Thermophiles

J Cell Sci, 1985 Oct, 78, 23 - 48
Effect of Cerophyl growth medium on exocytosis in Tetrahymena thermophila; Pesciotta DM et al.; Culturing the ciliate Tetrahymena thermophila in Cerophyl has provided an opportunity for studying the assembly and/or synthesis of the intramembrane particle array, the rosette, which marks the site of exocytosis in these cells . Cultures grown in this medium cease cell division after only 12h and enter 'stationary phase' earlier (12h of growth) relative to growth in standard medium (proteose peptone) . In addition, the cell changes from the normally observed pear-shaped body to a thinner more ellipsoid form . Despite the initial similarities to starving cells, several differences are observed in the Cerophyl-grown cells . One is that cell size remains constant for at least 72h in contrast to starved cells . Secondly, in spite of this block in cell division, results from freeze-fracture replicas of the cell membrane of these cells show that they continue to assemble rosettes, the number of which increases approximately six times, from 0.34 rosette/microgram2 to 2.1 rosettes/microgram2 . Addition of the protein synthesis inhibitor, cycloheximide (6h exposure), during growth in Cerophyl shows that 70% of rosettes can be assembled, despite the blockage of translation, by using pre-existing component(s) from a pool . The remaining 30% must involve de novo synthesis of one or more components; this percentage can be increased with longer exposure to the drug . Thirdly, an apparent increase in the number of mucocysts is observed by thin-section electron microscopy . At first (12-24h) only docked mucocysts seem to accumulate in the cell . However, by 36h a considerable increase seems to have taken place, particularly in the number of mucocysts located in the cytoplasm . In the cycloheximide-treated cells this increase in mucocysts begins to be blocked after 6h of exposure to the drug . These observations are in agreement with the results obtained from the freeze-fracture data on the concomitant increase in number of rosettes . This system therefore offers the first possibility of exploring the biosynthesis of these components.

Appl Environ Microbiol, 1985 Oct, 50(4), 906 - 13
Identification of thermophilic bacteria in solid-waste composting; Strom PF; The thermophilic microbiota of solid-waste composting, with major emphasis on Bacillus spp., was examined with Trypticase soy broth (BBL Microbiology Systems) with 2% agar as the initial plating medium . Five 4.5-liter laboratory units at 49 to 69 degrees C were fed a mixture of dried table scraps and shredded newspaper . The composting plants treating refuse at Altoona, Pa., and refuse-sludge at Leicester, England, were also sampled . Of 652 randomly picked colonies, 87% were identified as Bacillus spp . Other isolates included two genera of unidentified nonsporeforming bacteria (one of gram-negative small rods and the other of gram-variable coccobacilli), the actinomycetes Streptomyces spp . and Thermoactinomyces sp., and the fungus Aspergillus fumigatus . Among the Bacillus isolates, the following, in order of decreasing frequency, were observed: B . circulans complex, B . stearothermophilus, B . coagulans types A and B, B . licheniformis, B . brevis, B . sphaericus, Bacillus spp . types i and ii, and B . subtilis . About 15% of the Bacillus isolates could be assigned to species only by allowing for greater variability in one or more characteristics than has been reported by other authors for their strains . In particular, growth at higher temperatures than previously reported was found for strains of several species . A small number of Bacillus isolates (less than 2%) could not be assigned to any recognized species.

Appl Environ Microbiol, 1985 Oct, 50(4), 899 - 905
Effect of temperature on bacterial species diversity in thermophilic solid-waste composting; Strom PF; Continuously thermophilic composting was examined with a 4.5-liter reactor placed in an incubator maintained at representative temperatures . Feed was a mixture of dried table scraps and shredded newspaper wetted to 55% moisture . One run at 49 degrees C (run A) employed a 1:4 feed-to-compost ratio, while the other runs used a 10:1 ratio and were incubated at 50, 55, 60, or 65 degrees C . Due to self-heating, internal temperatures of the composting mass were 0 to 7 degrees C hotter than the incubator . Two full-scale composting plants (at Altoona, Pa., and Leicester, England) were also examined . Plate counts per gram (dry weight) on Trypticase soy broth (BBL Microbiology Systems) with 2% agar ranged from 0.7 X 10(9) to 5.3 X 10(9) for laboratory composting and 0.02 X 10(9) to 7.4 X 10(9) for field composting . Fifteen taxa were isolated, including 10 of genus Bacillus, which dominated all samples except that from run A . Species diversity decreased markedly in laboratory composting at 60 degrees C and above, but was similar for the three runs incubated at 49, 50, and 55 degrees C . The maximum desirable composting temperature based on species diversity is thus 60 degrees C, the same as that previously recommended based on measures of the rate of decomposition.

Appl Environ Microbiol, 1985 Oct, 50(4), 777 - 80
Galactokinase activity in Streptococcus thermophilus; Hutkins R et al.; ATP-dependent phosphorylation of {14C}galactose by 11 strains of Streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK) . Activities were 10 times higher in fully induced, galactose-fermenting (Gal+) strains than in galactose-nonfermenting (Gal-) strains . Lactose-grown, Gal- cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels . Gal+ S . thermophilus 19258 also released galactose into the medium, but when lactose was depleted growth on galactose commenced, and galK increased from 0.025 to 0.22 micromol of galactose phosphorylated per min per mg of protein . When lactose was added to galactose-grown cells of S . thermophilus 19258, galK activity rapidly decreased . These results suggest that galK in Gal+ S . thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal- strains.

Appl Environ Microbiol, 1985 Oct, 50(4), 772 - 6
Galactose transport in Streptococcus thermophilus; Hutkins R et al.; Although Streptococcus thermophilus accumulated {14C}lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate {14C}galactose unless an additional energy source was added to the test system . Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose . Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity . Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect . The results suggest that galactose transport in S . thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved . The galactose permease in S . thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells . Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.

Tohoku J Exp Med, 1985 Oct, 147(2), 135 - 44
Determination by enzyme-linked immunosorbent assay (ELISA) of specific IgG antibody activities for diagnosis of farmer's lung disease; Konishi K et al.; The specific IgG antibodies to thermophilic actinomycetes in the sera from patients with farmer's lung disease were quantitated by an ELISA and the results thereof were compared with those obtained by the standard double immunodiffusion assay (Ouchterlony's method) . All sera from patients with farmer's lung disease were precipitin positive to Thermoactinomyces vulgaris and/or Micropolyspora faeni by the Ouchterlony's method, and revealed significantly high levels of IgG antibody activities against these antigens by ELISA . We have found 18 precipitin-positive but asymptomatic subjects in a survey of the dairy farming population of the northern area of IWATE Prefecture . Their sera were precipitin positive to antigens related to the farmer's lung disease, however IgG antibody activities as determined by ELISA to these antigens was significantly lower in the group of asymptomatics than in the group of symptomatics . Precipitin negative dairy farming controls and normal controls living in the urban area revealed low antibody activities as determined by ELISA . From these studies, we concluded that the assay of specific IgG antibody activity to thermophilic actinomyces by ELISA is useful for the diagnosis and screening of farmer's lung disease.

