls (p less than 0.01) . Significant difference in F . rectivirgula IgG activity was also obtained between the precipitin-positive symptomatic group and the precipitin-positive asymptomatic group (p less than 0.05) . In strong contrast, the IgG antibody activity against T . thalpophilus was found to be uniformly low . A limited aeromicrobiological sampling of the dairy farm revealed S . viridis (55.8%) to be the commonest species followed by T . vulgaris (19.2%), T . thalpophilus (18.5%), F . rectivirgula (5%) and T . sacchari (1.5%) . On the basis of suggestive clinical and laboratory findings, farmer's lung disease was suspected in four dairy herd workers . A comprehensive clinical evaluation including pulmonary function studies on the dairy herd workers and their long-term follow-up is indicated to determine the extent of respiratory morbidity caused by F . rectivirgula, S . viridis, T . thalpophilus, T . sacchari and T . vulgaris in India.

Antonie Van Leeuwenhoek, 1991 Apr, 59(3), 147 - 54
Filament formation in Thermus species in the presence of some D-amino acids or glycine; Janssen PH et al.; A number of strains of Thermus spp . changed morphology from rods of about 6 to 8 microns long to multicellular filaments (unsheathed trichomes) up to many hundreds of micrometres long with the addition of glycine or certain D-amino acids to the growth medium . Associated with this change was the formation of braided trichomes and occasionally true knots . Filament formation was reversible by the removal of the causal agent, but only if growth was possible . Electron microscopy suggested that the wall structure was not changed, but only that cells did not separate due to the continuous nature of the outer membrane layer . The filaments were thus multicellular . The constituent cells were similar in length to the normal rod-shaped cells . Filament formation by Thermus spp . may have applications in industrial scale culture of these extracellular enzyme-producing thermophilic bacteria.

Int J Syst Bacteriol, 1991 Apr, 41(2), 306 - 9
Two cellulolytic Clostridium species: Clostridium cellulosi sp . nov . and Clostridium cellulofermentans sp . nov; He YL et al.; Two cellulolytic clostridia, one thermophilic and the other mesophilic, were isolated and characterized . Cells of the thermophile are gram-negative rods that are motile with lophotrichous flagella and spherical terminal endospores which swell the cells . The optimum growth temperature is 55 to 60 degrees C, with a range of 40 to 65 degrees C . The deoxyribonucleic acid composition is 35 mol% G + C . The name Clostridium cellulosi sp . nov . is proposed . The type strain is AS 1.1777 . Cells of the mesophile are gram negative and motile with peritrichous flagella and terminal oval or spherical spores which swell the cells . The deoxyribonucleic acid composition is 34 mol% G + C . The name Clostridium cellulofermentans sp . nov . is proposed . The type strain is AS 1.1775 . Both C . cellulosi AS 1.1777 and C . cellulofermentans AS 1.1775 are deposited in the China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Academia Sinica, Beijing, People's Republic of China.

J Vet Med Sci, 1991 Apr, 53(2), 229 - 32
Population of Salmonella serovar typhimurium in the cecum of gnotobiotic chickens with Escherichia coli and intestinal bacteria; Fukata T et al.; To test the interaction between various species of bacteria and Salmonella serovar typhimurium (S . typhimurium), the population of S . typhimurium was measured in the cecum of gnotobiotic chickens in the presence of Escherichia coli (E . coli) and one of the four intestinal bacteria; Lactobacillus acidophilus . Clostridium perfringens, Bifidobacterium thermophilum and Bacteroides vulgatus . Competitive exclusion of S . typhimurium by di-flora chicken was not demonstrated . But the population of S . typhimurium was temporarily suppressed in di-flora chickens with E . coli and L . acidophilus . In penta-flora chickens with E . coli and these four intestinal bacteria, the population of S . typhimurium was suppressed for only 2 days . In normalized chickens, the population of S . typhimurium was markedly suppressed.

J Biomol Struct Dyn, 1991 Apr, 8(5), 1045 - 55
Three-dimensional folding of Tetrahymena thermophila rRNA IVS sequence: a proposal; Benedetti G et al.; We studied the Tetrahymena thermophila rRNA IVS sequence with the aim of obtaining a model of the structure characterized by the bases proximity of the self-reactions sites . The considered sequence kept up those fragments essential for its catalytic activity as demonstrated by deletion mutants . The first step was the theoretical analysis with a computer method previously proposed, to find optimal free energy secondary structures with the required features, under the suitable constrains . Then we tried folding the obtained secondary structures, in low resolution tertiary models, which kept up the proximity of the catalytic sites also in the space . The proposed tertiary folding seems to provide for a better explanation to the transesterification mechanisms and moreover it is in good agreement with the experimental data (activity of mutants, enzymatic cleavages, phylogenetically conserved regions).

Rev Latinoam Microbiol, 1991 Apr-Sep, 33(2-3), 149 - 51
{Microbiological quality of spices consumed in Cuba}; Rodriguez M et al.; The microbiological quality of some widely consumed spices in Cuba was evaluated by means of aerobic mesophilic microorganisms count, filamentous fungi, yeasts, coliforms, thermophilic and thermoresistant microorganisms . Salmonella spp was looked for too . Black pepper and cumin resulted the most contaminated spices with values of total count and thermoresistant microorganisms at levels of 10(6) per gram, and coliform values up to 10(5) per gram . Oregano and cinnamon showed satisfactory microbiological quality; the contamination detected in these spices was lower than 10(4) per gram . Salmonella spp and yeasts were not detected.

Appl Microbiol Biotechnol, 1991 Apr, 35(1), 14 - 8
Lysosomal enzymes produced by immobilized Tetrahymena thermophila; Kiy T et al.; The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways . Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply . However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 x 10(7) cells/ml . These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily . This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells . Under these conditions alpha-glucosidase, beta-glucosidase, beta-hexosaminidase and acid phosphatase were produced for at least 4 weeks . The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time . Immobilized T . thermophila cells can thus serve as a good source for production of commercially interesting enzymes.

Biochem Biophys Res Commun, 1991 Mar 29, 175(3), 949 - 54
DNA-dependent RNA polymerase from an extremely thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum; Andera L et al.; Extremely thermophilic bacterium Calderobacterium hydrogenophilum contains DNA-dependent RNA polymerase with unusual properties . Purified enzyme is thermoresistant (40 min at 100 degrees C) and exhibits similar subunit composition as eubacterial RNA polymerases (e.g . Escherichia coli) . However, the enzyme is not susceptible to antibiotics which inhibit eubacterial RNA polymerases (rifampicin and streptolydigin) . The activity of the enzyme is inhibited by actinomycin D, daunomycin and heparin.

J Biol Chem, 1991 Mar 15, 266(8), 5025 - 35
Cytochrome oxidase genes from Thermus thermophilus . Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3; Mather MW et al.; Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c . The smaller subunit contains heme C and was termed the C-protein . We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene . The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc . The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase) . In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices . Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex . Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center . We compared the subunit IIc sequence with that of related proteins . N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands . By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.

Biochim Biophys Acta, 1991 Mar 4, 1073(2), 386 - 93
The identification of a structurally important cysteine residue in the glycerol dehydrogenase from Bacillus stearothermophilus; Spencer P et al.; Evidence is presented to demonstrate that the Zn2+ metallo-enzyme glycerol dehydrogenase from the thermophile Bacillus stearothermophilus has one cysteine residue per subunit which is only available for reaction with thiol reagents in the metal-depleted form of the enzyme . Modification of the metal-depleted enzyme by methyl methanethiosulphonate prevents the reactivation of the enzyme by Zn2+ ions and induces dissociation of the oligomer into subunits . The rate of reaction of the cysteine residue with the thiol reagent DTNB is limited by a factor other than reagent concentration and it is proposed that the reagent only reacts with the cysteine residue in dissociated monomers . The enzyme has been labelled at the single cysteine residue by radioactive iodo{2-3H}acetic acid . Two radiolabelled peptides have been isolated and sequenced; one peptide is a component of the other . Spectroscopic evidence suggests that the cysteine residue is not involved in ligation of the essential metal ion . Chemical modification studies using the reagent diethylpyrocarbonate have suggested that two histidines are involved in the ligation of the metal.

Appl Environ Microbiol, 1991 Mar, 57(3), 694 - 700
Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum"; Luthi E et al.; A lambda recombinant phage expressing beta-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis . The sequence of a 2.1-kb fragment containing the mannanase gene has been determined . One open reading frame was found which could code for a protein of Mr 38,904 . The mannanase gene (manA) was overexpressed in E . coli by cloning the gene downstream from the lacZ promoter of pUC18 . The enzyme was most active at pH 6 and 80 degrees C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan . The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the "C . saccharolyticum" genome by homologous recombination.

Acta Paediatr Scand, 1991 Mar, 80(3), 386 - 8
Riding-school lung? Allergic alveolitis in an 11-year-old girl; Kristiansen JD et al.; Farmer's lung is a rural disease, caused by inhalation of airborne moulds and actinomycetes in the environment (farms) . We describe to our knowledge the first case of farmer's lung in an 11-year-old girl briefly exposed to Thermophilic actinomycetes at a riding-school.

Mol Cell Biol, 1991 Mar, 11(3), 1729 - 33
Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes; Dedon PC et al.; The in vivo association of histone H1 with specific genes in Tetrahymena thermophila was studied by using a simplified cross-linking and immunoprecipitation technique . Four genes were analyzed whose activities vary in three different developmental states (logarithmic growth, starvation, and conjugation) . Hybridization of the immunoprecipitated DNA to cloned probes showed an inverse correlation between the level of immunoprecipitation with H1 antiserum and transcriptional activity . This represents the first demonstration of an alteration in histone H1-DNA interaction associated with developmental changes in transcriptional activity.

J Bacteriol, 1991 Mar, 173(6), 1987 - 91
Distribution of folates and modified folates in extremely thermophilic bacteria; White RH; Analyses were made of the structures and levels of folates and modified folates present in extremely thermophilic bacteria . These procedures involved the chemical analysis of products resulting from the oxidative cleavage of the 6-substituted, folatelike tetrahydropterins present in the cells . Air-oxidized cell extracts of extreme thermophiles from two members of the archaebacterial order Thermococcales, Thermococcus celer and Pyrococcus furiosus, contained only 7-methylpterin, indicating that these cells contain a modified folate with a methylated pterin . Cell extracts also contained 6-acetyl-7-methyl-7,8-dihydropterin, another product derived from the oxidative cleavage of a dimethylated folate, demonstrating that both the C-7 and C-9 carbons of the pterin were methylated . Extracts, however, contained neither p-aminobenzoylpolyglutamates nor methaniline, the oxidative cleavage products of folates and methanopterin, respectively, indicating that they contain a previously undescribed C1 carrier(s) . On the basis of the level of the 7-methylpterin isolated, the levels of modified folate were 2 to 10 times higher than those typically found in mesophilic bacteria and 10 to 100 times less than the level of methanopterin found in the methanogenic bacteria . Oxidized cell extracts of Sulfolobus spp . of the archaebacterial order Sulfolobales contained only pterin, and, like members of the order Thermococcales, they contained neither-p-aminobenzoylpolyglutamates nor methaniline . Oxidized cell extracts of the extreme thermophiles Pyrobaculum sp . strain H10 and Pyrodictium occultum, from the archaebacterial orders Thermoproteales and Pyrodictiales, respectively, and Thermotoga maritima from the eubacterial order Thermotogales, contained pterin and p-aminobenzoylpolyglutamates, indicating that these cells contained unmodified folates . The levels of p-aminobenzoylpolyglutamates in these archaebacterial cell extracts indicate that the folates were present in the cells at levels 4 to 10 times higher than generally found in those mesophilic eubacteria which do not folates in energy metabolism . The levels and chain lengths of the of p-aminobenzoylpolyglutamates present in Thermotoga maritima were typical of those found in mesophilic eubacteria.

J Biochem (Tokyo), 1991 Mar, 109(3), 444 - 9
Polyamine distributions in thermophilic eubacteria belonging to Thermus and Acidothermus; Hamana K et al.; Triamines such as norspermidine, spermidine, and homospermidine and tetraamines such as norspermine, spermine, thermospermine, and aminopropylhomospermidine were found to be distributed ubiquitously in the eight extremely thermophilic (growing at 70 degrees C) Thermus species tested . Three linear pentaamine (caldopentamine, homocaldopentamine, and thermopentamine), two linear hexaamines (caldohexamine and homocaldohexamine), two tertiary branched tetraamines (N4-aminopropylnorspermidine and N4-aminopropyl-spermidine), and quaternary branched pentaamines such as N4-bis(aminopropyl)norspermidine and N4-bis(aminopropyl)spermidine were detected in T . thermophilus HB8, T . filiformis Wai33 A1, T . flavus AT-62, and T . caldophilus GK24 . The linear hexaamines and branched polyamines were absent in T . aquaticus YT-1, T . sp . X-1, T . sp . T2, and T . sp . T351, in which linear pentaamines were minor components . Moderately thermophilic Thermus ruber and Thermus sp . K-2 contained putrescine, spermidine, norspermidine, homospermidine, spermine, norspermine, thermospermine, and aminopropylhomospermidine . No pentaamines, hexaamines, or branched polyamines were found in these two moderately thermophilic Thermus species . On the other hand, moderately thermophilic, acidophilic Acidothermus cellulolyticus was devoid of all the polyamines.

J Biochem (Tokyo), 1991 Mar, 109(3), 421 - 7
Unilateral aminoacylation specificity between bovine mitochondria and eubacteria; Kumazawa Y et al.; The present study shows unilateral aminoacylation specificity between bovine mitochondria and eubacteria (Escherichia coli and Thermus thermophilus) in five amino acid-specific aminoacylation systems . Mitochondrial synthetases were capable of charging eubacterial tRNA as well as mitochondrial tRNA, whereas eubacterial synthetases did not efficiently charge mitochondrial tRNA . Mitochondrial phenylalanyl-, threonyl-, arginyl-, and lysyl-tRNA synthetases were shown to charge and discriminate cognate E . coli tRNA species from noncognate ones strictly, as did the corresponding E . coli synthetases . By contrast, mitochondrial seryl-tRNA synthetase not only charged cognate E . coli serine tRNA species but also extensively misacylated noncognate E . coli tRNA species . These results suggest a certain conservation of tRNA recognition mechanisms between the mitochondrial and E . coli aminoacyl-tRNA synthetases in that anticodon sequences are most likely to be recognized by the former four synthetases, but not sufficiently by the seryl-tRNA synthetase . The unilaterality in aminoacylation may imply that tRNA recognition mechanisms of the mitochondrial synthetases have evolved to be, to some extent, simpler than their eubacterial counterparts in response to simplifications in the species-number and the structural elements of animal mitochondrial tRNAs.

J Biochem (Tokyo), 1991 Mar, 109(3), 371 - 6
Thermostable phenylalanine dehydrogenase of Thermoactinomyces intermedius: cloning, expression, and sequencing of its gene; Takada H et al.; The gene encoding the thermostable phenylalanine dehydrogenase {EC 1.4.1.-} of a thermophile, Thermoactinomyces intermedius, was cloned and its complete DNA sequence was determined . The phenylalanine dehydrogenase gene (pdh) consists of 1,098 nucleotides and encodes 366 amino acid residues corresponding to the subunit (Mr 41,000) of the hexameric enzyme . The amino acid sequence deduced from the nucleotide sequence of the pdh gene of T . intermedius was 56.0 and 42.1% homologous to those of the phenylalanine dehydrogenases of Bacillus sphaericus and Sporosarcina ureae, respectively . It shows 47.5% homology to that of the thermostable leucine dehydrogenase from B . stearothermophilus . The pdh gene was highly expressed in E . coli JM109, the amount of phenylalanine dehydrogenase produced amounting up to about 8.3% of that of the total soluble protein . We purified the enzyme to homogeneity from transformant cells in a day, with a 58% recovery.