Genetics, 1985 Oct, 111(2), 273 - 86
Isolation and genetic analysis of mutations at the SerH immobilization antigen locus of Tetrahymena thermophila; Doerder FP et al.; Multiple alleles at the SerH locus specify the major cell surface protein (immobilization antigen) of the ciliate Tetrahymena thermophila . Following mutagenesis of SerH1 homozygotes, two mutations, H1-1 and H1-2, were recovered in heterozygous form . Mutant homozygotes do not express H1 antigen, nor is H1 expressed in F1 progeny of crosses to wild-type strains homozygous for SerH2 or SerH3 . H1-1 and H1-2 segregate without recombination from these wild-type alleles in expected F2 and testcross Mendelian ratios . H1-1 and H1-2 do, however, complement each other to express H1 antigen . Experiments suggest this complementation is due neither to recombination during macronuclear development nor to interallelic complementation of defective SerH1 gene products . These results suggest that SerH1 is intact in one mutant, and possibly both, although no such allele has been segregated in testcross progeny (N = 205) . The hypothesis is presented that complementation between H1-1 and H1-2 is due to interaction between allele-specific regulators closely linked to the SerH1 gene.

J Bacteriol, 1985 Oct, 164(1), 421 - 4
Uracil-DNA glycosylase of thermophilic Thermothrix thiopara; Kaboev OK et al.; An activity which released free uracil from dUMP-containing DNA was purified approximately 1,700-fold from extracts of Thermothrix thiopara, the first such activity to be isolated from extremely thermophilic bacteria . The enzyme appeared homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . It had a native molecular weight of 26,000 and existed as a monomer protein in water solution . The enzyme had an optimal activity at 70 degrees C, between pH 7.5 and 9.0, and in the presence of 0.2% Triton X-100 . It had no cofactor requirement and was not inhibited by EDTA, but it was sensitive to N-ethylmaleimide . The purified enzyme did not contain any nuclease that acted on native or depurinated DNA . The Arrhenius activation energy was 76 kJ/mol between 30 and 50 degrees C and 11 kJ/mol between 50 and 70 degrees C . The rate of heat inactivation of the enzyme followed first-order kinetics with a half-life of 2 min at 70 degrees C . Ammonium sulfate and bovine serum albumin protected the enzyme from heat inactivation . One T . thiopara cell contains enough activity to release about 2 X 10(8) uracil residues from DNA during one generation time at 70 degrees C.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2789 - 94
Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains; Droffner ML et al.; Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles . Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10) . The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin . Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers . With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.

J Cell Sci, 1985 Oct, 78, 49 - 65
Protein secretion in Tetrahymena thermophila: characterization of the secretory mutant strain SB281; Maihle NJ et al.; The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion . Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210 . In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described . SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling . Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts . In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells . A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane . Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6908 - 12
Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila; Suprenant KA et al.; Most higher eukaryotic tubulins are separated into alpha- and beta-tubulin when electrophoresed in NaDodSO4- denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80) . By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two alpha-tubulins and one beta-tubulin . Moreover, at least five alpha- and two beta-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis . Three of these alpha isotypes and one beta isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro . Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related . Possible mechanisms for the generation and selection of these tubulin isotypes are discussed.

Appl Environ Microbiol, 1985 Oct, 50(4), 1103 - 6
Isolation and partial characterization of plasmid DNA from Streptococcus thermophilus; Herman RE et al.; Of 23 strains of Streptococcus thermophilus examined, 5 were found to contain a single small cryptic plasmid, designated pHM1 through pHM5 . Through analysis by restriction endonuclease mapping and DNA-DNA hybridization, the five plasmids were found to be closely related . They were present in 4 to 18 copies per cell and ranged in size from 1.4 to 2.2 megadaltons . Plasmids pHM1 and pHM5, as well as pHM2 and pHM4, were found to be identical . Single restriction endonuclease sites were observed on each plasmid for PvuII and MboI . Plasmids pHM2 and pHM3 each had an additional single site for HhaI, and pHM1 had an additional single site for HindIII . The characteristics of these plasmids may make them useful for the development of cloning vectors for use in S . thermophilus.

Eur J Cell Biol, 1985 Sep, 38(2), 306 - 11
Rapid and sensitive assays for phagosomal acidification in Paramecium and Tetrahymena; Fok AK et al.; Biochemical and cytochemical procedures were developed to measure the rate of phagosomal acidification for phagosomal pH ranging from 5 to 2.5 . These assays were based on the pH-dependent inactivation with time of horseradish peroxidase (HRP) activity, a result attributable to the dissociation of this enzyme to a colorless protein and ferriprotoporphyrin in acidic solutions . When preincubated in buffers of varying pH, the rate of HRP inactivation followed a sigmoid curve, with the highest rate of inactivation between 4.3 and 3.5 when using citrate-phosphate buffer and between pH 3.4 and 2.8 when using the universal ABC buffer . This inactivation was temperature but not concentration dependent . When Paramecium caudatum, members of the P . aurelia complex or Tetrahymena thermophila was pulsed briefly with HRP and small fluorescent beads, the loss of HRP activity, measured biochemically in cell homogenates and/or cytochemically in phagosomes, was rapid and followed the kinetics of a first-order rate reaction . Both assays gave similar values for the rate constant for acidification and similar rates of inhibition when P . caudatum was exposed to a proton ionophore, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone . These assays can readily be adapted to other phagocytic cells as long as a rapid procedure is available for removing all unphagocytosed HRP and latex beads . These procedures are sensitive and rapid thus allowing many samples to be quickly prepared and analyzed.

J Bacteriol, 1985 Sep, 163(3), 1275 - 8
Thermoanaerobacter ethanolicus in a comparison of the growth efficiencies of thermophilic and mesophilic anaerobes; Lacis LS et al.; Maintenance coefficients and theoretical maximum growth yields, with respect to both substrate and ATP, were estimated for Thermoanaerobacter ethanolicus growing in a glucose-limited, continuous culture . A comparison of these values with those for other bacteria showed that, contrary to predictions by others, anaerobic thermophiles had neither low observed growth yields nor high maintenance energy coefficients.

Ital J Biochem, 1985 Sep-Oct, 34(5), 341 - 55
Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe . Purification and characterization of intracellular beta-glucosidase; Bedino S et al.; Intracellular beta-glucosidase was extracted from the mycelium of Th . aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity . Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM . With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C . beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins . Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).

Arch Biochem Biophys, 1985 Sep, 241(2), 571 - 6
A specific L-asparaginase from Thermus aquaticus; Curran MP et al.; The L-asparaginase from an extreme thermophile, Thermus aquaticus strain T351, was highly substrate- and stereospecific, with no activity against glutamine or D-asparagine . It had a high Km of 8.6 mM . In these aspects it closely resembled the corresponding enzymes from thermophilic bacteria . The enzyme had a molecular weight of 80,000, an isoelectric point of 4.6, and a pH optimum of 9.5 . It showed some substrate inhibition above 20 mM asparagine and was also inhibited by L-aspartic acid, D- and L-lysine (Ki of 5.2 and 1.25 mM, respectively), and D- and L-serine . The half-life of the enzyme at 85 degrees C was 40 min . The Arrhenius plot showed a change in slope at 55 degrees C.