J Biochem (Tokyo), 1991 Mar, 109(3), 466 - 71
Purification by dye-ligand chromatography and a crystallization study of the F1-ATPase and its major subunits, beta and alpha, from a thermophilic bacterium, PS3; Shirakihara Y et al.; For a crystallization study, purification methods for F1-ATPase from a thermophilic bacterium, PS3, and its major subunits, beta and alpha, have been improved . The improvement depended on the introduction of dye-ligand chromatography columns to the previously adopted array of chromatography columns: a Blue-B (a blue dye bound to agarose) column was introduced for the F1 preparation, a Green-A column (a green dye attached to agarose) for the beta subunit, and a Blue-A (another blue dye, Cibacron Blue 3GA, bound to agarose) column for the alpha subunit . The improved preparations of all the proteins had purities of nearly 99% . Using the highly purified preparations of the proteins, crystallization conditions were searched for in a systematic way . Large plate crystals (0.2 X 0.5 X 0.5 mm) of F1 were grown from a polyethylene glycol solution . However, neither of the subunits was crystallized, in spite of extensive search for crystallization conditions.

Eur Cytokine Netw, 1991 Mar-Apr, 2(2), 137 - 40
Induction of 2'-5' A synthetase activity and interferon in humans by bacteria used in dairy products; Solis Pereyra B et al.; The production of interferon by fasted human subjects in response to lactic bacteria Lactobacillus bulgaricus and Streptococcus thermophilus was evaluated in vivo and in vitro . The 2'-5' A synthetase activity of blood mononuclear cells was used to estimate interferon production following a single ingestion of 10(11) bacteria in yoghurt or sterile milk (controls) . The level of the 2'-5' A synthetase of the yoghurt fed subjects was 83% (p = 0.002) higher than that of the milk fed controls 24 hours after ingestion . The baseline value remained unchanged in the control group . Blood mononuclear cells from a second group of subjects, were cultured with lactic bacteria for 48 hours, their cell-free supernatants contained gamma interferon . These results suggest that a transient production of interferon can be induced in healthy subjects by the lactic bacteria used in food processing.

J Bacteriol, 1991 Mar, 173(6), 1951 - 7
Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved; Leong-Morgenthaler P et al.; The genes coding for the lactose permease and beta-galactosidase, two proteins involved in the metabolism of lactose by Lactobacillus bulgaricus, have been cloned, expressed, and found functional in Escherichia coli . The nucleotide sequences of these genes and their flanking regions have been determined, showing the presence of two contiguous open reading frames (ORFs) . One of these ORFs codes for the lactose permease gene, and the other codes for the beta-galactosidase gene . The lactose permease gene is located in front of the beta-galactosidase gene, with 3 bp in the intergenic region . The two genes are probably transcribed as one operon . Primer extension studies have mapped a promoter upstream from the lactose permease gene but not the beta-galactosidase gene . This promoter is similar to those found in E . coli with general characteristics of GC-rich organisms . In addition, the sequences around the promoter contain a significantly higher number of AT base pairs (80%) than does the overall L . bulgaricus genome, which is rich in GC (GC content of 54%) . The amino acid sequences obtained from translation of the ORFs are found to be highly homologous (similarity of 75%) to those from Streptococcus thermophilus . The first 460 amino acids of the lactose permease shows homology to the melibiose transport protein of E . coli . Little homology was found between the lactose permease of L . bulgaricus and E . coli, but the residues which are involved in the binding and the transport of lactose are conserved . The carboxy terminus is similar to that of the enzyme III of several phosphoenolpyruvate-dependent phosphotransferase systems.

Appl Microbiol Biotechnol, 1991 Mar, 34(6), 707 - 14
A hyperthermostable pullulanase produced by an extreme thermophile, Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability; Suzuki Y et al.; A cell-associated pullulanase (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity . The molecular weight and isoelectric point were estimated to be about 55,000 and 7.0, respectively . The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr- Ala- Gly- . The enzyme was most active at pH 6.3 . The activities for 5% pullulan and 5% soluble starch were maximal at 75-80 degrees C and at 80-85 degrees C, respectively . The enzyme was stable up to 90 degrees C for 10 min at pH 6.8 . The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B . acidopullulyticus NCIB 11647 . A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K . pneumoniae----B . acidopullulyticus----B . flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.

Gene, 1991 Feb 15, 98(2), 161 - 7
Structure and evolution of the Tetrahymena thermophila gene encoding ribosomal protein L21; Rosendahl G et al.; The single-copy gene encoding ribosomal protein (r-protein) L21 in the macronucleus of the ciliate Tetrahymena thermophila was cloned and characterized . Sequencing of the L21 gene and a corresponding cDNA clone showed the gene to contain three introns, all located in the 3' half of the coding region . Primer extension and nuclease protection analyses revealed five transcription start points (tsp) 56-73 nucleotides upstream from the start codon . The uppermost tsp mapped to the first T in a sequence, TATAA, often found at tsp in T . thermophila . A comparison of amino acid sequences of r-proteins revealed that T . thermophila L21 belongs to a family of r-proteins that have been conserved in eubacteria, archaebacteria and eukaryotes . On the basis of structural and functional considerations, we suggest that the eukaryotic, like the prokaryotic, members of this protein family interact with the 5S RNA complex in ribosomes.

Biochem Pharmacol, 1991 Feb 15, 41(4), 561 - 6
In vitro inhibition of cell growth of MOLT-4 malignant human T-lymphoblasts by coenzyme F420; Raemakers-Franken PC et al.; The inhibitory effect of methanogenic coenzymes on the proliferation of MOLT-4 human malignant T-lymphoblasts was tested . Furthermore the effects of methanogenic coenzymes on dihydrofolate reductase activity (DHFR) from chicken liver have been examined . The results showed that heat-stable extracts of the hydrogenotrophs Methanobacterium thermoautotrophicum, Methanoculleus thermophilicum and Methanogenium tationis inhibit both proliferation of human T-lymphoblasts and DHFR activity . Heat-stable extract of the methylotroph Methanosarcina barkeri showed neither inhibitory nor stimulatory effects in both test systems . The present study proves coenzyme F420 to be the active, inhibitory component in methanogenic extracts.

J Biol Chem, 1991 Feb 15, 266(5), 3268 - 77
Methionyl-tRNA synthetase gene from an extreme thermophile, Thermus thermophilus HB8 . Molecular cloning, primary-structure analysis, expression in Escherichia coli, and site-directed mutagenesis; Nureki O et al.; The gene for the methionyl-tRNA synthetase (MetRS) from an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced . By expression of the T . thermophilus MetRS gene in Escherichia coli cells, thermostable MetRS was overproduced and purified to homogeneity by heat treatment and one-step column chromatography . The amino acid sequence of T . thermophilus MetRS showed low identities (approximately 25%) with those of MetRSs from E . coli, and cytoplasm and mitochondria of Saccharomyces cerevisiae . However, the amino acid residues in the binding sites for ATP and the anticodon and the 3' terminus of tRNA(Met) are highly conserved among the four MetRSs . T . thermophilus MetRS has a zinc finger-like sequence with all the three cysteine residues and a histidine residue . By site-directed mutagenesis of one of the cysteine residues (Cys127) of T . thermophilus MetRS, the SH group was found to be important for methionyl-tRNA synthesis . Just upstream of the structural gene for T . thermophilus MetRS there is a short open reading frame which codes for a methionine-rich peptide and is partly overlapped with an alternative terminator/antiterminator structure, suggesting that transcription of this gene is regulated by attenuation . Further upstream a region contains a nucleotide sequence homologous to that of the 5' half of T . thermophilus initiator tRNA(Met).

Nucleic Acids Res, 1991 Feb 11, 19(3), 547 - 52
Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro; Ueda T et al.; Phosphorothioate-containing RNAs were generated by transcription of coliphage T7 DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) and T7 RNA polymerase . RNAs in which a single nucleotide was substituted by the corresponding nucleoside phosphorothioate functioned as mRNA in the cell-free translation systems prepared from Escherichia coli and from an extreme thermophilic bacterium, Thermus thermophilus . This substitution increased the efficiency of protein synthesis by stabilizing the mRNAs in these systems . As the proportion of substituted nucleotides was increased, their mRNA activity was decreased accordingly . As judged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized using phosphorothioate-containing mRNAs as template were identical to those obtained with unsubstituted mRNAs . However, larger proteins which were barely detectable when unsubstituted mRNA was used were well represented when phosphorothioate-RNA was used instead . The advantages in using the phosphorothioate-mRNAs in the in vitro translation systems are discussed.

J Biol Chem, 1991 Feb 5, 266(4), 2567 - 72
Thermostable aspartate aminotransferase from a thermophilic Bacillus species . Gene cloning, sequence determination, and preliminary x-ray characterization; Sung MH et al.; The gene encoding aspartate aminotransferase of a thermophilic Bacillus species, YM-2, has been cloned and expressed efficiently in Escherichia coli . The primary structure of the enzyme was deduced from nucleotide sequences of the gene and confirmed mostly by amino acid sequences of tryptic peptides . The gene consists of 1,176 base pairs encoding a protein of 392 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 42,661 daltons . The active site lysyl residue that binds the coenzyme, pyridoxal phosphate, was identified as Lys-239 . Comparison of the amino acid sequence with those of aspartate aminotransferases from other organisms revealed very low overall similarities (13-14%) except for the sequence of the extremely thermostable enzyme from Sulfolobus solfataricus (34%) . Several amino acid residues conserved in all the compared sequences include those that have been reported to participate in binding of the coenzyme in three-dimensional structures of the vertebrate and E . coli enzymes . However, the strictly conserved arginyl residue that is essential for binding of the distal carboxyl group of substrates is not found in the corresponding region of the sequences of the thermostable enzymes from the Bacillus species and S . solfataricus . The Bacillus aspartate aminotransferase has been purified from the E . coli clone cell extracts on a large scale and crystallized in the buffered ammonium sulfate solution by the hanging drop method . The crystals are monoclinic with unit cell dimensions a = 121.2 A, b = 110.5 A, c = 81.8 A, and beta = 97.6 degrees, belonging to space group C2, and contain two molecules in the asymmetric unit . The crystals of the enzyme-alpha-methylaspartate complex are isomorphous with those without the substrate analog.

Biochemistry, 1991 Feb 5, 30(5), 1335 - 41
Nitrogen ligation to manganese in the photosynthetic oxygen-evolving complex: continuous-wave and pulsed EPR studies of photosystem II particles containing 14N or 15N; DeRose VJ et al.; The possibility of nitrogen ligation to the Mn in the oxygen-evolving complex from photosystem II was investigated with electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopies using 14N- and 15N-labeled preparations . Oxygen-evolving preparations were isolated from a thermophilic cyanobacterium, Synechococcus sp., grown on a medium containing either 14NO3- or 15NO3- as the sole source of nitrogen . the substructure on the "multiline" EPR signal, which arises from Mn in the S2 state of the enzyme, was measured with continuous-wave EPR . No changes were detected in the substructure peak positions upon substitution of 15N for 14N, indicating that this substructure is not due to superhyperfine coupling from nitrogen ligands . To detect potential nitrogen ligands with superhyperfine couplings of lesser magnitude than could be observed with conventional EPR methods, electron spin-echo envelope modulation experiments were also performed on the multiline EPR signal . The Fourier transform of the light-minus-dark time domain ESEEM data shows a peak at 4.8 MHz in 14N samples which is absent upon substitution with 15N . This gives unambiguous evidence for weak hyperfine coupling of nitrogen to the Mn of the oxygen-evolving complex . Possible origins of this nitrogen interaction are discussed.

J Biochem Biophys Methods, 1991 Feb-Mar, 22(2), 101 - 18
Test and calibration processes for microcalorimeters, with special reference to heat conduction instruments used with aqueous systems; Briggner LE et al.; Calorimeters are normally calibrated by use of electrical heaters, but the calibration values derived may not always be representative for the chemical or biological process investigated . It is therefore important to have available chemical test processes by which the electrical calibration values can be controlled . In some cases, electrical calibration should be replaced by chemical calibrations . With particular reference to needs in work with thermophile heat conduction microcalorimeters, a number of test and calibration processes are presented . Some calibration problems for various types of reaction vessel are discussed.

Genetics, 1991 Feb, 127(2), 327 - 34
A mutation in the large subunit ribosomal RNA gene of Tetrahymena confers anisomycin resistance and cold sensitivity; Sweeney R et al.; Anisomycin, an antibiotic that specifically inhibits the peptidyl transfer function of eukaryotic ribosomes, has been used to select resistant mutants in Tetrahymena thermophila . A mutation conferring anisomycin resistance (an-r) has been localized to a 1.2-kb fragment of the large subunit ribosomal RNA (rRNA) gene by transformation via microinjection . A single base pair change was detected within this region . Nine independently isolated an-r mutants had the same base pair change . T . thermophila strains that are homozygous for this mutation are cold sensitive, unable to mate and grossly abnormal in cell morphology.

J Gen Microbiol, 1991 Feb, 137 ( Pt 2), 369 - 80
Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences; Schroeder CJ et al.; The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S . thermophilus ATCC 19258 . Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length . When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons . Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon . The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da . Comparison of the S . thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E . coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively . Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S . thermophilus, E . coli and L . bulgaricus were compared.

Trends Biochem Sci, 1991 Feb, 16(2), 46 - 50
Tracing origins with molecular sequences: metazoan and eukaryotic beginnings; Lake JA; Milestones in the evolution of the eukaryotic cell are being discovered through the analysis of molecular sequences . As sequence data become increasingly plentiful, our ability to reconstruct the most distant evolutionary branchings of evolutionary trees is limited only by the mathematics of phylogenetic reconstruction . Analysis of ribosomal RNAs agrees with traditional analyses of morphological and developmental characters that all multicellular animals probably arose from a common ancestor, but highlights one of the major limitations of the various mathematical algorithms used . Refined methods of sequence analysis also suggest a previously unsuspected sister relationship between the eukaryotic nucleus and eocytes, a group of extremely thermophilic, sulfur-metabolizing bacteria, that questions the classical eukaryote/prokaryote division.

Biochem J, 1991 Feb 1, 273 ( Pt 3), 645 - 50
Purification and properties of an aryl beta-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli; Hudson RC et al.; An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 ('Caldocellum saccharolyticum') . It has a pI of 4.3 and shows optimal activity at pH 5.7 . The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside . It does not act on xylobiose . The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM . Xylose is a competitive inhibitor with Ki 40 mM . Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively . The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.

J Biochem (Tokyo), 1991 Feb, 109(2), 199 - 203
Phosphoenolpyruvate-insensitive phosphofructokinase isozyme from Thermus thermophilus HB8; Xu J et al.; In the previous paper {Xu, J., Oshima, T., & Yoshida, M . (1990) J . Mol . Biol . 215, 597-606}, we reported that phosphofructokinase from Thermus thermophilus is allosterically inhibited by phosphoenolpyruvate, which induces dissociation of the active four-subunit enzyme into an inactive two-subunit form . When T . thermophilus was cultured in a glucose-containing medium, another phosphofructokinase (PFK2) appeared in addition to the reported one (PFK1) . The molecular weights of the native PFK2 molecule (132,000) and its subunit (34,500), which are slightly smaller than those of PFK1, suggest that PFK2 is also composed of four identical subunits . However, the hyperbolic kinetics and molecular form of PFK2 are not affected at all by phosphoenolpyruvate . The NH2-terminal amino acid sequences of subunits of PFK1 and PFK2 revealed that they are composed of very similar but different polypeptides.

Protein Expr Purif, 1991 Feb, 2(1), 43 - 50
Effect of temperature on the expression of wild-type and thermostable mutants of kanamycin nucleotidyltransferase in Escherichia coli; Liao HH; The expression of kanamycin nucleotidyltransferase (KNTase) in Escherichia coli results in different forms of the protein, depending on the temperature; soluble active enzyme is synthesized at 23 degrees C but the protein is mostly aggregated and inactive in inclusion bodies when made at 37 degrees C . However, active enzyme can be recovered by solubilization of the inclusion bodies with 8 M urea followed by dilution of the denaturant, indicating that the polypeptide is not damaged covalently but is present in a misfolded state . The availability of thermostable mutants of KNTase allows a test of the hypothesis that formation of inclusion bodies when proteins are highly expressed in E . coli is due to the accumulation of a folding intermediate that is prone to temperature-dependent aggregation . Because these mutants were isolated by cloning the KNTase gene into the thermophile Bacillus stearothermophilus and selecting for kanamycin resistance at high growth temperatures, they must be thermostable for both synthesis and activity and must have folding intermediates that are less susceptible to the formation of aggregates . Indeed, whereas decreasing the temperature from 37 to 23 degrees C increased the KNTase specific activity 10-fold in cells expressing the wild-type enzyme, this change resulted in only a 2.1-fold increase for the TK1 (Asp80----Tyr) mutant and a 1.7-fold increase for the TK101 (Asp80----Tyr and Thr130----Lys) double mutant . The strategy of cloning in thermophiles and selecting or screening for mutants that fold correctly to yield biological activity at high growth temperatures may be useful in overcoming the problem of the insolubility of some proteins when expressed in heterologous hosts.

Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 1028 - 34
Tetrahymena 14-nm filament-forming protein has citrate synthase activity; Numata O et al.; The Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein involved in oral morphogenesis and in pronuclear behavior during conjugation . Cloning the 49K protein gene from a Tetrahymena thermophila cDNA library, we found that its primary structure exhibits a high sequence identity (51.5%) with porcine heart citrate synthase and retains functional domains . The 49K protein actually possesses citrate synthase activity, and is detected in mitochondria . These results suggest that the 49K protein has dual functions as both a respiratory enzyme and a structural protein in the cytoskeleton.

Biochem Biophys Res Commun, 1991 Jan 31, 174(2), 667 - 72
Identification of the NADH-binding subunit of energy-transducing NADH-quinone oxidoreductase (NDH-1) of thermus thermophilus HB-8; Xu XM et al.; The energy-transducing NADH--quinone oxidoreductase (NDH-1) isolated from Thermus thermophilus HB-8 is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN and at least three iron-sulfur clusters {Yagi, T., Hon-nami, K., and Ohnishi, T . (1988) Biochemistry 27, 2008-2013} . When NDH-1 of T . thermophilus HB-8 was irradiated by short UV light in the presence of {adenylate-32P}NADH or {adenylate-32P}NAD, radioactivity was incorporated into a single polypeptide of Mr 47,000 . The labeling of the Mr 47,000 polypeptide was diminished when UV irradiation of the enzyme complex with {adenylate-32P}NAD was carried out in the presence of NADH or deamino-NADH which act as substrates for the NDH-1, but not in the presence of NADP(H) or AMP which act neither as substrates nor as competitive inhibitors . These results strongly suggest that the Mr 47,000 polypeptide is an NADH-binding subunit of the NDH-1 of T . thermophilus HB-8.

Science, 1991 Jan 25, 251(4992), 401 - 7
Visualizing the higher order folding of a catalytic RNA molecule; Celander DW et al.; The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry . The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions . Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed . Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition . The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active . Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

Biochim Biophys Acta, 1991 Jan 23, 1073(1), 142 - 8
Purification and properties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus; Schinkinger MF et al.; Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3.) of the extreme thermophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity by (NH4)2SO4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose . The purified native enzyme had a Mr of about 270,000 and was shown to be a hexamer of subunit Mr of 44,000 . It was active from 30 to 95 degrees C, with a maximum activity at 85 degrees C . No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90 degrees C for 60 min, or in the presence of 4 M urea or 0.1% SDS . The enzyme was catalytically active with both NADH and NADPH as coenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates . With respect to coenzyme utilization the Sulfolobus solfataricus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.

Biochim Biophys Acta, 1991 Jan 8, 1076(1), 161 - 3
Intermediates in guanidine-HC1 unfolding of glutamine synthetase from the extreme thermophile, Bacillus caldolyticus; Wedler FC et al.; Glutamine synthetase is expressed in Bacillus caldolyticus as two isoforms that differ in physico-chemical and regulatory properties . Biphasic kinetics of thermal denaturation of E-I and E-II (Merkler, D.J., et al (1987) Biochemistry 26, 7805), suggested the formation of intermediates . CD spectral changes of E-II induced by guanidine-HC1 clearly indicate a three-state pathway for unfolding (N----I----D) . Refolding of E-II from 6 M GuHCl led to only 15% recovery of activity, compared to greater than or equal to 90% with E-I.

Free Radic Res Commun, 1991, 12-13 Pt 1, 259 - 68
Structure-function relationships in iron and manganese superoxide dismutases; Stallings WC et al.; Using the complete sequences for MnSOD from Thermus thermophilus and for FeSOD from E . coli, structural models for both oxidized enzymes have been refined, the Mn protein to an R of 0.186 for all data between 10.0 and 1.8 A, and the Fe protein to an R of 0.22 for data between 10.0 and 2.5 A . The results of the refinements support the presence of a solvent as a fifth ligand to Mn(III) and Fe(III) and a coordination geometry that is close to trigonal bipyramidal . The putative substrate-entry channel is comprised of residues from both subunits of the dimer; several basic residues that are conserved may facilitate approach of O2-, while other conserved residues maintain interchain packing interactions . Analysis of the azide complex of Fe(III) dismutase suggests that during turnover O2- binds to the metal at a sixth coordination site without displacing the solvent ligand . Because crystals reduced with dithionite show no evidence for displacement of the protein ligands, the redox-linked proton acceptor (C . Bull and J.A . Fee (1985), Journal of the American Chemistry Society 107, 3295-3304) is unlikely to be one of the histidines which bind the metal ion . Structural, kinetic, titration, and spectroscopic data can be accommodated in a mechanistic scheme which accounts for the differential titration behaviour of the Fe(III) and Fe(II) enzymes at neutral and high pH.

Zentralbl Mikrobiol, 1991, 146(3), 193 - 6
Production of cellulase(s) by Myriococcum albomyces; el-Gindy AA; The ability of the facultative thermophilic fungi Myriococcum albomyces (Cooney et Emerson) to produce extracellular cellulase(s) in a stationary liquid medium was studied . Diverse cellulosic carbon sources were used, assessing the different cellulolytic activities and enzymes different celluloytic activities and enzymes of the fungus (expressed in the classic terms C1-, Cx-activity) and their temperature and pH optima . The optimum temperature for "C1"-"Cx"-activity was 45 degrees C . The optimum pH for "C1"-activity was pH 6 while that for "Cx"-activity was pH 5.

Mikrobiol Zh, 1991 Jan-Feb, 53(1), 84 - 9
{The antimicrobial properties of thermophilic bacilli}; Pavlova IN et al.; Lytic activity and antagonistic properties of 11 thermophilic strains in different species of the genus Bacillus have been studied . It is shown that the spectra of lytic and antagonistic effect of the studied thermophiles do not coincide: filtrates of cultural liquids of these strains implement the degradation of living cells of Gram-negative bacteria and yeast but inhibit the growth of Gram-positive bacteria . Both the intensities of lytic and antagonistic actions of the extracellular products of the studied strains of bacilli do not correlate . An analysis of the obtained data shows that the lysis of test cultures takes place as a result of degrading action of extracellular hydrolases of thermophiles but not as a result of activation of autolytic systems of test cultures by the substances of antibiotic nature synthesized by the studied sporeforming bacteria.

Mikrobiol Zh, 1991 Jan-Feb, 53(1), 44 - 8
{The effect of the carbon sources and calcium ions on the activity of thermostable alpha-amylase in Bacillus sp . 86}; Kichakova NA; Amylolytic activity of Bacillus sp . 86 thermophilic strain was studied as affected by carbon sources . Glucose and saccharose being added to the nutrition medium with baker's yeast as the basic source of carbon the activity of alpha-amylase decreased . Introduction of native or soluble starch to the medium promoted a sharp increase in the enzymic activity . Bacillus sp . 86 being periodically cultivated in the optimized medium containing 4% of soluble starch and 0.5% of CaCl2, an eight-fold increase of amylolytic activity of cultural liquid was achieved as compared to the enzyme activity in the primary medium.

Microbiologica, 1991 Jan, 14(1), 31 - 5
Observations made on strains of Campylobacter spp . isolated in 1989 in northern Italy; Varoli O et al.; Over a 12-month period (1989), 192 thermophilic campylobacters (190 from stool and two from blood specimens) were isolated . All the strains were identified as far as species and biotype was concerned and 89 were also serotyped according to the Lior method . C . jejuni (90.10%) was the species most frequently isolated (mainly biotype I) while the most frequent serogroup was LIO 4 . Erythromycin-resistance was found in only 15 strains, but while it was very low for the species C . jejuni (1.16%), it was frequent for C . Coli (68.42%), mainly for biotype I (73.33%) . Moreover, eight strains of thermophilic campylobacters resistant to nalidixic acid (three strains of C . jejuni and five strains of C . coli) were found.

Biol Chem Hoppe Seyler, 1991 Jan, 372(1), 27 - 33
Polyamine biosynthesis in arginine-starved and refed rats; Schertel B et al.; Growth of rats fed with a synthetic diet was studied under control conditions (arginine-rich), arginine starvation, and arginine starvation/refeeding . Hepatic polyamine concentrations and ornithine decarboxylase (ODC-)activity were determined for each population . In the livers of arginine-starved rats putrescine was decreased to half the control content within 8 days; upon refeeding, it returned to control levels within another 8 days . Spermidine content in liver tissue of arginine-starved rats remained rather stable for 7 days, but thereafter dropped to half the original value within two days . Refeeding for a period of 11 days was not enough to restore the spermidine content . The effects of arginine starvation/refeeding on spermine were very similar to those of spermidine . ODC specific activity, when correlated with growth, was higher in livers of arginine-starved rats than in control animals . Refeeding caused a decrease in ODC-activity although growth arrest was completely released . This apparent uncoupling of growth and ODC stimulation supports the theory that ODC in rat liver is regulated at three levels: first the growth-related component which is observed after stimulation by growth-hormone; second the known feed back control by polyamines, e.g . via antizyme; third the regulation at the level of the substrate supply which has been shown in this work . This is not a unique finding since very similar results have been obtained in previous experiments with the protozoan Tetrahymena thermophila . A remarkable observation of these assays was that L-ornithine, when added to the arginine-free diet was not able to substitute for L-arginine in directing growth and growth related processes.

Berl Munch Tierarztl Wochenschr, 1991 Jan 1, 104(1), 4 - 7
{Yersinia pseudotuberculosis in New World monkeys}; Brack M et al.; It is reported on four cases of pseudotuberculosis in 2 Saimiri sciureus, 1 Callithrix jacchus/penicillata hybrid and 1 S . oedipus . The animals came from two different groups, one being infected with Y . pseudotuberculosis serotype I, the other with Y . pseudotuberculosis serotype II . All cases showed intestinal infection of thermophile Campylobacter . Remarkable were severe haemorrhagic components and the distinct RHS-proliferation, especially in the mesenteric lymphatic nodes.

Cancer Lett, 1991 Jan, 56(1), 37 - 43
Antitumor activity of Streptococcus thermophilus against fibrosarcoma: role of T-cells; Kaklij GS et al.; Antitumor activity induced by a heat-killed preparation of S . thermophilus against mouse fibrosarcoma was investigated . The treatment of Swiss mice with S . thermophilus prior to transplantation could not prevent tumors . However, animals cured by treatment with a S . thermophilus preparation failed to take up the tumor when rechallenged with fibrosarcoma . S . thermophilus did not induce antitumor activity in animals immunosuppressed by sublethal whole body gamma-irradiation (4 Gy) or hydrocortisone treatment prior to transplantation . Suppression of activity of macrophages by carrageenan had no effect on antitumor activity of the heat inactivated preparation of S . thermophilus . The intravenous administration of sera from cured animals was ineffective in curing the tumours . Spleen cells from cured animals could effectively transfer the antitumor activity to recipients transplanted with the tumor . This effect was abolished when the T-lymphocyte population in the inoculum was specifically depleted . The results thus suggest the involvement of T-lymphocytes in antitumor activity exhibited by S . thermophilus.

Eur J Biochem, 1991 Jan 1, 195(1), 55 - 63
Thymidine kinase from Tetrahymena thermophila . Purification and immunological analysis; Kinyanjui PW et al.; Thymidine kinase is an enzyme involved in DNA precursor metabolism and DNA replication . The synthesis of this enzyme is highly regulated during the cell cycle and the activity of the enzyme is also regulated by feedback inhibition . Genes encoding thymidine kinase have been extremely useful as selectable markers for introducing DNA into a number of cells . In order to study cell cycle regulation of thymidine kinase, the gene which encodes this enzyme, as well as aspects of DNA replication in the ciliated protozoan Tetrahymena thermophila, we have purified thymidine kinase from Tetrahymena . Two forms of thymidine kinase with native molecular masses of 59 kDa and 80 kDa have been identified and purified 6800- and 4600-fold, respectively . The 59-kDa enzyme, a homodimer of 30-kDa subunits, has been purified to near homogeneity and polyclonal antibodies have been raised against the 30-kDa subunit . Serological studies indicate that the two enzymes are antigenically distinct . The antibody against the Tetrahymena protein cross-reacts with a polypeptide in Chinese hamster ovary (CHO) cell extracts of 26 kDa which corresponds to the reported size of Chinese hamster thymidine kinase protein.

J Bacteriol, 1991 Jan, 173(2), 929 - 32
Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila; Raybuck SA et al.; The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of {1-14C} acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of {3'-32P}CoA with acetyl-CoA . Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1 . CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme . Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum . Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.

Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 184 - 8
Reconstitution of a group I intron self-splicing reaction with an activator RNA; van der Horst G et al.; The self-splicing rRNA intron of Tetrahymena thermophila belongs to a subgroup of group I introns that contain a conserved extra stem-loop structure termed P5abc . A Tetrahymena mutant precursor RNA lacking this P5abc is splicing-defective under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5) in vitro . However, the mutant precursor RNA by itself is capable of performing the self-splicing reaction without P5abc under different conditions (15 mM MgCl2/2 mM spermidine, pH 7.5) . We have investigated the functional role of the P5abc in the mechanism of the self-splicing reaction . When an RNA consisting of the P5abc but lacking the rest of the Tetrahymena intron is incubated with the mutant precursor, the self-splicing reaction proceeds highly efficiently under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5) . Two steps of the bimolecular self-splicing reaction can be performed accurately by a shortened precursor RNA containing all essential components required in the self-splicing reaction and an activator RNA consisting of the P5abc . Gel-mobility-shift assays suggest that two molecules associate by a direct RNA-RNA interaction during the splicing reaction . The results imply that there might exist other small RNAs whose role is to activate ribozymes.