Nucleic Acids Res, 1985 Aug 26, 13(16), 5817 - 31
Macronuclear DNA of Tetrahymena thermophila exists as defined subchromosomal-sized molecules; Altschuler MI et al.; Using the method of orthogonal-field-alternation gel electrophoresis, we have resolved the macronuclear DNA of Tetrahymena thermophila into a series of distinct bands . Using electrode switching intervals ranging from 10 to 70 seconds we have resolved DNA bands ranging in size from about 21 kb up to and beyond the size of yeast chromosomes VII and XV . Hybridization of Southern blots from these gels to both unique and repetitive DNA sequences shows that the macronuclear genome of T . thermophila has a precise organization . The unique sequences tested each hybridize to only one band of macronuclear DNA and the hybridization patterns seem to be identical in several inbred strains examined.

J Biol Chem, 1985 Aug 25, 260(18), 10013 - 8
Regulation of galactokinase gene expression in Tetrahymena thermophila . II . Identification of 3,4-dihydroxyphenylalanine as a primary effector of adrenergic control of galactokinase expression; Ness JC et al.; Intracellular concentrations of catecholamines were determined in wild-type and mutant Tetrahymena thermophila, using the highly sensitive techniques of high-performance liquid chromatography and electro-chemical detection . Catecholamines were determined in these cell strains grown under various steady-state conditions, including those which initiate and maintain repression of galactokinase gene expression . Wild-type cells grown in defined minimal medium supplemented with 1% glycerol, exhibiting derepressed galactokinase synthesis, were found to contain considerable quantities of dopa (3,4-dihydroxyphenylalanine) and dopamine, but no detectable levels of either norepinephrine or epinephrine . Analyses of wild-type cells revealed a strong positive correlation between the internal concentration of dopa and expression of the galactokinase gene, both of which are regulated by exogenous carbohydrates, catecholamine agonists, or dibutyryl-cAMP; an analogous relationship between intracellular dopamine concentrations and galactokinase activity was not found . In addition, a correlation between intracellular dopa content and the phenotypic expression of galactokinase in various mutants deficient in the catecholamine biosynthetic pathway or in glucokinase further confirms the role of dopa as a primary effector in the regulation of galactokinase gene expression.

J Biol Chem, 1985 Aug 25, 260(18), 10001 - 12
Regulation of galactokinase gene expression in Tetrahymena thermophila . I . Intracellular catecholamine control of galactokinase expression; Ness JC et al.; The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) . The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose . The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity . Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations . The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations . Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase . Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period . Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited . Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose . This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation . These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose . A possible model is discussed.

J Protozool, 1985 Aug, 32(3), 517 - 9
An organofluorophosphate-hydrolyzing activity in Tetrahymena thermophila; Landis WG et al.; An enzymatic activity that hydrolyzes O,O-diisoproplyphosphofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoridate (Soman) was discovered in the ciliate protozoan Tetrahymena thermophila . The enzymatic activity classifies the protein as Mazur-type similar to that found in hog kidney and Escherichia coli . The rate of hydrolysis of Soman by the Tetrahymena-extract is the highest, on a per gram of extract basis, of any eucaryote . The molecular weight is approximately 75,400 as determined by Sephacryl column chromatography . A maximum fifteen-fold purification has been achieved . Potential exists for the detoxification and one-step detection of common organofluorophosphate pollutants . Additionally, Tetrahymena should prove an easier subject for manipulation than mammalian or squid sources . Protozoa may be a potentially important source of detoxification and degradation enzymes for other environmental contaminants.

J Dairy Sci, 1985 Aug, 68(8), 1910 - 6
Use of microbial beta-lactamase to destroy penicillin added to milk; Korycka-Dahl M et al.; A simple method is described for destruction of penicillin residues in bulk milk to an undetectable amount (less than .003 U/ml) with commercially available crude beta-lactamase enzyme . Milk containing .1 or .5 U/ml penicillin G was treated with .01 to 1.0 mU/ml of beta-lactamase (Bacillus cereus) for up to 96 h . The Bacillus stearothermophilus var . calidolactis assay was used to quantify penicillin in milk between .003 to 1.0 U/ml . The .5 U/ml of penicillin G was reduced to an undetectable amount within 18 h at 4 degrees C by 1.0 mU/ml of beta-lactamase . The development of titratable acidity over 5 to 6 h in contaminated milks treated with beta-lactamase and inoculated with Streptococcus thermophilus GH, Streptococcus cremoris, Streptococcus lactis, or a commercial starter culture was the same as for control milk samples containing no additives or only enzyme . Pilot-scale manufacture of Swiss and Cheddar cheeses from contaminated milks treated with beta-lactamase yielded cheeses of comparable quality, to control cheeses produced from penicillin-free milk . There were no delays in acid production as judged from pH measurements during production and ripening of the cheeses . About 50% of beta-lactamase activity added to milk remained after pasteurization at 63 degrees C for 30 min . The safety for human consumption of cheese containing small quantities of penicillin degradation products from milk treated with beta-lactamase remains to be established.

Mol Cell Biol, 1985 Aug, 5(8), 1925 - 32
Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila; Williams NE et al.; A temperature shift from 40 to 28 degrees C rapidly induced expression of a specific immobilization antigen at the cell surface in Tetrahymena thermophila . This transformation was inhibited by actinomycin D and cycloheximide but not by colchicine or cytochalasin B . The major surface antigen expressed at 28 degrees C in cells homozygous for the SerH3 allele was partially purified, and an antiserum against this preparation was raised in rabbits . Electrophoresis, immunoblot, and {35S}methionine incorporation studies are reported which support the conclusion that the H3 antigen is an acidic protein with an Mr of approximately 52,000 daltons . An induced synthesis of the H3 immobilization antigen was detected within 30 min after a shift from 40 to 28 degrees C . This protein appeared to be synthesized in the microsomal fraction and transferred without cleavage to the cell surface, where it was inserted first into nonciliated regions.

Mol Cell Biol, 1985 Aug, 5(8), 2061 - 9
Induction of acquired thermotolerance in Tetrahymena thermophila: effects of protein synthesis inhibitors; Hallberg RL et al.; When Tetrahymena thermophila cells growing at 30 degrees C are shifted to either 40 or 43 degrees C, the kinetics and extent of induction of heat shock mRNAs in both cases are virtually indistinguishable . However, the cells shifted to 40 degrees C show a typical induction of heat shock protein (HSP) synthesis and survive indefinitely (100% after 24 h), whereas those at 43 degrees C show an abortive synthesis of HSPs and die (less than 0.01% survivors) within 1 h . Cells treated at 30 degrees C with the drugs cycloheximide or emetine, at concentrations which are initially inhibitory to protein synthesis and cell growth but from which cells can eventually recover and resume growth, are after this recovery able to survive a direct shift from 30 to 43 degrees C (ca . 70% survival after 1 h) . This induction of thermotolerance by these drugs is as efficient in providing thermoprotection to cells as is a prior sublethal heat treatment which elicits the synthesis of HSPs . However, during the period when drug-treated cells recover their protein synthesis ability and simultaneously acquire the ability to subsequently survive a shift to 43 degrees C, none of the major HSPs are synthesized . The ability to survive a 1-h, 43 degrees C heat treatment, therefore, does not absolutely require the prior synthesis of HSPs . But, as extended survival at 43 degrees Celsius depends absolutely on the ability of cells to continually synthesize HSPs, it appears that a prior heat shock as well as the recovery from protein synthesis inhibition elicits a change in the protein synthetic machinery which allows the translation of HSP mRNAs at what would otherwise be a nonpermissive temperature for protein synthesis.