Mol Cell Biol, 1991 Jan, 11(1), 166 - 74
Macronuclei and micronuclei in Tetrahymena thermophila contain high-mobility-group-like chromosomal proteins containing a highly conserved eleven-amino-acid putative DNA-binding sequence; Schulman IG et al.; HMG (high-mobility-group protein) B and HMG C are abundant nonhistone chromosomal proteins isolated from Tetrahymena thermophila macronuclei with solubilities, molecular weights, and amino acid compositions like those of vertebrate HMG proteins . Genomic clones encoding each of these proteins have been sequenced . Both are single-copy genes that encode single polyadenylated messages whose amounts are 10 to 15 times greater in growing cells than in starved, nongrowing cells . The derived amino acid sequences of HMG B and HMG C contain a highly conserved sequence, the HMG 1 box, found in vertebrate HMGs 1 and 2, and we speculate that this sequence may represent a novel, previously unrecognized DNA-binding motif in this class of chromosomal proteins . Like HMGs 1 and 2, HMGs B and C contain a high percentage of aromatic amino acids . However, the Tetrahymena HMGs are small, are associated with nucleosome core particles, and can be specifically extracted from macronuclei by elutive intercalation, properties associated with vertebrate HMGs 14 and 17, not HMGs 1 and 2 . Thus, it appears that these Tetrahymena proteins have features in common with both of the major subgroups of higher eucaryotic HMG proteins . Surprisingly, a linker histone found exclusively in transcriptionally inactive micronuclei also has several HMG-like characteristics, including the ability to be specifically extracted from nuclei by elutive intercalation and the presence of the HMG 1 box . This finding suggests that at least in T . thermophila, proteins with HMG-like properties are not restricted to regions of transcriptionally active chromatin.

J Hyg Epidemiol Microbiol Immunol, 1991, 35(3), 309 - 16
Circulating antibodies against thermophilic actinomycetes in farmers and mushroom workers; Shen YE et al.; Sera from individuals exposed to moldy hay, mushroom compost and unexposed individuals, were studied for antibodies against thermophilic actinomycetes and Aspergillus fumigatus . Antigens extracted from Thermoactinomyces vulgaris strains isolated from China were used in the study along with antigens from standard strains to screen the sera of individuals exposed to farms and unexposed controls from both China and the United States . It was found that very little antibody response to A . fumigatus and F . rectivirgula was demonstrated in the sera of both individuals exposed to farm environments and unexposed controls . On the other hand, antibodies to Thermoactinomyces vulgaris and T . candidus strains were seen in the sera of both exposed and unexposed individuals from China, indicating that these organisms are prevalent in all the environments of China . The results also indicate that antigenicity of the strains vary considerably in their reactivity with sera.

Arch Microbiol, 1991, 155(6), 593 - 600
Purification and properties of N5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri; Ma K et al.; Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110 degrees C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogens known so far . N5,N10-Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO2, was purified from this hyperthermophile . The apparent molecular mass of the native enzyme was found to be 300 kDa . Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa . The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group . The reductase was specific for reduced coenzyme F420 as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin . The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots . Vmax at 65 degrees C and pH 6.8 was 435 U/mg (kcat = 275 s-1) and the Km for methylenetetrahydromethanopterin and for reduced F420 were 6 microM and 4 microM, respectively . From Arrhenius plots an activation energy of 34 kJ/mol was determined . The Q10 between 40 degrees C and 90 degrees C was 1.5 . The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate . Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M . Sodium-, potassium-, and ammonium salts of these anions were equally effective . Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity . The thermostability of the reductase was found to be very low in the absence of salts . In their presence, however, the reductase was highly thermostable . Salt concentrations between 0.1 M and 1.5 M were required for maximal stability . Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity . The anion was of less importance . The N-terminal amino acid sequence of the reductase from M . kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri . Significant similarity was found.

Appl Biochem Biotechnol, 1991 Spring, 28-29, 267 - 75
Growth kinetics of Bacillus stearothermophilus BR219; Worden RM et al.; Bacillus stearothermophilus BR219, a phenol-resistant thermophile, can convert phenol to the specialty chemical catechol . The growth kinetics of this organism were studied in batch, continuous, and immobilized-cell culture . Batch growth was insensitive to pH between 6.0 and 8.0, but little growth occurred at 5.5 . In continuous culture on a dilute medium supplemented with 10 mM phenol, several steady states were achieved between dilution rates of 0.25 and 1.3 h-1 . Phenol degradation was found to be uncoupled from growth . Immobilized cells grew rapidly in a rich medium, but cell viability plummeted following a switch to a dilute medium supplemented with 5 mM phenol.

Parasitol Res, 1991, 77(6), 469 - 74
Species identification and characterization of an Acanthamoeba strain from human cornea; Matias R et al.; The isoenzyme pattern of an Acanthamoeba, stock H-1, isolated from a patient with keratitis (Krankenhaus Heidberg, Hamburg) was compared with that of two strains of A . quina-A . lugdunensis (302-2, 312-1), two stocks of A . lenticulata (45, 89-1) and one strain of A . rhysodes (302-1) . The isolated stock showed glucose-6-phosphate dehydrogenase (G-6-PDH), beta-hydroxybutyric dehydrogenase (beta-HBDH), alcohol dehydrogenase (ADH) and superoxide dismutase (SOD) isoenzyme patterns similar to those of A . quina-A . lugdunensis but their acid phosphatase (AP) patterns differed . Furthermore, cyst morphology showed that the patient-isolated stock belongs to group II of the taxonomic classification of Acanthamoeba . This stock was not thermophilic and exhibited non-pathogenic properties after its intranasal instillation into NMRI mice, whereas it killed BALB/c mice . Immunofluorescent studies revealed the presence of antibodies against Acanthamoeba in the patient's serum . Immunoblotting experiments showed that a 45-kDa protein reacted with this serum . Such an antigen was also detected in A . quina-A . lugdunensis and A . lenticulata . Lectin reactions with Canavalia ensiformis, Ricinus communis-120, Lotus tetragonolobus, Ulex europaeus I, Helix pomatia, Arachis hypogaea, Triticum vulgaris, Glycine maxima, Bauhinia purpurea and Mycoplasma gallisepticum demonstrated that only the A . lenticulata stocks could not be distinguished and that the H-1 stock was more similar to the A . lugdunensis 302-2 strain than to the other acanthamoebae.

Glas Srp Akad Nauka {Med}, 1991, (41), 1 - 10
{Immunologic response to microorganisms from a polluted ventilation system in a working environment}; Cernelc S et al.; Authors point out the morbidity of employees working in ventilation systems contaminated with various microorganisms . They analysed 96 workers exposed to air conditioning system (Group A), and 71 workers (Group B) breathing normal ambient air . The workers of both groups were subjected clinically by functionally and immunologically . Preparation of antigens "MMM" (Monday morning miseries) was used as an original method by Ajello et al . for producing antigens from systemic mycotic agents and subsequently modified . The aim of the present study is to evaluate the possibility of using ELISA in clinical practice for respiratory allergy diagnosis, and especially Hypersensitivity pneumonitis . Atopic status was determined by skin prick tests with common airborne allergens including Dermatophagoides pteronyssinus, ragweed, grasses and Aspergillus fumigatus., by Enzygnost--IgE (Behringwerke AG, Marburg) and for specific IgE by RAST technique (Pharmacia, Uppsala) . The skin prick tests were performed with "MMM"-antigens . PEFR (Peak Expiratory Flow-Rate) was measured by using a Wright's peak flow meter . PEFR was recorded on Monday (first day at work) and Friday (the end of the working week) . Measured values of PEFR in both groups of employees from Monday to Friday were elaborated by the Wilcoxon test . Results and discussion: Culture of scrapings from air conditioning vents were obtained and water from the humidifier system also cultured . They were grown: T . vulgaris, Aspergillus fumigatus, Thermoactinomyces vulgaris and others . Results of questionnaires, clinical evaluation and diagnostical procedures in employees of Group A and B are as follows: Thirty eight workers in Group A had a positive clinical history of "Monday illness" . In the symptomatic Group A we found in 8 cases abnormal chest roentgenogram . Further, there was no correlation between the presence of antibodies (ELISA) against MMM and pulmonary function abnormalities, as measured by either spirometry or DLCO . Further, we found good agreement between ELISA and prick test results with antigen MMM . Wilcoxon test showed a statistically significant difference between the two groups (0.01) . The median or central value of PEFR reduction in Group A is 10.23 per cent, and in Group B 1.49 per cent . A 30 per cent reduction of PEFR was observed in 5.21 per cent of subjects in Group A . Exposure to ventilation systems contaminated with Thermophilic actinomyces may be responsible for increased morbidity and reduced performance of employees working in air conditioning systems . Particularly the main filter should be checked regularly . Moreover, regular microbiologic examinations of dust and water from air preventing chronic obstructive lung diseases in employees working in areas served by contaminated air conditioning systems.

Biochem Int, 1991 Jan, 23(2), 343 - 8
The effects of beta-mercaptoethanol and sodium dodecyl sulfate on the Humicola insolens beta-glucosidase; Rao US et al.; Hydrolysis of p-nitrophenyl-beta-D-glucoside by the beta-glucosidase of a thermophilic and cellulolytic fungus, Humicola insolens was stimulated by two-fold in the presence of high concentrations of beta-mercaptoethanol . This enzyme did not have any free sulfhydryl groups and high concentrations of beta-mercaptoethanol (5% v/v) reduced all of the three disulfide bonds present in the enzyme . In contrast, the hydrolysis of cellobiose and cellulose polymers was inhibited by 50% under the same conditions . Sodium dodecyl sulfate (1% w/v) even in combination with beta-mercaptoethanol did not show any significant effects on this enzyme . These unusual properties suggest that this enzyme may be of significant importance for understanding the structure of the enzyme.

Biosystems, 1991, 25(1-2), 1 - 11
Cytoskeletal origins in sulfur-metabolizing archaebacteria; Searcy DG et al.; Several of the thermophilic acidopholic sulfur-metabolizing archaebacteria lack rigid cell walls . Their irregular shapes were maintained by an internal mechanism, presumably a cytoskeleton . Apparently this is an adaptation for respiration upon elemental sulfur, which requires cell contact since sulfur is insoluble in water . Also, we speculate that there could be additional functions of the cytoskeleton, such as prevention of osmotic cell lysis, thermal stabilization of enzymes, and improvements in metabolic efficiency through specific enzyme positioning . Such a well-developed cytoskeleton, evolving first in thermophilic archaebacteria, could have been a preadaptation for the evolution of eukaryotic cells.

Biofactors, 1991 Jan, 3(1), 29 - 35
Structural characteristics of methanogenic cofactors in the non-methanogenic archaebacterium Archaeoglobus fulgidus; Gorris LG et al.; Archaeoglobus fulgidus is an extremely thermophilic, sulphate reducing archaebacterium thought to represent a biochemical missing-link between sulphur-metabolizing bacteria and methanogenic bacteria . Whereas the phylogenetic position of A.fulgidus is closer to the sulphur-metabolizing bacteria, there is a partial overlap in the biochemical machinery of A.fulgidus with both groups of bacteria . In particular, the presence of a number of aberrant cofactors up to now thought to be involved exclusively in the process of methanogenesis in methanogenic archaebacteria, i.e . coenzyme F420, methanofuran and methanopterin, has been indicated by previous studies . Here we present evidence for the structural identity of the methanopterin cofactor of A.fulgidus with the methanopterin isolated from Methanobacterium thermoautotrophicum and show that this non-methanogenic bacterium contains two as yet unknown analogues of coenzyme F420 . The levels of the various cofactors were determined in cultures grown either on formate or lactate as the carbon source and sulphate or thiosulphate as the sulphur source.

Antonie Van Leeuwenhoek, 1991 Jan, 59(1), 1 - 7
Production and properties of xylanases from thermophilic actinomycetes; Holtz C et al.; 30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the genera Thermomonospora, Saccharomonospora, Microbispora, Streptomyces and Actinomadura . Screening of these strains for extracellular xylanase indicated that strains of Saccharomonospora and Microbispora generally were poor xylanase producers (0.5-1.5 U/ml) whereas relatively high activities were observed in cultures of Streptomyces and Actinomadura (4-12 U/ml) . A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains of Actinomadura exhibited higher thermostabilities than those of Streptomyces . To evaluate the potential of thermophilic Actinomadura for industrial applications, xylanases of three strains were studied in more detail . The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran . The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80 degrees C . The enzymes exhibited considerable thermostability at their optimum temperature . The half-lives at 75 degrees C were in the range from 6.5 to 17 h . Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products . Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65% . Complete utilization of xylan is presumably achieved by beta-xylosidase activities which could be shown to be largely cell-associated in the 3 Actinomadura strains.

J Photochem Photobiol B, 1991 Jan, 8(2), 189 - 97
Isolation and protein chemical characterization of the B806-866 antenna complex of the green thermophilic bacterium Chloroflexus aurantiacus; Wechsler TD et al.; The B806-866 antenna complex was isolated from cytoplasmic membranes of the green thermophilic bacterium Chloroflexus aurantiacus . The membranes were treated with 7 M urea at 50 degrees C, the B806-866 antenna complex was solubilized with a mixture of Noni-fjdet P-40 (octylphenoxypolyethoxyethanol (Sigma)) and sodium dodecylsulphate (2:1) and isolated by sucrose density gradient centrifugation . This antenna complex was characterized by reversed-phase chromatography (fast polypeptide and polynucleotide liquid chromatography), amino acid and sequence analyses . The B806-866 antenna of Chloroflexus aurantiacus consists of two polypeptides: the B806-866-alpha and B806-866-beta polypeptides in an apparent stoichiometric ratio of 1:1, which may be equivalent to the structural elementary unit found in the antenna systems of many species of Rhodospirillaceae.

J Dairy Sci, 1991 Jan, 74(1), 87 - 95
Influence of nonfermented dairy products containing bacterial starter cultures on lactose maldigestion in humans; Lin MY et al.; The effect of nonfermented dairy products containing yogurt or acidophilus cultures on lactose utilization by lactose-maldigesting humans was investigated . Yogurt and acidophilus milk containing 10(7) or 10(8) of Streptococcus thermophilus and Lactobacillus bulgaricus, or Lactobacillus acidophilus, respectively, were prepared using commercially processed 2% low fat milk . Immediately following inoculation, products were refrigerated . Lactose maldigestion was monitored by measuring breath hydrogen excretion at hourly intervals for 8 h following consumption of 400 ml of each test meal containing approximately 20 g of lactose . The yogurt milk containing 10(8) cfu/ml was shown to contain significant concentrations of microbial beta-galactosidase (EC 3.2.1.23; approximately 3 U/ml), which remained stable for at least 14 d at refrigerator temperatures . Breath hydrogen peaks were delayed and significantly lower (approximately 20 ppm at 5 to 7 h) than control values (approximately 70 ppm at 4 h), and intolerance symptoms were eliminated in all subjects . Yogurt milk containing 10(7) cfu/ml demonstrated intermediate breath hydrogen values and was marginally significantly different from control values . Lactobacillus acidophilus strains with varying resistance to bile and total beta-galactosidase-producing potential were also tested . Only one strain, LA-1, which demonstrated low bile resistance and intermediate beta-galactosidase activity, was capable of significantly decreasing breath hydrogen values when 10(8) cfu/ml of milk was consumed.