Mol Cell Biol, 1985 Aug, 5(8), 2039 - 50
Reproducible and variable genomic rearrangements occur in the developing somatic nucleus of the ciliate Tetrahymena thermophila; Howard EA et al.; We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila . To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events . This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome . Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs . We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development . The variable somatic genomes so produced may be the source of phenotypically variant cell lines.

J Protozool, 1985 Aug, 32(3), 480 - 5
Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron-containing species; Nielsen H et al.; The extrachromosomal rDNA molecules from a number of Tetrahymena strains were characterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis . Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region . The evolutionary relationship among the species of the T . pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T . thermophila and T . pigmentosa . Examination of a large number of T . pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA . It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species.

J Mol Biol, 1985 Jul 20, 184(2), 257 - 77
X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures; Schirmer T et al.; The structure of the biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 3 A resolution by X-ray diffraction methods . Phases have been obtained by the multiple isomorphous replacement method . The electron density map could be improved by solvent flattening and has been interpreted in terms of the amino acid sequence . The protein consists of three identical (alpha-beta)-units which are arranged around a threefold symmetry axis to form a disc of approximate dimensions 110 A X 30 A with a central channel of 35 A in diameter . This aggregation form is supposed to be the same as that found in the rods of native phycobilisomes . Both subunits, alpha and beta, exhibit a similar structure and are related by a local twofold rotational axis . Each subunit is folded into eight helices and irregular loops . Six helices are arranged to form a globular part, whereas two helices stick out and mediate extensive contact between the subunits . The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent C alpha-atoms have a root-mean-square deviation of 2 X 9 A . The chromophores attached to cystein 84 of the alpha- and beta-subunits are topologically equivalent to the haem . All three chromophores of C-phycocyanin, open-chain tetrapyrroles, are in an extended conformation . alpha 84 and beta 84 are attached to helix E (globin nomenclature), beta 155 is linked to the G--H loop . The shortest centre-to-centre distance between chromophores in trimer is 22 A.

Acta Odontol Scand, 1985 Jul, 43(3), 147 - 53
Secretory and serum antibodies against Streptococcus lactis, Streptococcus thermophilus, and Lactobacillus bulgaricus in relation to ingestion of fermented milk products; Carlsson P et al.; Serum, saliva, and urine were analyzed for the presence of IgA, IgG, and IgM antibodies reactive with the yoghurt bacteria Streptococcus thermophilus and Lactobacillus bulgaricus . A comparison was made between four subjects who frequently ate yoghurt and four subjects who never ate yoghurt . Salivary IgA and serum IgG activity against the milk-fermenting bacterium S . lactis was studied in five other subjects before, during, and after a period of ingestion of a fermented milk product, filmjolk . All analyses were carried out by an enzyme-linked immunosorbent assay method . Antibody activity against the yoghurt bacteria was found in saliva, serum, and urine . No difference in antibody activity between yoghurt eaters and non-yoghurt eaters was measured for salivary IgA, but for serum IgG a lower activity against S . thermophilus was present among the yoghurt eaters . Antibody activity against S . lactis was present already before the ingestion of filmjolk began, and the activity was not altered during the period of ingestion . It is concluded that in adult subjects, the ingestion of milk-fermenting bacteria does not result in a significant change in the antibody activity against these bacteria.

Biochimie, 1985 Jul-Aug, 67(7-8), 841 - 7
A modified two primer approach to oligonucleotide-directed in vitro mutagenesis; Sims PF et al.; Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E . coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L . casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila . We have used a modification of the method described by Norris et al . {1} . A mutagenic primer and an M13 universal sequencing primer are annealed simultaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors . We have analysed the effect on the frequency of mutation of: the temperature used for strand extension; the class of base change attempted; the host mismatch repair system . A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies.

Vet Res Commun, 1985 Jul, 9(3), 167 - 98
The occurrence and significance of Campylobacter jejuni in man and animals; Shane SM et al.; Campylobacter jejuni, which is now recognized as a discrete species, is a gram negative, microaerophilic, thermophilic, nalidixic acid sensitive, hippurate positive pathogen requiring special selective media for propogation . The organism is widely distributed in avian species, experimental and companion animals and in humans . Mammalian campylobacteriosis is characterized by an enterocolitis of variable severity . The prevalence of the condition is relatively high in young individuals, in underdeveloped countries and in subjects with diarrhea . Food animals, especially poultry, are reservoirs of the organism and infection occurs following consumption of untreated surface water, unpasteurized milk, incompletely cooked meat or other contaminated food products . Close contact with infected immature companion animals is a significant cause of campylobacteriosis in children and direct intrafamilial transmission and occupational infection have been documented . Campylobacteriosis attributable to C . jejuni is a condition of emerging significance which arises principally from deficiencies in hygiene inherent in the environment and in the food chain which extends from domestic animals to the consumer.

J Biochem (Tokyo), 1985 Jul, 98(1), 95 - 103
Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites; Ihara H et al.; The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B . stearothermophilus was determined . The NH2-terminal amino acid sequence analysis of the B . stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids) . The B . stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E . coli . The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation . Comparison of the amino acid sequence inferred from the B . stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.

Can J Microbiol, 1985 Jul, 31(7), 614 - 9
Genetic relationship between pUB110 and antibiotic-resistant plasmids obtained from thermophilic bacilli; Hoshino T et al.; The molecular relationship between pUB110 (Kmr, 4.4 kilobases (kb} and antibiotic-resistant plasmids from thermophilic bacilli, pTHT15 (Tcr, 4.5 kb) and pTHN1 (Kmr, 4.8 kb), were studied by blot hybridization . Extensive homology was observed between pUB110 and pTHT15 at the region which includes the replication origin . Incompatibility studies revealed that pTHT15 and pUB110 were slightly incompatible in Bacillus subtilis but that they were apparently compatible in B . stearothermophilus . This difference in incompatibility between pTHT15 and pUB110 in the two host cells might be due to a difference in the copy number of pTHT15 in the two organisms . From the results of blot hybridization, mode of kanamycin inactivation, and DNA sequencing, it was determined that pTHN1 encoded the identical gene for kanamycin nucleotidyl transferase as that of pUB110 . All three plasmids pTHT15, pTHN1, and pUB110 shared a common DNA homology at the in vitro membrane-binding region.