J Biochem (Tokyo), 1991 Jan, 109(1), 1 - 2
Crystallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HB8 chimeric 3-isopropylmalate dehydrogenase; Onodera K et al.; A chimeric gene was constructed by fusing the Bacillus subtilis and Thermus thermophilus genes coding for 3-isopropylmalate dehydrogenase, and expressed in Escherichia coli . The chimeric enzyme was crystallized in a size suitable for X-ray structure analysis . The crystal has a space group of P3(1)21 or P3(2)21, a = b = 77.1 A and c = 158.3 A, which is isomorphous with that of the native enzyme from T . thermophilus.

J Mol Evol, 1991 Jan, 32(1), 53 - 63
A molecular phylogeny of dinoflagellate protists (pyrrhophyta) inferred from the sequence of 24S rRNA divergent domains D1 and D8; Lenaers G et al.; The sequence of two divergent domains (D1 and D8) from dinoflagellate 24S large subunit rRNA was determined by primer extension using total RNA as template . Nucleotide sequence alignments over 401 bases have been analyzed in order to investigate phylogenetic relationships within this highly divergent and taxonomically controversial group of protists of the division Pyrrhophyta . Data are provided confirming that dinoflagellates represent a monophyletic group . For 11 out of the 13 investigated laboratory grown species, an additional domain (D2) could not be completely sequenced by reverse transcription because of a hidden break located near its 3'-terminus . Two sets of sequence alignments were used to infer dinoflagellate phylogeny . The first {199 nucleotides (nt)} included conservative sequences flanking the D1 and D8 divergent domains . It was used to reconstruct a broad evolutionary tree for the dinoflagellates, which was rooted using Tetrahymena thermophila as the outgroup . To confirm the tree topology, and mainly the branchings leading to closely related species, a second alignment (401 nt) was considered, which included the D1 and D8 variable sequences in addition to the more conserved flanking regions . Species that showed sequence similarities with other species lower than 60% on average (Knuc values higher than 0.550) were removed from this analysis . A coherent and convincing evolutionary pattern was obtained for the dinoflagellates, also confirmed by the position of the hidden break within the D2 domain, which appears to be group specific . The reconstructed phylogeny indicates that the early emergence of Oxyrrhis marina preceded that of most Peridiniales, a large order of thecate species, whereas the unarmored Gymnodiniales appeared more recently, along with members of the Prorocentrales characterized by two thecal plates . In addition, the emergence of heterotrophic species preceded that of photosynthetic species . These results provide new perspectives on proposed evolutionary trees for the dinoflagellates based on morphology, biology, and fossil records.

Nucleic Acids Symp Ser, 1991, (24), 19 - 20
Diadenosine oligophosphates: peculiarities of synthesis by phenylalanyl-tRNA synthetases from E . coli MRE-600 and Thermus thermophilus HB8; Biryukov AI et al.; Temperature and other factors affecting synthesis of bis(5'-adenosyl) tetraphosphate (Ap4A) and bis(5'-adenosyl)triphosphate (Ap3A) catalyzed by phenylalanyl-tRNA synthetases (PheRSs) from Escherichia coli MRE-600 and Thermus thermophilus HB8 have been investigated . Those two synthetases exhibited different temperature-dependent rates of the Ap4A and Ap3A synthesis . However, with respect to the effects of such effectors of the Ap4A synthesis as Zn2+, Mg2+, tRNA and Ap4A phosphonate analogues, as well as some inhibitors of aminoacyl-tRNA synthetase, those two enzymes were apparently undistinguishable.

J Basic Microbiol, 1991, 31(5), 391 - 8
Thermophilic actinomycetes associated with agro-environment of Punjab state (India); Singh S et al.; Thermophilic actinomycetes (TAs) are unique high temperature aerobic bacteria which belong to the group actinomycetes . While working on hypersensitivity pneumonitis (HP), an immunological disorder, resulting from the inhalation of spores of thermophilic actinomycetes, it was considered worthwhile to record their occurrence in the environment of Punjab . Thermophilic actinomycetes isolated by spread plate method from nine different substrates of the Punjab environment were identified as 8 different species . Atmospheric occurrence of TAs was detected by exposing Petri dishes containing soy agar medium which were exposed in six different environments of the state . Seven thermophilic species were recorded . Thermoactinomyces vulgaris was the predominant species with 87.5, 87.1, and 100% prevalence in the soil, natural manure and the wheat field, respectively . The study is the first report of its kind from this region.

Acta Microbiol Hung, 1991, 38(2), 117 - 20
Thermophilic and thermotolerant fungi of animals' hair; Bagy MM et al.; Nine thermophilic genera and 17 species in addition to one variety of Aspergillus flavus, Malbranchea pulchella and Humicola grisea were collected from hair samples in Riyadh, Saudi Arabia at 45 degrees C . Fifty-one hair specimens of rabbit, sheep, camel and horse were examined for the presence of thermophilic fungi . The most frequent species were Aspergillus fumigatus, Aspergillus niger, Thermoascus aurantiacus and Malbranchea pulchella var . sulfurea . In low frequency, Aspergillus flavus, Aspergillus quadrilineatus, Paecilomyces variotii, Paecilomyces aerugineus, Mucor pusillus and Rhizopus stolonifer were also recovered . The hair samples tested in the present study were completely free from any keratinolytic fungi at 45 degrees C.

Arch Microbiol, 1991, 156(1), 49 - 55
Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri; Rospert S et al.; Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110 degrees C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogen known so far . Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile . The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two alpha-, beta- and gamma-subunits and that it contained the nickel porphinoid coenzyme F430 as prosthetic group . The purified reductase was inactive . The N-terminal amino acid sequence of the gamma-subunit was determined . A comparison with the N-terminal sequences of the gamma-subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity . Besides methyl-coenzyme M reductase cell extracts of M . kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO2 (apparent Vmax at 65 degrees C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran:tetrahydro-methanopterin formyltransferase, 13 U/mg; N5,N10-methylenetetrahydromethanopterin cyclohydrolase, 14U/mg; N5,N10-methenyltetrahydromethanopterin dehydrogenase (H2-forming), 33 U/mg; N5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420 dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F420-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg . Apparent Km values for these enzymes and the effect of salts on their activities were determined . The coenzyme F420 present in M . kandleri was identified as coenzyme F420-2 with 2-gamma-glutamyl residues.

Arch Microbiol, 1991, 156(1), 43 - 8
N5, N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming) from the extreme thermophile Methanopyrus kandleri; Ma K et al.; Methanopyrus kandleri is a novel abyssal methanogenic archaebacterium growing at 110 degrees C on H2 and CO2 . The N5, N10-methylenetetrahydromethanopterin dehydrogenase, an enzyme involved in methanogenesis from CO2 and H2, was purified from this hyperthermophile and characterized . The dehydrogenase was found to be composed of only one polypeptide of apparent molecular mass 44 kDa . The UV/Vis spectrum was similar to that of albumin . The protein catalyzed the reversible dehydrogenation of N5, N10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5, N10-methenyltetrahydromethanopterin (CH identical to H4MPT+) and molecular hydrogen: CH2 = H4MPT H+ in equilibrium CH identical to H4MPT+ +H2 . The rate of CH2 = H4MPT dehydrogenation (apparent Vmax) at 65 degrees C and pH 5.8 was 1500 U/mg, the apparent Km for CH2 = H4MPT was 50 microM, the Arrhenius activation energy was 52 kJ/mol, and the Q10 between 30 degrees C and 70 degrees C was 2.0 . The specific activity increased hyperbolically with the proton concentration between pH 7 and pH 4.5 . The purified dehydrogenase did not catalyze the reduction of viologen dyes, of coenzyme F420, and of pyridine nucleotides with either CH2 = H4MPT or H2 . For activity the CH2 = H4MPT dehydrogenase required the presence of salts . Fifty percent of maximal activity was reached at salt concentrations of 100 mM, potassium phosphate, potassium chloride, and sodium chloride being almost equally effective in stimulating the enzyme activity . Cell extracts of M . kandleri did not loose CH2 = H4MPT dehydrogenase activity when incubated at 90 degrees C for 60 min . The purified enzyme, however, proved very thermolabile . The purified enzyme, however, proved very thermolabile.(ABSTRACT TRUNCATED AT 250 WORDS)

Comp Biochem Physiol B, 1991, 100(1), 31 - 5
Species-specific antibodies of Tetrahymena acid alpha-glucosidase; Banno Y et al.; 1 . Tetrahymena acid alpha-glucosidases A and B were purified from T . pyriformis W and T . thermophila 399, respectively . The acid alpha-glucosidases A and B were different in immunological properties and thermostability . 2 . The acid alpha-glucosidases A and B reflected specific distribution between T . pyriformis and T . thermophila . 3 . Type A and B of taurolipid showed a species-specific distribution pattern between T . pyriformis and T . thermophila.

Nucleic Acids Symp Ser, 1991, (25), 49 - 50
Relation between functions and conformational characteristics of modified nucleosides found in tRNAs; Kawai G et al.; Conformational characteristics of N4-acetyl-2'-O-methylcytidine (ac4Cm), 5-methyl-2'-O-methylcytidine (m5Cm) and N2-dimethyl-2'-O-methylguanosine (m2(2)Gm) found in tRNAs from extremely thermophilic archaebacteria were analyzed by proton NMR spectroscopy . The 2'-O-methylation of ac4C, m5C and m2(2)G was found to stabilize the C3'-endo form and therefore cause "conformational rigidity" . In particular, the ac4Cm was found to be extremely rigid due to additive effects of the N4-acetylation and 2'-O-methylation . Therefore, tRNAs from the extremely thermophilic archaebacteria use the base modifications in combination with the 2'-O-methylation, resulting in stabilization of the A-type conformation at specific positions in the tRNAs even at very high temperatures . In contrast, mesophile tRNAs use for a given site only one of these ribose and base modifications each of which is effective enough by itself at ordinary temperatures . These findings are consistent with our previous findings that roles of a variety of post-transcriptional modifications are to regulate the conformational rigidity/flexibility which is essential for the tRNA functions.

Biochem Cell Biol, 1991 Jan, 69(1), 66 - 71
Timing of the appearance of ubiquitinated histones in developing new macronuclei of Tetrahymena thermophila; Davie JR et al.; Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inert micronucleus . During vegetative growth, macronuclear histones H2A and H2B and micronuclear H2A are ubiquitinated . Despite differences in function, macro- and micro-nuclei are related . During conjugation (the sexual phase of the life cycle in Tetrahymena), postzygotic division products of micronuclei give rise to new micro- and macro-nuclei . Using an anti-ubiquitin antibody in Western blotting experiments, we determined the levels of ubiquitinated histones in new macro- and micro-nuclei at various times during conjugation . Very soon after the second postzygotic division (approximately 8 h) when new macronuclei begin to synthesize RNA, ubiquitinated H2B and polyubiquitinated H2A are present . At this time micronuclei have only low levels of ubiquitinated H2A . During later stages of conjugation (15 h), the level of polyubiquitinated H2A decreases, while ubiquitinated H2B increases in developing new macronuclei, attaining levels of ubiquitinated H2B approaching that of parental macronuclei . Ubiquitinated histones are not detectable in the 15-h micronuclei . These results show that ubiquitination of H2B coincides with the transformation of an inert germinal nucleus into that of a transcriptionally active somatic nucleus, suggesting that ubiquitinated H2B has a role in maintaining the transcriptionally active chromatin state.

Agric Biol Chem, 1991 Jan, 55(1), 221 - 7
Xylose (glucose) isomerase gene from the thermophile Clostridium thermohydrosulfuricum; cloning, sequencing, and expression in Escherichia coli; Dekker K et al.; The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned, using a fragment of the Bacillus subtilis gene as a probe . The complete nucleotide sequence of the gene was analyzed . C . thermohydrosulfuricum is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized . Comparison with amino acid sequences from other xylose isomerases showed that amino acids involved in substrate binding and isomerization are well conserved . Purification of the enzyme produced in E . coli was done by heating a cell-free extract at 85 degrees C for 10 min, giving a 20-fold purified enzyme . The native enzyme is a homomeric tetramer with a molecular weight of 200,000.

Eur J Biochem, 1990 Dec 27, 194(3), 839 - 44
Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilium . Cloning and sequencing of the gene; Sutherland KJ et al.; The gene encoding the citric acid cycle enzyme, citrate synthase, has been cloned from the thermoacidophilic archaebacterium, Thermoplasma acidophilum . We report the sequencing of this gene and its flanking regions, and the derived amino acid sequence of the enzyme is compared by multiple-sequence alignment analysis with those of citrate synthases from eubacterial and eukaryotic organisms . The similarity is less than 30% between the archaebacterial and non-archaebacterial sequences, although the majority of residues implicated in the catalytic action of the enzyme have been conserved across all three kingdoms . The cloned archaebacterial gene has been expressed in Escherichia coli to produce catalytically active citrate synthase . This is the first reported sequence of citrate synthase from the archaebacteria.

J Biol Chem, 1990 Dec 15, 265(35), 21946 - 50
Thermus thermophilus membrane-associated ATPase . Indication of a eubacterial V-type ATPase; Yokoyama K et al.; An ATPase with Mr of 360,000 was purified from plasma membranes of a thermophilic eubacterium Thermus thermophilus, and was characterized . ATP hydrolytic activity of the purified enzyme was extremely low, 0.07 mumol of Pi released mg-1 min-1, and it was stimulated up to 30-fold by bisulfite . The following properties of the enzyme indicate that it is not a usual F1-ATPase but that it belongs to the V-type ATPase family, another class of ATPases found in membranes of archaebacteria and eukaryotic endomembranes . Among its four kinds of subunits with approximate Mr values of 66,000 (alpha), 55,000 (beta), 30,000 (gamma), and 12,000 (delta), the alpha subunit had a similar molecular size to the catalytic subunits of the V-type ATPases but was significantly larger than the alpha subunit of F1-ATPases . ATP hydrolytic activity was not affected by azide, an inhibitor of F1-ATPases, but was inhibited by nitrate, an inhibitor of the V-type ATPase . N-terminal amino acid sequences determined for the purified alpha and beta subunits showed much higher similarity to those of the V-type ATPases than those of F1-ATPases . Thus the distribution of the V-type ATPase in the prokaryotic kingdom may not be restricted to archaebacteria.

Nucleic Acids Res, 1990 Dec 11, 18(23), 6915 - 9
Complete sequence of the extrachromosomal rDNA molecule from the ciliate Tetrahymena thermophila strain B1868VII; Engberg J et al.; The recent development of rDNA vectors for transformation of Tetrahymena combined with improved microinjection technology should lead to a renewed interest in this organism . In particular, the rDNA itself constitutes an attractive system for biochemical studies . The rDNA is amplified to a level of 2% of the total DNA and exists as extrachromosomal molecules . Furthermore, the rDNA is homogeneous in sequence because it is derived from a single gene during sexual reorganization . In order to facilitate studies of this molecule, we report here a compilation of previously published sequence information together with new sequence data that completes the entire sequence of the 21 kb rDNA molecule.

Nucleic Acids Res, 1990 Dec 11, 18(23), 6889 - 93
Interaction of the isolated domain II/III of Thermus thermophilus elongation factor Tu with the nucleotide exchange factor EF-Ts; Peter ME et al.; The middle and C-terminal domain (domain II/III) of elongation factor Tu from Thermus thermophilus lacking the GTP/GDP binding domain have been prepared by treating nucleotide-free protein with Staphylococcus aureus V8 protease . The isolated domain II/III of EF-Tu has a compact structure and high resistance against tryptic treatment and thermal denaturation . As demonstrated by circular dichroism spectroscopy, the isolated domain II/III does not contain any alpha-helical structure . Nucleotide exchange factor, EF-Ts, was found to interact with domain II/III, whereas the binding of aminoacyl-tRNA, GDP and GTP to this EF-Tu fragment could not be detected.