Proc Natl Acad Sci U S A, 1985 Jul, 82(14), 4758 - 62
Tetrahymena thermophila glutamine tRNA and its gene that corresponds to UAA termination codon; Kuchino Y et al.; Nucleotide sequence analysis of one of several tRNA genes cloned from Tetrahymena thermophila macronuclear DNA indicated that it corresponds to a tRNA species having TTA as anticodon . Subsequently, the tRNA species corresponding to that gene was isolated and its nucleotide sequence was determined by post-labeling techniques . The nucleotide sequence was found to be pG-G-U-U-C-C-A-U-A-m2G-U-A-psi-A-G-D-G-G-D- D-A-G-U-A-C-U-G-G-G-G-A-Cm-U-Um-U-A-i6A-A-psi-C-C-C-U-U-G-A-C- m5C-U-G-G-G-U-psi-C-G-m1A-A-U-C-C-C-A-G-U-G-G-G-A-C-C-U-C-C-AOH . This tRNA sequence exactly matched the DNA sequence of the corresponding tRNA gene . The first position of anticodon is 2'-O-methyluridine (Um), forming UmUA as the anticodon, which presumably recognizes the ochre termination codon UAA . This tRNA species is aminoacylated with glutamine by a Tetrahymena crude aminoacyl tRNA synthetase fraction, suggesting that ochre termination codon is used as a glutamine codon during cytoplasmic protein synthesis in Tetrahymena.

Eur J Biochem, 1985 Jul 1, 150(1), 61 - 6
Nucleotide sequence of the Escherichia coli gap gene . Different evolutionary behavior of the NAD+-binding domain and of the catalytic domain of D-glyceraldehyde-3-phosphate dehydrogenase; Branlant G et al.; A 1523-base-pair DNA fragment, spanning the gap gene from Escherichia coli, has been sequenced . It contains an open-reading frame whose length (330 amino acids) is in agreement with D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) molecular mass . This coding sequence is preceded by a Shine-Dalgarno complementary sequence and by two overlapping promoter-like structures . The codon usage within gap is consistent with that expected for a gene which is strongly expressed . The amino acid sequence of the E . coli GAPDH, deduced from the DNA sequence, contains all the amino acids postulated to play a functional role in GAPDH . Comparison of the E . coli enzyme with enzymes from other species reveals different evolutionary behaviour of the NAD+-binding domain and of the catalytic domain of GAPDH . The E . coli enzyme is found to be more similar to eucaryotic enzymes than to enzymes from thermophilic bacteria . This observation is discussed in terms of adaptation to growth at high temperature.

J Appl Biochem, 1985 Jun, 7(3), 192 - 201
Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus stearothermophilus; Okuno H et al.; Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) from the thermophilic bacteria, Bacillus stearothermophilus, was purified and its properties were examined . The enzyme was shown to consist of four identical subunits, each of about Mr 50,000 . This enzyme utilized both NADP+ and NAD+ as cofactors, and the maximum velocity for both cofactors was similar . However, the Km values were quite different from each other, being 0.016 and 1.64 mM for NADP+ and NAD+, respectively . From the analysis of sulfhydryl groups it was shown that there is one sulfhydryl group and one disulfide bridge per subunit . This sulfhydryl group had no reactivity with 5,5'-dithiobis(2-nitrobenzoic acid) in the absence of guanidine hydrochloride . The enzyme showed a remarkable thermostability as well as storage stability.

Biochem Int, 1985 Jun, 10(6), 955 - 62
Thermophilic NAD-dependent glutamate dehydrogenase from Bacillus stearothermophilus; Mantsala P; A rapid purification procedure for glutamate dehydrogenase (GDH) from Bacillus stearothermophilus var calidolactis was developed . The homogeneous enzyme with a total molecular weight of approximately 240,000 daltons, contained 6 identical subunits . No high molecular weight form of GDH present in crude extracts was found after elution of the enzyme from a 5'AMP-Sepharose column with 4 M urea . The purified enzyme functions in both directions i.e . amination and deamination and is strictly specific for NAD . 2-Oxo glutarate, glutamate or 2-mercaptoethanol protects against heat inactivation . NADH or ammonia, on the other hand, makes GDH more sensitive to heat . The purified enzyme undergoes thermal inactivation process.

Mol Cell Biol, 1985 Jun, 5(6), 1260 - 7
Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA; Yao MC et al.; Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development . In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction . The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development . The breakage event is associated with DNA elimination and telomeric sequence addition . Similar results were also found previously for the 3' junction of this gene . These results could explain how the extrachromosomal rDNA is first generated . Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found . These sequences might serve as signals for site-specific breakage . In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene . The repeats are 42 nucleotides long and are separated by 28 nucleotides . The existence of this structure provides a simple explanation for the formation of the palindromic rDNA.

Anal Biochem, 1985 Jun, 147(2), 419 - 27
A cellulase assay coupled to cellobiose dehydrogenase; Canevascini G; An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented . Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result . The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition . The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity . With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated . An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out.

J Cell Sci, 1985 Jun, 76, 179 - 87
Infection and maintenance of Holospora obtusa, a macronucleus-specific bacterium of the ciliate Paramecium caudatum; Fujishima M et al.; The gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum, which invades the host cell via a food vacuole, infects its macronucleus and grows exclusively in the nucleus . From infection experiments, we showed that a property of the macronucleus that is necessary for it to be recognized and infected by H . obtusa is commonly provided by P . caudatum, P . multimicronucleatum and 14 species of the P . aurelia complex, but not by P . jenningsi, P . bursaria, P . trichium, P . duboscqui, Didinium nasutum, Blepharisma japonicum, Pseudourostyla levis, seven species of Euplotes or Tetrahymena thermophila . Furthermore, it was also shown that the bacteria that infect the macronuclei of P . multimicronucleatum and the P . aurelia species complex always disappear from the nuclei within 5 days and the infected bacteria are maintained stably in the host nuclei in only 13 out of 22 strains of P . caudatum . The results indicate that the species specificity of the habitat of H . obtusa is not simply a matter of its ability to penetrate the host nuclear membrane but depends on unknown factors that exist only in certain strains of P . caudatum.

Cell, 1985 Jun, 41(2), 541 - 51
A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin; Bonven BJ et al.; Topoisomerase I is associated with DNAase I hypersensitive sites in the nontranscribed spacers flanking the rRNA genes in Tetrahymena thermophila . The endogenous topoisomerase I introduces site and strand specific single-strand cleavages in the rDNA spacers in situ . The cleavages occur base specifically within a hexadecameric sequence element present in two or three direct repeats at the hypersensitive sites . The sequence specificity and polarity of the cleavage reaction are identical when the enzyme is reacted with naked rDNA, indicating that the repetitive element functions as a high-affinity topoisomerase I attraction site in the r-chromatin . The biological mechanism associated with this phenomenon appears to be widespread among eukaryotes, since the topoisomerase I recognition sequence is conserved in the rDNA spacers of phylogenetically remote organisms, such as fungi, slime molds, ciliates, and insects.

Biochim Biophys Acta, 1985 May 31, 807(3), 293 - 9
Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP; Bar-Zvi D et al.; 3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1) . As with the CF1-ATPase (Bar-Zvi, D . and Shavit, N . (1984) Biochim . Biophys . Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase . BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax . In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound . Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase . Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1 . Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation . Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz{3H}ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit.

J Biol Chem, 1985 May 25, 260(10), 6334 - 40
The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin; Spangler EA et al.; We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila . The gene encodes an RNA molecule which is 1753 nucleotides in length . The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs . We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin . In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure . Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function.

Eur J Biochem, 1985 May 15, 149(1), 41 - 6
DNA polymerases from the extremely thermophilic bacterium Thermus thermophilus HB-8; Ruttimann C et al.; Three DNA polymerase isoenzymes, which have been called A, B and C, were purified from Thermus thermophilus HB-8 . These enzymes can be separated by chromatography (pH 7.5) on phosphocellulose and DNA-agarose . Their relative molecular masses, as determined by glycerol gradient centrifugation, fall in the range of 110000-120000 . The three of them are devoid of exonuclease activity . Species A, B and C differ in their sensitivity towards N-ethylmaleimide (A greater than B greater than C) and urea (A greater than B = C) and also in their stability at high temperature (90 degrees C) (B greater than C greater than A) . In addition, these enzymes can be distinguished utilizing various templates under different conditions . Thus, with activated DNA and Mg2+ as a cofactor, the highest incorporation is obtained at 50 degrees C with enzyme A and at 63 degrees C with enzymes B and C . If Mg2+ is replaced by Mn2+, the optimal temperatures remain unchanged, but enzyme A is stimulated twofold, while the activities of enzymes B and C decrease to one-half . On the other hand, with either poly(dA) X (dT)10 or poly(dA-dT) and Mg2+, enzyme A is inactive and enzyme C is severalfold more active than enzyme B . With the former synthetic template, optimal temperatures are 50 degrees C (enzyme C) and 40 degrees C (enzyme B), while with poly(dA-dT) they both work best at 63 degrees C . In turn, only enzyme C is able to utilize poly(rA) X (dT)10, although only with Mn2+ as a cofactor.

Biochim Biophys Acta, 1985 May 14, 815(2), 268 - 80
The effect of growth temperature on the thermotropic behavior of the membranes of a thermophilic Bacillus . Composition-structure-function relationships; Reizer J et al.; The following study was carried out with the aim of widening our understanding of the thermoadaptive mechanisms of the membrane of thermophiles, using Bacillus stearothermophilus var . nondiastaticus as test-organism . The phospholipids and their acyl chain composition of this Bacillus studied in relation to the physical properties of its membrane from bacteria grown at various temperatures . Phospholipids account for 68-75 weight% of the total lipid in cells grown at 45, 55 or 65 degrees C . Phosphatidylglycerol and diphosphatidylglycerol constitute up to 90% of the total phospholipids; no amino phospholipids were found . Increasing the growth temperatures from 45 degrees to 65 degrees C caused an approximately 4-fold decrease in the proportion of the branched-chain fatty acids and a 2-fold increase in the amount of the saturated acyl chains . The reduced proportion of the branched fatty acids was mainly due to a decrease in their anteiso forms . Unsaturated fatty acids were not produced by cells grown at 65 degrees C . In accordance with the fatty acid composition, the molecular packing of phospholipids in monolayers was more expanded with phospholipids from 45 degrees C grown cells as compared with cultures grown at 55 degrees C . The thermotropic gel to liquid-crystalline phase transition of the membrane lipids was monitored by differential scanning calorimetry and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene . With increase of the growth temperature the phase transition was progressively shifted to higher but narrower range of temperatures . Completion of the lipid melting occurred always at temperatures below those employed for growth . A constructed phase diagram enabled to relate the growth temperature, the fatty acid composition and the lipid apparent microviscosity at temperatures not used in the present study for growth of the thermophile . The minimum temperature for growth and the upper boundary temperature of the least saturated lipid crystallization were extrapolated in this manner; they correspond to the experimentally determined minimal growth temperature . The apparent microviscosity, a measure of membrane order, decreased gradually and conspicuously as the growth temperature was elevated . The delimiting apparent microviscosity values, at the maximal (65 degrees C) and minimal (41 degrees C) growth temperatures were 0.8 and 1.8 poise, respectively . This lack of rigorous homeostatic control of the bulk lipid viscosity prompted reevaluation of the physiological significance of 'homeoviscous adaptation' in Bacillus stearothermophilus.

J Biochem (Tokyo), 1985 May, 97(5), 1521 - 4
Thermophilic microspheres of peptide-like polymers and silicates formed at 250 degrees C; Yanagawa H et al.; We examined the possibility of chemical evolution in superheated hydrothermal environments and found the formation of microspheres at 250 degrees C and above from a mixture of glycine, alanine, valine, and aspartic acid . The microspheres did not form at lower temperatures and consisted of silicates and peptide-like polymers that contained imide bonds and amino acid residues having an abundance of valine . The results show the possibility of thermophilic cellular structures, which might be adopted by the extremely thermophilic organisms, if they exist, reported by Baross and Deming.

Exp Cell Res, 1985 May, 158(1), 244 - 56
Effect of the antitubulin drug nocodazole on meiosis and postmeiotic development in Tetrahymena thermophila . Induction of achiasmatic meiosis; Kaczanowski A et al.; Nocodazole (ND), a potent antitubulin drug, can be used to dissect the steps of meiosis in Tetrahymena, presumably by interfering with the assembly of microtubules . Its effects depend upon the time during conjugation at which the drug is applied . When applied prior to the elongation of the micronucleus into the characteristic 'crescent' configuration, no crescent is formed and the chromosomes of prepachytene and pachytene condense into spherical nuclei . If ND is applied after micronuclear elongation has begun, but before it is fully elongated, the chromosomes fail to synapse and appear in metaphase I as unpaired monovalents . In contrast, the metaphase I chromosomes appear as bivalents when ND is applied later, during or after the crescent has reached its maximum elongation . Still later, application of ND inhibits chromosome movements during anaphase and telophase of either meiotic division, but does not prevent separation of kinetochores . In some of the blocked restitutive nuclei an additional round of chromosome replication occurs, corresponding to the third pregamic division in normal conjugation . The hyperploid micronuclei produced by such treatment may be useful in certain genetic manipulations and in studying the regulation of nuclear DNA content.

Exp Cell Res, 1985 May, 158(1), 159 - 69
Deciliation interferes with cell-cycle progression in Tetrahymena; Seyfert HM et al.; The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied . It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation . Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later . This was shown by pulse-labelling the cells with {3H}thymidine at various times after deciliation . Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose . At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment . This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division . The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure . The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells . This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation . Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.

Chest, 1985 May, 87(5), 658 - 61
Pyridoxine deficiency in children treated with isoniazid; Pellock JM et al.; Isoniazid-induced deficiency of pyridoxine (vitamin B6) is reportedly not uncommon in adults but rare in children . In the present study, 38 children had serum levels of pyridoxine tested while receiving therapy with isoniazid . A biologic assay using the protozoan Tetrahymena thermophila determined pyridoxine status after 2 to 18 months of therapy with isoniazid . Five children (13 percent) were deficient . None had definitive clinical symptoms or signs consistent with pyridoxine deficiency . Three had normal nerve conduction velocity . Children receiving isoniazid in dosages greater than 10 mg/kg/day had a higher incidence of deficiency . Present recommendations for withholding pyridoxine prophylaxis from children receiving isoniazid therapy must be reconsidered in light of these findings, particularly in those children who are debilitated or have a poor nutritional history with a known pyridoxine deficit prior to therapy with isoniazid.