J Mol Biol, 1990 Dec 5, 216(3), 501 - 2
Crystals of protein L1 from the 50 S ribosomal subunit of Thermus thermophilus . Preliminary crystallographic data; Agalarov SC et al.; Crystals have been obtained of protein L1 from the large ribosomal subunit of an extreme thermophile . Thermus thermophilus, using a mixed solution of ammonium sulphate/methane pentanediol . The crystals belong to the space group P2(1)2(1)2, with cell parameters a = 82.7 A, b = 63.4 A, c = 44.7 A . They diffract X-rays to 2.3 A resolution.

J Bacteriol, 1990 Dec, 172(12), 7306 - 9
Isolation and characterization of Bacillus stearothermophilus 30S and 50S ribosomal protein mutations; Schnier J et al.; Bacillus stearothermophilus mutations which confer resistance to or dependence on a variety of ribosome-targeted antibiotics have been isolated . Many of these mutations produce ribosomal proteins with altered mobilities in a two-dimensional gel electrophoresis system . This collection of altered thermophilic ribosomal proteins will be useful in examining ribosomal structure and function.

Biotechniques, 1990 Dec, 9(6), 680, 682 - 3
pUC18rspL: a plasmid vector for indirect selection and direct sequencing of promoter-bearing DNA fragments; Vockley JG et al.; Plasmid pUC18rspL is a 3.788-kilobase pair vector, derived from pUC18, pKK232-8 and pHSG664, which identifies promoter-bearing DNA fragments functional in StrA E . coli by activation of a promoterless streptomycin-sensitive gene cartridge (rspL) . Expression of the plasmid-borne rspL gene leads to sensitivity dominance and death of the cell . Promoter-bearing DNA fragments can be cloned within a synthetic polylinker containing 12 unique restriction nuclease target sequences . After transformation of StrA E . coli TB1, ampicillin-resistant transformants are replica plated on medium containing ampicillin and streptomycin to identify promoters cloned in their functional orientation . These elements can be sequenced without additional subcloning steps from the M13 universal forward primer hybridization site located 5' of the polylinker in the pUC18 contribution . Transcriptional terminators are cloned 3' of the rspL gene to maintain a balance between transcription and replication when high signal strength promoters such as the E . coli Tac promoter are analyzed . pUC18rspL is used to clone transcriptional promoters from the extreme thermophile T . aquaticus . Promoter signal strength can be estimated by determining the extent of sensitivity dominance conferred to the host.

J Bacteriol, 1990 Dec, 172(12), 6803 - 8
Reverse gyrase, a hallmark of the hyperthermophilic archaebacteria; Bouthier de la Tour C et al.; Investigation of the presence of a reverse gyrase-like activity in archaebacteria revealed wide distribution of this activity in hyperthermophilic species, including methanogens and sulfur-dependent organisms . In contrast, no reverse gyrase activity was detected in mesophilic and moderately thermophilic organisms, which exhibited only an ATP-independent activity of DNA relaxation . These results suggest that the presence of reverse gyrase in archaebacteria is tightly linked to the high growth temperatures of these organisms . With respect to antigenic properties, the enzyme appeared similar among members of the genus Sulfolobus . In contrast, no close antigenic relatedness was found between the reverse gyrase of members of the order Sulfolobales and that of the other hyperthermophilic organisms.

Dtsch Tierarztl Wochenschr, 1990 Dec, 97(12), 511 - 5
{The suitability of selected humic acid batches as culture media additives for isolating thermophilic Campylobacter species}; Weinrich K et al.; Using freshly poured respectively five days under light and air stored Campylobacter media blood or lignite humic acids containing Mueller-Hinton-Agar proved to be better for isolation of a Campylobacter field strain than unsupplemented or peat humic acids containing media . The observed differences between the used media are probably caused by three different ability to prevent the accumulation of a photochemically induced toxicity by storing of media under light and air . The storage of blood respectively lignite humic acids containing media over fourteen days under dark and air leads to a time and trend dependent decrease of the colony counts of the Campylobacter field strain . By means of equations of spline functions the observed decrease in ability of media to support the growth of thermophilic Campylobacter can be described mathematically . By using the mathematical model the theoretical extend of growth of Campylobacter on the media can be determinate with respect to the applied inoculum and to the time of storage of media . The cause of decrease in the isolations rate with respect to the time of storage and the cause of different effectiveness of the used humic acids in the media are discussed.

J Gen Microbiol, 1990 Dec, 136 ( Pt 12), 2569 - 76
Cloning, sequencing and homologies of the cbh-1 (exoglucanase) gene of Humicola grisea var . thermoidea; Azevedo Mde O et al.; Studies on the enzymes of the cellulase complex of the thermophilic fungus Humicola grisea var . thermoidea are described . A genomic library was constructed in the phage vector EMBL 4, and from this library two clones were isolated using as a probe the cloned cbh-1 (exoglucanase, EC 3.2.1.91) gene of Phanerochaete chrysosporium, a cellulolytic basidiomycete fungus . These clones were analysed by restriction mapping and Southern blotting, and one of them (lambda 3) was sub-cloned into the M13 phage vectors mp18 and mp19 . The gene sequence was determined by the dideoxy chain-termination method . Sequence comparison with the equivalent genes from P . chrysosporium and Trichoderma reesei was made: in terms of primary sequence there is about 60% homology between the three species . Secondary structure prediction of the H . grisea sequence was also computed.

J Bacteriol, 1990 Dec, 172(12), 7145 - 50
Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila; Abbanat DR et al.; The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C . The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex . The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0 . The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA . Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture . CO could be replaced with CO2 and titanium(III) citrate . When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2 . Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide.

J Bacteriol, 1990 Dec, 172(12), 6669 - 72
Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain; Nanmori T et al.; We isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase . Based on taxonomical research, this bacterium was identified as Bacillus stearothermophilus . Each extracellular enzyme was separated by hydrophobic chromatography by using a Toyopearl HW-65 column, followed by gel filtration with a Sephacryl S-200 column . Each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatography system with a Mono Q HR 5/5 column . The optimum temperatures were 60 degrees C for xylanase and 70 degrees C for beta-xylosidase . The isoelectric points and molecular masses were 5.1 and 39.5 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively . Heat treatment at 60 degrees C for 1 h did not cause inhibition of the activities of these enzymes . The action of the two enzymes on xylan gave only xylose.

Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9083 - 7
N epsilon-acetyl-beta-lysine: an osmolyte synthesized by methanogenic archaebacteria; Sowers KR et al.; Methanosarcina thermophila, a nonmarine methanogenic archaebacterium, can grow in a range of saline concentrations . At less than 0.4 M NaCl, Ms . thermophila accumulated glutamate in response to increasing osmotic stress . At greater than 0.4 M NaCl, this organism synthesized a modified beta-amino acid that was identified as N epsilon-acetyl-beta-lysine by NMR spectroscopy and ion-exchange HPLC . This beta-amino acid derivative accumulated to high intracellular concentrations (up to 0.6 M) in Ms . thermophila and in another methanogen examined--Methanogenium cariaci, a marine species . The compound has features that are characteristic of a compatible solute: it is neutrally charged at physiological pH and it is highly soluble . When the cells were grown in the presence of exogenous glycine betaine, a physiological compatible solute, N epsilon-acetyl-beta-lysine synthesis was repressed and glycine betaine was accumulated . N epsilon-acetyl-beta-lysine was synthesized by species from three phylogenetic families when grown in high solute concentrations, suggesting that it may be ubiquitous among the methanogens . The ability to control the biosynthesis of N epsilon-acetyl-beta-lysine in response to extracellular solute concentration indicates that the methanogenic archaebacteria have a unique beta-amino acid biosynthetic pathway that is osmotically regulated.

J Dairy Sci, 1990 Dec, 73(12), 3379 - 84
Antimutagenic activity of milk fermented by Streptococcus thermophilus and Lactobacillus bulgaricus; Bodana AR et al.; Antimutagenic activity of acetone or ethylacetate extracts of skim milk fermented by Streptococcus thermophilus, Lactobacillus bulgaricus, or a combination of both the organisms was studied using Salmonella typhimurium (TA 98 and TA 100) . Mutagens used were 4-nitroquinoline-N-oxide (a direct-acting mutagen) and 2-aminofluorene (a mutagen requiring S9 activation) . Extracts from all fermented milks showed significant (P less than .05) dose response in suppressing the number of revertants caused by NQNO and 2-aminofluorene in both tester strains, whereas extracts from unfermented milk had no effect . Extracts prepared from milk fermented by L . bulgaricus plus S . thermophilus showed significantly (P less than .05) more antimutagenic activity than extracts prepared from milk fermented by S . thermophilus alone . Solvent (acetone vs . ethyl acetate) effect was not significant with 4-nitroquinoline-N-oxide as mutagen . However, in the case of 2-aminofluorene, acetone extracts showed significantly (P less than .05) higher antimutagenic activity . The results of this and related studies strongly indicate that antimutagenic compounds are produced in milk during fermentation by S . thermophilus and L . bulgaricus.

Biochimie, 1990 Dec, 72(12), 855 - 62
Characterization and comparison of virulent bacteriophages of Streptococcus thermophilus isolated from yogurt; Benbadis L et al.; Seven virulent bacteriophages of Streptococcus thermophilus were characterized at the molecular level and classified into 2 subgroups (A and B) by DNA/DNA hybridization experiments and analysis of their structural proteins . Two representatives of subgroups A and B were compared to 3 representatives of Neve's subgroups I, II and III (Neve et al, 1989) by Southern blot experiments . These isometric-headed phages possess a double-stranded DNA genome varying between 30-44 kilobase (kb) pairs . Subgroup A is composed of 3 phages (phi 57 as representative) with similar structural proteins as determined by sodium dodecyl sulfate-poly-acrylamide gel (SDS-PAGE) electrophoresis (estimated molecular weights of 31,000 and 27,500 for phage phi 57 and 32,000 and 27,000 for the 2 others) . A common structural protein of 43,000 was found for phages of subgroup B . Phages phi 57 (subgroup A) and a10/J9 or PO (Neve's subgroups I or II, respectively) belonged to the same subgroup as determined by DNA/DNA hybridization experiments . Partial DNA homology was detected among all the phages tested except for phage phi ST27 of AW Jarvis . Phage-host interactions were also investigated by cross-propagation of the 7 studied phages on different indicator strains . A complete lack of correlation existed between the DNA homology grouping of the phages and their host range . Various restriction-modification systems were detected in some of the Streptococcus thermophilus strains.

Eur J Clin Microbiol Infect Dis, 1990 Dec, 9(12), 895 - 7
Isolation of a urease positive thermophilic variant of Campylobacter lari from a patient with urinary tract infection; Bezian MC et al.; An unusual campylobacter strain, a urease positive thermophilic variant of Campylobacter lari, was isolated from the urine of a patient with urinary tract infection who was hospitalized because of cirrhosis and haemorrhage . The strain was isolated from urine specimens on three separate occasions . A significant serological response to the organism was also detected . This is the first documented case of extra-intestinal infection due to this group of organisms.

Mol Cell Probes, 1990 Dec, 4(6), 435 - 43
Amplification of DNA sequences of Epstein-Barr and human immunodeficiency viruses using DNA-polymerase from Thermus thermophilus; Glukhov AI et al.; Using thermophilic DNA-polymerase from Thermus thermophilus we have amplified by polymerase chain reaction (PCR) specific DNA sequences of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV) . DNA-polymerase from Thermus thermophilus (molecular mass of 80-86 kDa) differs in its physico-chemical properties from DNA-polymerase from Thermus aquaticus (molecular mass of 62-68 kDa) . To amplify the specific EBV DNA sequence, oligonucleotide primers for the virus replicon region (oriP region) were used . As a result of amplification, a specific 405-bp DNA fragment was produced.

FEBS Lett, 1990 Nov 26, 275(1-2), 130 - 4
Engineering thermostability in archaebacterial glyceraldehyde-3-phosphate dehydrogenase . Hints for the important role of interdomain contacts in stabilizing protein conformation; Biro J et al.; Construction of hybrid enzymes between the glyceraldehyde-3-phosphate dehydrogenases from the mesophilic Methanobacterium bryantii and the thermophilic Methanothermus fervidus by recombinant DNA techniques revealed that a short C-terminal fragment of the Mt . fervidus enzyme contributes largely to its thermostability . This C-terminal region appears to be homologous to the alpha 3-helix of eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenases which is involved in the contacts between the two domains of the enzyme subunit . Site-directed mutagenesis experiments indicate that hydrophobic interactions play an important role in these contacts.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6637 - 9
Transcriptional regulation of gene expression in Tetrahymena thermophila; Stargell LA et al.; The only well-characterized study of gene expression in Tetrahymena thermophila (1) demonstrates that the temperature dependent expression of the Ser H3 gene is regulated at the level of mRNA stability . A run-on transcription assay was developed to determine if regulation of RNA stability was a major mechanism regulating gene expression in Tetrahymena or if transcriptional regulation dominates . The relative transcriptional activities of 14 Tetrahymena genes were determined in different physiological/developmental states (growing, starved and conjugating) in which many of the genes showed striking differences in RNA abundance . In every case except Ser H3, changes in transcription accompanied changes in RNA abundance . Thus differential transcription, not differential RNA degradation, is the major mechanism regulating RNA abundance in Tetrahymena.

J Chromatogr, 1990 Nov 23, 521(2), 169 - 78
Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources; Maas E et al.; The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane . A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described . This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique . Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T . aquaticus . The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.

Biochemistry, 1990 Nov 20, 29(46), 10573 - 6
The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification; Walstrum SA et al.; When the circular form of the self-splicing intervening sequence of Tetrahymena thermophila was purified by denaturing polyacrylamide gel electrophoresis by standard methods, the rate of its reaction with tetrauridylate decreased 150-fold at 30 degrees C and at least 1000-fold at 0 degrees C . The activity of the self-splicing RNA was restored by heating it to high temperature and letting it renature in the presence of Mg2+ . The rate of reaction of tetrauridylate with the self-splicing RNA flanked by exons was also greatly decreased by gel purification . The difference in activation energies for the reaction of native and denatured intervening sequences suggests that a substantial conformational rearrangement of the gel-purified RNA occurs prior to reaction.

J Mol Biol, 1990 Nov 20, 216(2), 239 - 41
Characterization and preliminary crystallographic studies on large ribosomal subunits from Thermus thermophilus; Volkmann N et al.; Diffracting crystals, suitable for X-ray crystallographic analysis, have been obtained from large (50 S) ribosomal subunits from Thermus thermophilus . These crystals, with P4(1)2(1)2 symmetry and a unit cell of 495 A x 495 A x 196 A, reach typically a size of 0.15 mm x 0.25 mm x 0.35 mm . Using synchrotron radiation at cryo-temperature, these crystals diffract X-rays to better than 9 A resolution, and do not show any measurable decay after a few days of irradiation . They complete a series of crystals, grown by us, from ribosomal particles of the same source, including a 30 S subunits, 70 S ribosomes and complexes of the latter with: (1) an oligomer of 35 uridine residues and (2) the same oligonucleotide together with approximately two Phe-tRNA(Phe) molecules . Crystallographic analysis of the various members of this series should provide information for investigating the conformational changes that take place upon the association of ribosomes from their subunits as well as upon binding of non-ribosomal components that participate in protein biosynthesis.