Mutat Res, 1985 May, 145(3), 157 - 64
Repair of 8-methoxypsoralen induced DNA interstrand cross-links in Tetrahymena thermophila . The effect of inhibitors of macromolecular synthesis; Nielsen PE et al.; The effect of several growth-inhibiting compounds on the repair of 8-methoxypsoralen-UVA-light-induced DNA interstrand cross-links has been studied in the protozoan Tetrahymena thermophila . The repair process was analyzed by the alkaline elution technique and could be divided into 3 phases: a protein-DNA complexing phase, a DNA-incision phase and finally a DNA-ligation phase . The incision was found to be completely inhibited by novobiocin (50 micrograms/ml), nalidixic acid (150 micrograms/ml), n-butyrate (15 mM) and cycloheximide (1 microgram/ml), while no effect was observed for cytosine-1-beta-D-arabinofuranoside (10 mM), puromycin (1 mM), hydroxyurea (5 mM) or 3-aminobenzamide (2.5 mM) . None of the compounds showed any effect on the protein-DNA complexing step, and the ligation was partly inhibited only by nalidixic acid (150 micrograms/ml) . The involvement of topoisomerases in the repair of psoralen-induced DNA interstrand cross-links is suggested.

J Bacteriol, 1985 May, 162(2), 768 - 72
Buffering capacity of bacilli that grow at different pH ranges; Krulwich TA et al.; Cytoplasmic buffering capacities and buffering by whole cells were examined in six bacterial species: Bacillus acidocaldarius, Bacillus stearothermophilus, Escherichia coli, Bacillus subtilis, Bacillus alcalophilus, and Bacillus firmus RAB . Acid-base titrations were conducted on whole cells and cells permeabilized with Triton X-100 or n-butanol . In all of the species examined, the buffering capacity of intact cells was generally a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied from species to species . Over the entire range of pH values from 4 to 9.5, B . subtilis exhibited a cytoplasmic buffering capacity that was much higher than that of B . stearothermophilus, B . acidocaldarius, or E . coli . The latter three species had comparable cytoplasmic buffering capacities at pH 4 to 9.5, as long as optimal conditions for cell permeabilization were employed . All of the nonalkalophiles exhibited a decrease in cytoplasmic buffering capacity as the external pH increased from pH 5 to 7 . At alkaline pH values, the two thermophiles in the study had particularly low cytoplasmic buffering capacities, and the two alkalophilic bacteria had appreciably higher cytoplasmic buffering capacities than any of the other species studied . Cytoplasmic buffering capacities as high as 1,100 nmol of H+ per pH unit per mg of protein were observed in alkalophilic B . firmus RAB . Since previous studies have shown that immediate cytoplasmic alkalinization occurs upon loss of the active mechanisms for pH homeostasis in the alkalophiles, the very high buffering capacities apparently offer no global protection of internal pH . Perhaps, the high buffering capacities reflect protective mechanisms for specific macromolecules or process rather than part of the mechanisms for bulk pH homeostasis.

Dev Biol, 1985 May, 109(1), 1 - 8
Modulation of linker histones during development in Tetrahymena: selective elimination of linker histone during the differentiation of new macronuclei; Chicoine LG et al.; Macronuclei of Tetrahymena thermophila contain a typical H1 which has been shown to be missing from micronuclei . Instead, micronuclei contain three unique polypeptides, alpha, beta, and gamma, which are associated with linker regions of micronuclear chromatin . In this report polyclonal antibodies raised against macronuclear H1 are shown to react with alpha, beta, and gamma by immunoblotting analyses . This result suggests that these polypeptides share some common structural feature(s) . Also consistent with this result is the finding that both macro- and micronuclei in growing and mating cells stain positively with H1 antibodies by in situ indirect immunofluorescence . However, these analyses demonstrate that the level of linker histone is greatly reduced in the micronucleus of starved cells and in young macronuclear anlagen . These results are in agreement with earlier biochemical studies and together provide strong evidence that dramatic changes in linker histone accompany nuclear differentiation (and dedifferentiation) in Tetrahymena.

Mycopathologia, 1985 May, 90(2), 81 - 3
Amylase and growth characteristics of Papulaspora thermophilia; Adams PR; Mycelial dry weight of Papulaspora thermophilia reached a maximum of about 96 mg after six days of growth in a starch-yeast medium . Extracellular amylase activity was not measurable during this growth period and remained thereafter only about 0.1 unit per ml for 30 days, yet starch concentration reduced rapidly, and reducing sugar appeared in the extracellular medium within the first few days of incubation.

Biochem Biophys Res Commun, 1985 Apr 30, 128(2), 781 - 7
Amino acid sequence of cytochrome c-552 from Thermus thermophilus HB8; Titani K et al.; The complete amino acid sequence of thermophilic cytochrome c-552 from Thermus thermophilus HB8 is presented . The 131-residue sequence was derived by analysis of three cyanogen bromide fragments of the S-carboxymethylated apo-protein and their subpeptides . The sequence is homologous to c-type cytochromes, especially in the heme-binding region.

Biochim Biophys Acta, 1985 Apr 29, 828(3), 369 - 74
Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin . Possible new fingerprints for three iron clusters; Hagen WR et al.; We describe two new characteristics of the EPR of the seven-iron containing ferredoxin from Thermus thermophilus . First, the reduced state of the 3Fe center, which has traditionally been considered to be EPR-silent, has been found to exhibit a delta m = 4 transition, which is unique for Fe-S centers . This signal is similar to that of high-spin Fe2+-EDTA and supports the suggestion that the ground electronic state of the 3Fe cluster is S = 2 . Second, we have recorded the EPR spectrum of the fully reduced protein at 9 and 15 GHz and found that changes occur in the signal which are consistent with a weak electronic spin-spin interaction between the {4Fe-4S}+ (S = 1/2) and the reduced 3Fe center . A theoretical explanation is given for the observation of interaction signals with constant effective g values.

Nucleic Acids Res, 1985 Apr 11, 13(7), 2661 - 80
Conserved arrangements of repeated DNA sequences in nontranscribed spacers of ciliate ribosomal RNA genes: evidence for molecular coevolution; Challoner PB et al.; We have analyzed the nucleotide sequences of the nontranscribed spacer (NTS) and transcription initiation and termination regions of the extrachromosomal rDNAs of the ciliated protozoans Tetrahymena thermophila and Glaucoma chattoni . The sequences surrounding the sites of transcription initiation and termination are highly conserved . The only extensive homologies of the NTS regions occur in five sets of dispersed repetitive sequences . Type I, II and III repeats in the 5' NTS are strongly conserved in sequence between Tetrahymena and Glaucoma in the case of the type I and III repeats, and in location relative to the transcription initiation site in the case of type I and II repeats . We identify two new repeat types, designated IV and V, in the 3' NTS . The sequence of type IV repeats, and the location relative to the transcription termination site of type IV and V repeats, are conserved . All five types of repeats are interspersed with nonconserved DNA sequences . These results suggest that the five repeat types in the 5' and 3' NTSs are important in rRNA gene function; the sequence organization, and the differing rates of divergence between species of the repeat types, provide strong evidence for their functional selection through the process of molecular coevolution.