Cell, 1990 Nov 16, 63(4), 763 - 72
The controlling sequence for site-specific chromosome breakage in Tetrahymena; Yao MC et al.; Site-specific chromosome breakage occurs in many ciliated protozoa during nuclear differentiation . We have determined the cis-acting sequence that controls this process in Tetrahymena thermophila . The Tetrahymena ribosomal RNA gene is bounded by two breakage sites . Injection of this gene into developing macronuclei leads to breakage at these sites . Deletion analysis has localized the sequences essential for breakage to a 28 bp region that includes a 15 bp sequence (Cbs) known to be present in other breakage sites . Insertions of Cbs allow breakage to occur at new sites, which is accompanied by elimination of surrounding DNAs and formation of telomeric sequences, as it is at natural sites . Thus, Cbs is the necessary and sufficient sequence signal for chromosome breakage in Tetrahymena.

Biochim Biophys Acta, 1990 Nov 15, 1041(2), 172 - 7
Chemical modification of xylanases: evidence for essential tryptophan and cysteine residues at the active site; Deshpande V et al.; N-Bromosuccinimide (NBS) completely inactivated xylanases from Chainia and alkalophilic and thermophilic (AT) Bacillus with a concomittant decrease in absorption at 280 nm and with second-order rate constants of 10,500 and 5000 M-1.min-1, respectively at pH 6.0 and 25 degrees C . The kinetic analysis of inactivation indicated that one and three tryptophan residues were essential for the xylanase activity from Chainia and Bacillus, respectively . The xylanases were also inhibited by 2-hydroxy-5-nitrobenzyl bromide (HNBB) . The modification of cysteine residues by p-hydroxymercurybenzoate (PHMB) and N-ethylmaleimide did not cause a loss in activity of the xylanase from Bacillus, whereas that from Chainia was completely inactivated . The kinetics of inactivation revealed the involvement of one cysteine residue for xylanase from Chainia with a second-order rate constant of 50,000 M-1.min-1 . The PHMB-modified enzyme failed to show the presence of titrable -SH groups . Xylan afforded complete protection against inactivation by NBS, HNBB and PHMB, indicating the involvement of tryptophan and cysteine residues at the substrate-binding region of the enzyme.

Biochim Biophys Acta, 1990 Nov 15, 1041(2), 97 - 100
Substrate inhibition or activation kinetics of the beta-galactosidase from the extreme thermoacidophile archaebacterium Caldariella acidophila; Pulvin S et al.; The kinetics of the hydrolysis of p-nitrophenyl-beta-D-galactopyranoside (pNPG) by a thermophile, beta-galactosidase, was studied at different temperatures . This enzyme was isolated from the thermophilic microorganism archaebacterium Caldariella acidophila . The hydrolysis of pNPG by beta-galactosidase does not follow Michaelis-Menten law . This enzyme is inhibited by excess substrate at low temperatures and it is activated by excess substrate at high temperatures . A minimum mechanistic model is proposed to explain the behaviour . This model assumes the binding of an additional substrate molecule on the glycosidyl enzyme intermediate . This model is in good agreement with the postulated mechanism for beta-galactosidase from Escherichia coli . The kinetic parameters are calculated at six different temperatures.

Biochemistry, 1990 Nov 6, 29(44), 10172 - 80
Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme . 2 . Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site; Herschlag D et al.; The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme {G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R . (1990) Biochemistry (preceding paper in this issue)} . The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU . Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7) . This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product . With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting . The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate . This can be described as "uniform binding" of the base at position -3 in the ground state and transition state {Albery, W.J., & Knowles, J . R . (1976) Biochemistry 15, 5631-5640} . Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 Nov 6, 29(44), 10159 - 71
Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme . 1 . Kinetic description of the reaction of an RNA substrate complementary to the active site; Herschlag D et al.; A ribozyme derived from the intervening sequence (IVS) of the Tetrahymena preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine) . This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5'-exon . The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50 degrees C and neutral pH in the presence of 10 mM Mg2+ . The value of kcat/Km = 9 x 10(7) M-1 min-1 for the oligonucleotide substrate with saturating G represents rate-limiting binding . This rate constant for binding is of the order expected for formation of a RNA.RNA duplex between oligonucleotides . (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS) . The value of kcat = 0.1 min-1 represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU . The product GA5 dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU . The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUA5, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pA5 portion of the substrate makes no net contribution to binding . Both the substrate and product bind approximately 10(4)-fold (6 kcal/mol) stronger than expected from base pairing with the 5'-exon binding site . Thus, tertiary interactions are involved in binding . Binding of G2CCCUCU and binding of G are independent . These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent . The rate constant for reaction of the ternary complex is calculated to be kc approximately equal to 350 min-1, a rate constant that is not reflected in the steady-state rate parameters with saturating G . The simplest interpretation is adopted, in which kc represents the rate of the chemical step . A site-specific endonuclease reaction catalyzed by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, kc(-G) = 0.7 min-1, is approximately 500-fold slower than that with saturating guanosine . The value of kcat/Km = 6 x 10(7) M-1 min-1 for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1990 Nov 6, 29(44), 10147 - 58
Melting and chemical modification of a cyclized self-splicing group I intron: similarity of structures in 1 M Na+, in 10 mM Mg2+, and in the presence of substrate; Jaeger JA et al.; C IVS is the cyclized form of the intron from the RNA precursor of the Tetrahymena thermophila large subunit (LSU) ribosomal RNA . C IVS was mapped by chemical modification in 1 M Na+, 0.05 M Na+ and 10 mM Mg2+ (Na+/Mg2+), and Na+/Mg2+ with CUCU substrate . The results suggest the secondary structure is similar for all three conditions . Optical melting curves were also measured for C IVS in 1 M Na+ and Na+/Mg2+ and indicate the secondary structures have similar stabilities under both conditions . Computer predictions of secondary structure and stability are in good agreement with observations . The results suggest that many of the approximations used for computer prediction of secondary structure by free energy minimization are reasonable.

J Biol Chem, 1990 Nov 5, 265(31), 19082 - 90
Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes . Characterization of the structural gene and function of active site histidine; Lee CY et al.; The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined . The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474 . The deduced amino acid sequence of thermophilic C . thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%) . Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes . To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C . thermosulfurogenes enzyme were individually modified by site-directed mutagenesis . Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity . When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes . The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis . The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5 . Deuterium isotope effects by D-{2-2H}glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes . These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism . Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.

Biochim Biophys Acta, 1990 Nov 5, 1020(2), 207 - 12
Monomer-dimer structure of cytochrome-c oxidase and cytochrome bc1 complex from the thermophilic bacterium PS3; Sone N et al.; Molecular weights of three membrane proteins have been measured in the presence of 0.1% octaethylene glycol n-dodecyl ether (C12E8) by the measuring system in which a membrane protein eluted from a gel chromatography column is monitored sequentially for ultraviolet absorption, light scattering and refractive index . The relative molecular mass (Mr) and amount of bound detergent per protein (delta) can be calculated from these data, if instrumental constants are measured using a set of appropriate water-soluble proteins which does not bind nonionic surfactants . The molecular masses of cytochrome c oxidase and cytochrome bc1 complex from the thermophilic bacterium PS3 were determined to be 127 kDa and 185 kDa, respectively, indicating that the oxidase is monomeric, while the bc1 complex dimeric in the presence of C12E8 . The larger apparent molecular mass of about 310 kDa of the PS3 oxidase obtained from the retention time of the gel chromatography (Sone, N., Sekimachi, M . and Kutoh, E . (1987) J . Biol . Chem . 262, 15386-15391) turned out to be due to a high binding ability with the detergent (delta = 1.25 g/g) of this very hydrophobic protein . Analyses of bovine heart cytochrome oxidase, on which monomer/dimer properties have been reported, showed that the enzyme is mainly dimeric (Mr = 374,000), while a small portion is monomeric (Mr = 191,000) . Mild alkaline treatment of this enzyme caused monomerization of the enzyme with accompanying aggregate formation . These results, thus, show that this method is suitable to analyze monomer/dimer conversion of membrane protein as well as to estimate structure of membrane proteins.

J Appl Bacteriol, 1990 Nov, 69(5), 758 - 64
Thermophilic campylobacters in surface waters around Lancaster, UK: negative correlation with Campylobacter infections in the community; Jones K et al.; The incidence of campylobacter enteritis in Lancaster City Health Authority is three times the UK average for similar sizes of population and has marked seasonal peaks in May and June . Environmental monitoring of surface waters around Lancaster showed that thermophilic campylobacters were absent from drinking water from the fells and from the clean upper reaches of the River Conder but were present in the main rivers entering Morecambe Bay, the lower reaches of the River Conder, the Lancaster canal, and seawater from the Lune estuary and Morecambe Bay . All the surface waters tested showed the same seasonality, namely, higher numbers in the winter months and low numbers or none in May, June and July . The absence of thermophilic campylobacters in the summer months may be due to high sunshine levels because experiments on the effects of light showed that campylobacters in sewage effluent and seawater were eliminated within 60 and 30 min of daylight respectively but survived for 24 h in darkness . As the concentrations of campylobacters in surface waters were at their lowest precisely at the time of peak infections in the community it is unlikely that surface waters form Lancaster's reservoir of campylobacter infection for the community.

Biochem Cell Biol, 1990 Nov, 68(11), 1292 - 6
Purification and some properties of a thermostable DNA polymerase from a Thermotoga species; Simpson HD et al.; A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp . strain FjSS3-B.1 by a five-step purification procedure . First, the crude extract was treated with polyethylenimine to precipitate nucleic acids . The endonuclease activity coprecipitated . DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation . As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used . The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography . Its pH optimum was in the range pH 7.5-8 . When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C . The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate . Several additives such as Triton X-100 enhanced thermostability . During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.

Biochem J, 1990 Nov 1, 271(3), 839 - 41
Rubredoxin from Clostridium thermosaccharolyticum . Amino acid sequence, mass-spectrometric and preliminary crystallographic data; Meyer J et al.; Rubredoxin isolated from the thermophilic bacterium Clostridium thermosaccharolyticum has been sequenced and crystallized . The 52-residue sequence is similar to those of rubredoxins occurring in other anaerobic bacteria, but displays some unique features, including a tryptophan residue in position 4, two consecutive proline residues in positions 25 and 26, and an aspartic acid residue in position 41 . The molecular mass (5988 Da) of the native rubredoxin has been measured by electrospray-ionization m.s., thus establishing the applicability of the technique to this type of iron-sulphur protein . C . thermosaccharolyticum rubredoxin crystallizes as dark-red elongated prisms with a flat diamond cross-section . The X-ray diffraction shows symmetry consistent with space group P2(1)2(1)2(1) . Cell parameters are: a = 2.73 nm, b = 2.98 nm, c = 6.49 nm.

Mol Cell Biol, 1990 Nov, 10(11), 6091 - 6
Molecular characterization of SerH3, a Tetrahymena thermophila gene encoding a temperature-regulated surface antigen; Tondravi MM et al.; The DNA sequences of a cDNA clone and the macronuclear genomic fragment corresponding to the functional copy of the SerH3 surface antigen gene of Tetrahymena thermophila were determined . Primer extension and nuclease protection assays show that the SerH3 transcription unit is 1,425 nucleotides long and contains no introns . The predicted polypeptide encoded by the SerH3 gene has a molecular mass of 44,415 daltons; one-third of its 439 residues are either cysteine, serine, or threonine . The central half of the polypeptide consists of three homologous domains in tandem array; within these domains, the cysteine, proline, and tryptophan residues occur in highly regular patterns.

J Protozool, 1990 Nov-Dec, 37(6), 471 - 2
Chromosomal localization of an exocytosis mutant in Tetrahymena thermophila; Bleyman LK et al.; Exocytosis mutants of Tetrahymena thermophila are deficient in mucus release . Experiments to chromosomally locate two of these mutants are described, using the technique of deletion mapping with nullisomic strains . One exo locus has been assigned to chromosome 5.

J Biochem (Tokyo), 1990 Nov, 108(5), 737 - 40
Resonance Raman studies on the structure of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus; Nozawa T et al.; Resonance Raman spectra of chlorosomes isolated from the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus have been obtained with several excitation wavelengths from 441.6 to 514.5 nm . Resonance Raman spectra of bacteriochlorophyll (BChl) c isolated from C . aurantiacus cells have also been observed . The C=C stretching frequencies of BChl c in the chlorosomes were found to be at 1,556 (strong) and 1,544 (shoulder) cm-1, which correspond to those expected for the 5-coordinated BChl c . The C-9 carbonyl resonance Raman frequency was found at 1,642 cm-1, indicating that this group is either hydrogen-bonded to an Mg-coordinated hydroxyl group or coordinated to the Mg ion.

Genetika, 1990 Nov, 26(11), 1915 - 25
{Molecular cloning and structural-functional analysis of the arginine biosynthesis genes of the thermophilic bacterium Bacillus stearothermophilus}; Sakanian VA et al.; Genes encoding arginine biosynthesis of Bacillus stearothermophilus strain 718 were cloned in the mutant argA strain of the Escherichia coli K-12 . The arg genes were shown to be located on the 3.7 kb DNA fragment in the following order: argA--argE--argB . The expression of the argA gene of B . stearothermophilus on the multicopy vehicle is twofold higher in argR- strain of E . coli K-12 than is isogenic argR+ strain . According to hybridization analysis argA genes of B . stearothermophilus and B . subtilis have low level of homology, which is the evidence of their evolutional divergence.

J Biochem (Tokyo), 1990 Nov, 108(5), 866 - 73
Nucleotide sequence of the gene coding for four subunits of cytochrome c oxidase from the thermophilic bacterium PS3; Ishizuka M et al.; The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced . The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame . The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing . Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 {Sone et al . (1988) J . Biochem . 103, 606-610}, although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct . PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart . PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments . The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes . PS3 CO3 and CO4 are much more similar to E . coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.

Appl Microbiol Biotechnol, 1990 Nov, 34(2), 214 - 9
Overproduction of an acetylxylan esterase from the extreme thermophile "Caldocellum saccharolyticum" in Escherichia coli; Luthi E et al.; The xynC gene coding for an acetylxylan esterase from the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli strain RR28 by cloning the gene downstream from the lacZ promoter region of pUC18 (pNZ1447) or downstream from the temperature-inducible lambda pRpL promoters of pJLA602 (pNZ1600) . The protein formed high molecular weight aggregates in induced cells of RR28/pNZ1600 but not in RR28/pNZ1447 . The enzyme constituted up to 10% of the total cell protein and was located in the cytoplasmic fraction of RR28/pNZ1447 . The acetyl esterase was most active at pH 6.0 and 70-75 degrees C with a half-life of 64 h at 70 degrees C and 30 h at 80 degrees C, respectively.

FEBS Lett, 1990 Oct 29, 273(1-2), 59 - 62
The primary structure of DNA binding protein II from the extreme thermophilic bacterium Thermus thermophilus; Zierer R et al.; The primary structure of DNA binding protein II (DNA bp II) from the extreme thermophilic bacterium Thermus thermophilus has been established by combination of manual and automated techniques . The protein has 95 residues and a molecular mass of 11,843 . Comparison of the primary structure with the known sequence data of DNA bp II from Clostridium pasteurineum, Baccillus stearothermophilus, Escherichia coli, Rhizobium meliloti, Anabena, Thermoplasma acidophilum, Pseudomonas aeruginosa and Bacillus caldolyticus reveals a clear homology among these small basic proteins . In particular, two short sequences in the middle and C-terminal part of the proteins (residues N-Gly-Phe-Gly-X-Phe and Pro-X-Thr at positions 46-51 and 63-65, respectively) are completely conserved.

FEBS Lett, 1990 Oct 29, 273(1-2), 211 - 4
A comparative study of ribosomal and DNA binding protein II from two thermophilic bacteria, Bacillus caldolyticus strain EP 00275 and Bacillus stearothermophilus; Zierer R et al.; Ribosomal and DNA binding proteins (DNA bp II) from an extreme thermophilic bacterium, B . caldolyticus strain EP 00275, were investigated for stability and crystallization and compared to the homologous proteins from B . stearothermophilus . Two-dimensional gel electrophoresis of both types of proteins, the amino acid composition and the sequences of some of the peptides of DNA bp II revealed a close relationship between each other . The physico-chemical characteristics of DNA bp II were similar but different from homologous proteins from T . thermophilus and C . pasteurineum . From our results we conclude: B . stearothermophilus and B . caldolyticus strain EP 00275 are similar organisms with regard to their ribosomal and DNA binding proteins.

Nucleic Acids Res, 1990 Oct 25, 18(20), 6061 - 8
DNA sequences required for yeast actin gene transcription do not include conserved CCAAT motifs; Munholland JM et al.; Sequences required for Saccharomyces cerevisiae actin gene transcription were mapped and compared to the regulatory region of the actin gene from a thermophilic fungus, Thermomyces lanuginosus . Two CCAAT motifs conserved in position in these two species could be mutated without affecting promoter activity, regardless of whether the yeast were grown in fermentable or non-fermentable carbon sources . Two TATA-like sequences and an upstream activation sequence (UAS) composed of multiple elements were identified . The contribution of sequence motifs within these elements to UAS activity varied depending on the carbon source . The Thermomyces gene contains sequences highly homologous to this UAS, but in the opposite orientation.

J Biol Chem, 1990 Oct 25, 265(30), 18207 - 12
Free amino acid turnover in methanogens measured by 15N NMR spectroscopy; Roberts MF et al.; Turnover of the nitrogen moiety from free amino acid pools in two thermophilic methanogens, Methanobacterium thermautotrophicum delta H and Methanococcus thermolithotrophicus SN1, has been monitored with 15N NMR spectroscopy . In cells growing exponentially on 15NH4Cl, glutamate was the major soluble 15N-labeled species in both organisms . When the Mb . thermoautotrophicum cells were harvested, washed, and resuspended into medium containing 14NH4Cl, the resonance for {15N}glutamate decreased with a half-life of 0.5 h . This is considerably faster than the turnover rate for the carbon side chain of glutamate (7 h) obtained when a 13CO2 pulse followed by a 12CO2 chase was incorporated into the 15N/14N-labeling experiment . Such behavior is consistent with recycling of the glutamate carbon skeleton via alpha-ketoglutarate after transamination reactions remove the 15N for biosynthesis of other amino acids, nucleic acids, etc . When the cells were in stationary phase, 15N turnover was considerably slower indicating that transaminase activity had also decreased . Mc . thermolithotrophicus has a much more fragile cell wall and easily lyses . To avoid cell loss in the 15N/14N experiment, 15NH+4 growth followed by 14NH4+ dilution was used . In this organism the glutamate-labeled nitrogen turns over quite rapidly (t1/2 approximately 9 min), at a rate comparable to that for the carbon skeleton (t1/2 approximately 10 min) . Beta-Glutamate, the second major carbon and nitrogen pool in this organism, turns over its 15N label very slowly . Therefore, this beta-amino acid does not appear to serve as a nitrogen donor in Mc . thermolithotrophicus.

J Mol Biol, 1990 Oct 20, 215(4), 597 - 606
Tetramer-dimer conversion of phosphofructokinase from Thermus thermophilus induced by its allosteric effectors; Xu J et al.; Phosphofructokinase (PFKase) was purified from an extreme thermophile . Thermus thermophilus . Allosteric natures of T . thermophilus PFKase is similar to those of Bacillus stearothermophilus PFKase, that is, hyperbolic plots of the activity versus concentration of fructose 6-phosphate (F6P) were changed into a sigmoidal shape by the addition of phosphoenolpyruvate (PEP), while further addition of ADP caused it to revert to a hyperbolic shape . The native T . thermophilus PFKase has an Mr of 148,000 consisting of four 36,500 subunits . However, it exists as a two-subunit form of Mr 74,000 in the presence of PEP . The two-subunit form was catalytically inactive . The four-subunit enzyme was regenerated by addition of either F6P or Mg.ADP, or by removal of PEP from the solution . This reversible dissociation was observed within a wide range of pH (6.5 to 8.4) and temperature (4 degrees C to 65 degrees C) . Thus, unlike PFKase from other sources, the allosteric kinetics of T . thermophilus PFKase can be explained well, at least qualitatively, by the dynamic equilibrium between the active four-subunit form and inactive two-subunit form that is modulated by PEP, F6P and Mg.ADP . Parallel suppression of the PEP-induced conversion in molecular form and kinetics by high concentrations of sulfate and phosphate supports the above explanation . Also, the observation that the degree of PEP inhibition was dependent on the protein concentration of the PFKase in the assay solution is consistent with the presence of this equilibrium.

Biochim Biophys Acta, 1990 Oct 15, 1055(1), 19 - 26
Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature; Lepock JR et al.; Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus . The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth . A low temperature, lipid transition was detected in B . stearothermophilus and B . megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax . The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax . The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation . The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg) . Tmax for B . stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively . For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level . A similar pattern holds for B . megaterium and B . psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively . Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus . This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

FEMS Microbiol Lett, 1990 Oct, 60(1-2), 97 - 101
A plasmid vector for an extreme thermophile, Thermus thermophilus; Koyama Y et al.; The host-vector system for an extreme thermophile, Thermus thermophilus HB27, was developed . The host strain has a mutation in tryptophan synthetase gene (trpB), and the mutation was determined to be a missense mutation by DNA sequence analysis . A Thermus-E . coli shuttle vector pYK109 was constructed . pYK109 consists of Thermus cryptic plasmid pTT8, tryptophan synthetase gene (trpB) of Thermus T2 and E . coli plasmid vector pUC13 . pYK109 transformed T . thermophilus HB27 trpB5 to Trp+ at a frequency of 10(6) transformants per microgram DNA.

J Gen Microbiol, 1990 Oct, 136 ( Pt 10), 1967 - 71
Purification and properties of an extracellular endoglucanase from Myceliophthora thermophila D-14 (ATCC 48104); Roy SK et al.; An extracellular endoglucanase (1,4-beta-glucanohydrolase, EC 3.2.1.4) produced by Myceliophthora thermophila D-14 (ATCC 48104) has been purified to homogeneity by ammonium sulphate precipitation and two consecutive ion-exchange chromatographic steps on DEAE-Sephadex A-50 columns . The enzyme was purified 13.8-fold and was homogeneous by analytical PAGE and SDS-PAGE . It has a high apparent Mr, of about 100,000 . The pH and temperature optima for its activity were 4.8 and 65 degrees C respectively . The Km of the purified enzyme for CMC (sodium salt) was 3 mg ml-1 . The enzyme displayed low activity toward salicin and p-nitrophenyl beta-D-glucoside . The activity was enhanced in the presence of Na+, K+ and Ca2+ but effectively inhibited by Hg2+, Fe2+, Mg2+, Cu2+ and NH4+ . Inhibition studies indicated that the enzyme may be a metalloprotein and/or that it requires metal ions for its optimum activity.

Eur J Clin Microbiol Infect Dis, 1990 Oct, 9(10), 760 - 2
Improved medium for storage and transportation of thermophilic campylobacters; Rogol M et al.; A medium for storage and transportation of thermophilic campylobacter cultures was developed . The medium contains brucella broth with 0.16% or 0.3% agar, 5% human blood, a growth supplement which enhances aerotolerance and an antibiotic supplement . Using this medium all cultures tested were recovered after 48 days at 4 degrees C, 56 days at 37 degrees C and 32 days at room temperature . Some of the cultures even remained viable for up to three months at 4 degrees C and 56 days at room temperature . The blackening of the medium by H2S positive cultures offers an indication as to the biotype.

Cryobiology, 1990 Oct, 27(5), 569 - 75
Freeze-drying of Streptococcus thermophilus: a comparison between the vacuum and the atmospheric method; Wolff E et al.; Frozen suspensions of Streptococcus thermophilus were freeze-dried in a vacuum or a fluidized adsorbent bed at atmospheric pressure . Optimum operating conditions for each process were defined . For the duration of processing and survival rate of bacteria, in each case vacuum freeze-drying seemed more satisfactory than atmospheric pressure freeze-drying . The use of reconstituted skimmed milk as a suspension medium provides good protection for S . thermophilus.

J Bacteriol, 1990 Oct, 172(10), 6098 - 105
Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes; Morag E et al.; Xylanase activity of Clostridium thermocellum, an anaerobic thermophilic cellulolytic bacterium, was characterized . The activity was localized both in the cellulosome (the principal multienzyme, cellulose-solubilizing protein complex) and in noncellulosomal fractions . Each of these fractions contained at least four major polypeptide bands which contributed to the xylanolytic activity . In both cases, pH and temperature optima, product pattern, and other features of the xylanase activity were almost identical . The main difference was in the average molecular weights of the respective polypeptides which appeared responsible for the activity . In the noncellulosomal fraction, xylanases with Mrs ranging from 30,000 to 65,000 were detected . Distinct from these were the cellulosomal xylanases, which exhibited much larger Mrs (up to 170,000) . The cellulosome-associated xylanases corresponded to known cellulosomal subunits, some of which also exhibited endoglucanase activity, and others which coincided with subunits which appeared to express exoglucanaselike activity . In contrast, the noncellulosomal xylanases hydrolyzed xylan exclusively . beta-Glucosidase and beta-xylosidase activities were shown to be the action of different enzymes; both were associated exclusively with the cell and were not components of the cellulosome . Despite the lack of growth on and utilization of xylan or its degradation products, C . thermocellum produces a highly developed xylanolytic apparatus which is interlinked with its cellulase system.

FEBS Lett, 1990 Oct 1, 271(1-2), 111 - 5
Electron microscopy of the reconstituted complexes of the F1-ATPase with various subunit constitution revealed the location of the gamma subunit in the central cavity of the molecule; Fujiyama Y et al.; The subunit structure of the F1-ATPase from the thermophilic bacterium PS3 was probed by comparing electron microscopic images of the subunit complexes reconstituted to contain different subunit compositions . The following structural features were observed . (1) The alpha 3 beta 3 complex has a hexagonal, apparently symmetrical arrangement of masses with a central cavity . (2) The projections of the alpha 3 beta 3 delta complexes are similar to those of the alpha 3 beta 3 complexes . (3) In contrast, the alpha 3 beta 3 gamma complex has an additional mass in the centre which is similar to that found in the native enzyme (alpha 3 beta 3 gamma delta epsilon) . From these observations, it is concluded that the central mass in the F1-ATPase is comprised mostly of the gamma subunit.

Appl Environ Microbiol, 1990 Oct, 56(10), 3117 - 24
celB, a gene coding for a bifunctional cellulase from the extreme thermophile "Caldocellum saccharolyticum"; Saul DJ et al.; "Caldocellum saccharolyticum" is an obligatory anaerobic thermophilic bacterium . A gene from this organism, designated celB, has been cloned in Escherichia coli as part of a bacteriophage lambda gene library . This gene produces a thermostable cellulase that shows both endoglucanase and exoglucanase activities on test substrates and is able to degrade crystalline cellulose to glucose . The sequence of celB has homology with both exo- and endoglucanases described by others . It appears to have a central domain without enzymatic activity which is joined to the enzymatic domains by runs of amino acids rich in proline and threonine (PT boxes) . Deletion analysis shows that the exoglucanase activity is located in the amino-terminal domain of the enzyme and that endoglucanase activity is located in the carboxy-terminal domain . There are internal transcriptional and translational start sites within the gene . The intact gene has been cloned into a temperature-inducible expression vector, pJLA602, and overexpressed in E . coli . Polyacrylamide gel electrophoresis showed that celB produced a protein with a molecular weight of 118,000 to 120,000 . A number of smaller proteins with activity against carboxymethyl cellulose and 4-methyl umbelliferyl-beta-D-cellobioside were also produced . These are believed to be the result of alternative translational start sites and/or proteolytic degradation products of the translated gene product.

Poult Sci, 1990 Oct, 69(10), 1670 - 4
Influence of whey and probiotic-supplemented withdrawal feed on the retention of Salmonella intubated into market age broilers; Bilgili SF et al.; Broilers (6 wk of age) were crop-intubated with a nalidixic acid-resistant strain of Salmonella typhimurium and given four withdrawal feeds: 1) control feed (corn and soybean meal base, 18% CP and 3,200 kcal ME/kg); 2) control plus 5% whey (61% lactose); 3) control plus probiotic (6.8 x 10(6) cfu of Bifidobacterium pseudolongum, Bifidobacterium thermophilium, and Lactobacillus acidophilus); and 4) control plus 5% whey plus probiotic . In Experiment 1, male broilers received 9.8 x 10(7) cfu of S . typhimurium per bird midway through a 20-h fast . The withdrawal feeds were continuously offered through a subsequent period of 6 to 7 days . Live performance of broilers during this time was similar among treatments . Ceca from birds consuming whey were significantly (P less than .05) increased in weight and distended with gas . Recovery of S . typhimurium from the ceca was low with all treatments, and the effects of whey or probiotic at reducing the organism could not be determined . In Experiment 2, female broilers received the same four experimental feeds for 48 h . Salmonella typhimurium was intubated 24 h after feed access (1.2 x 10(7) cfu per bird) . Again, no differences (P greater than .05) in performance occurred, but the inclusion of whey led to increased cecal weight and size (P less than .05) . Large numbers of S . typhimurium were recovered from the ceca 24 h after intubation, but the levels were similar among treatments . Whey and probiotic in the withdrawal feed did not appear to affect the Salmonella level in ceca when the organism was consumed prior to marketing.

EMBO J, 1990 Oct, 9(10), 3067 - 75
Ordered arrays of the photosystem I reaction centre after reconstitution: projections and surface reliefs of the complex at 2 nm resolution; Ford RC et al.; We present an electron microscopical analysis of the photosystem I reaction centre, the membrane complex involved in the second light-driven step of photosynthetic electron transfer in plants and cyanobacteria . To this end, ordered two-dimensional arrays were reconstituted from detergent solubilized photosystem I reaction centres and phospholipids, and studied by electron microscopy and digital image processing . Small (P1) and large (P3) hexagonal lattices obtained with reaction centres of the thermophilic cyanobacterium Phormidium laminosum had unit cell sizes of a = b = 8.8 nm and 15.8 nm, respectively . Reaction centres of a second thermophilic strain, Synechococcus sp . OD24, gave square lattices (a = b = 14.5 nm; P2(1)) . Irrespective of the packing arrangement, projections of negatively stained photosystem I complexes showed elongated asymmetric shapes with a large domain at one end which was tilted with respect to a small domain forming the tip of the other end . Such features were also found in averaged projections of solubilized reaction centre trimers . Surface reliefs reconstructed from freeze-dried metal-shadowed P2(1) lattices revealed that reaction centres had a ridge of 2.5 nm height projecting from one side of the membrane while their other side was rather flat and exhibited a shallow, central indentation.

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