Nucleic Acids Res, 1985 Apr 11, 13(7), 2647 - 59
Autonomously replicating sequences from the non transcribed spacers of Tetrahymena thermophila ribosomal DNA; Amin AA et al.; The non-transcribed spacer (NTS) regions of the linear extrachromosomal palindromic rDNA from the ciliated protozoan Tetrahymena thermophila contain, in addition to sequences regulating transcription, the origin(s) of bidirectional replication as well as telomere associated sequences . These NTS regions function as autonomous replication sequences (ARS) in the yeast Saccharomyces cerevisiae (Kiss, G.B., Amin, A.A . and Pearlman, R.E., Mol . Cell Biol . 1: 535-543, 1981) . We have now identified in the 5' NTS two adjacent but non-overlapping restriction fragments which function as ARS in yeast . These fragments contain 200 bp of duplicated sequence and include the in vivo origin of rDNA replication . The ARS in the 3' NTS has been subdivided into a sequence which allows high frequency transformation of yeast but with transformants extremely unstable when grown either under selective or non-selective conditions, and a sequence which appears to be required for plasmid maintenance yet not to be essential for high frequency transformation . Detailed analysis of the DNA sequences in these regions gives rise to interesting information about structural and functional features of the molecule.

Microbiologica, 1985 Apr, 8(2), 191 - 6
Chemoresistances among 80 Campylobacter strains isolated from childhood gastroenteritis cases; Figura N et al.; The antimicrobial susceptibility of 80 thermophilic Campylobacter strains, 61 C . jejuni and 19 C . coli, isolated from childhood gastroenteritis cases has been studied . Gentamicin and chloramphenicol were effective against all strains; beta-lactams, except carbenicillin and ticarcillin, had on the whole little activity . 26.2% of strains proved to be resistant to tetracycline and 7.5% to erythromycin; erythromycin-resistance was found significantly more often in the species C . coli and always associated to clindamycin-resistance . The high prevalence of strains resistant to erythromycin suggests chloramphenicol and gentamicin as possible alternative drugs in the treatment of life-threatening Campylobacter infections.

J Clin Microbiol, 1985 Apr, 21(4), 553 - 7
Thermophilic bacteria: a new cause of human disease; Rabkin CS et al.; We studied a group of 31 bacterial isolates from clinical specimens, received by the Centers for Disease Control since 1961, which have been denoted thermophilic for their unusual ability to grow at 50 degrees C . Microbiological characteristics were determined for the group, and an assessment of their clinical significance was made based on retrospective chart review . These bacteria are all gram-negative, nonfermentative, nonsporulating rods, most of which grow better at 42 or 50 degrees C than at 35 degrees C . Some of the bacteria could be implicated as the etiological agents for meningitis, endocarditis, and septicemia . Thermophilic bacteria should be considered potential pathogens when isolated from appropriate clinical specimens.

J Bacteriol, 1985 Apr, 162(1), 102 - 5
Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum; Beguin P et al.; The nucleotide sequence of the celA gene, encoding the extracellular endoglucanase A of Clostridium thermocellum, was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme . The mature protein appeared to be extended by a signal sequence of 32 amino acids . A segment of 23 amino acids was duplicated at the COOH-terminal end of the protein . The putative GUG initiation codon was preceded by an AGGAGG sequence, typical of procaryotic ribosomal binding sites . The segment of DNA presumably specifying transcriptional initiation contained a high percentage of adenine and thymine residues, including an adenine-thymine tract extending over 54 base pairs.

Exp Cell Res, 1985 Apr, 157(2), 315 - 21
A transmissible developmental block in Tetrahymena thermophila; Kaney AR; When the amicronucleate mutant BI3840 of Tetrahymena thermophila is mated with normal micronucleate cells, it receives a pronucleus from its partner but there is no further nuclear development and the conjugants separate, retaining their original macronuclei . Both of these sexually mature exconjugants and any cells with which they are mated show an unconditional block in macronuclear development . Although prezygotic nuclear divisions, nuclear transfer and post-zygotic nuclear divisions appeared normal upon cytological analysis of Giemsa-stained conjugants, macronuclear development was invariably aborted . Since the original macronucleus was resorbed, the cells were rendered amacronucleate and they died . When no macronuclear development was initiated, as in crosses with the aneuploid strain A* (III), the exconjugants were viable and retained their original macronuclei . This pattern was invariant with three different strains serving as the original micronuclear source, and in the case of one non-BI3840 exconjugant, persisted for over 200 cell generations . Exconjugants from a cross of one of the micronuclear donors with strain A* (III) did not show arrested development when crossed . It thus appears likely that there is conjugal transfer of non-nuclear information originating in BI3840 which is self-replicating and which causes an arrest in macronuclear development.

Can J Microbiol, 1985 Apr, 31(4), 339 - 45
Isolation and characterization of antibiotic-resistance plasmids in thermophilic bacilli; Hoshino T et al.; Four antibiotic-resistance plasmids isolated from thermophilic bacilli were characterized in detail . Three tetracycline-resistance (Tc1) plasmids were designated as pTHT9 (7.7 kilobases (kb}, pTHT15 (4.5 kb) and pTHT22 (8.4 kb) . From the results of restriction endonuclease analysis and the subsequent Southern hybridization, these were found to possess extensive genetic homology in the regions that include the replication origin and the Tcr gene . Detailed restriction maps of the smallest Tcr plasmid pTHT15 and a kanamycin-resistance (Kmr) plasmid pTHN1 (4.8 kb) were constructed . The positions of antibiotic-resistance loci and regions essential for plasmid replication were determined by cloning plasmid fragments in Bacillus subtilis . These four plasmids were found to replicate and express the resistance genes stably in both B . subtilis and B . stearothermophilus.

Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2452 - 5
An unusual genetic code in nuclear genes of Tetrahymena; Horowitz S et al.; We have cloned and partially sequenced two histone H3 genes of Tetrahymena thermophila . The DNA sequences strongly suggest that both genes are active in the vegetatively growing cell . Comparison of the derived amino acid sequences of these two genes with the actual sequence of Tetrahymena histone H3 results in the surprising conclusion that TAA codes for glutamine . This represents the first demonstration of a coding function for this termination codon of the "universal" code . This observation has important implications for the evolution of ciliates and of the genetic code.

Biochem Int, 1985 Apr, 10(4), 655 - 62
Isolation and partial characterization of BstVI, a thermostable isoschizomer of XhoI; Vasquez C; A type II restriction endonuclease, which has been named BstVI, was isolated and partially purified from a spore-forming, gram-positive thermophilic bacilli . On the basis of its digestion patterns on various DNA's, it was concluded that this enzyme is an isoschizomer of XhoI, isolated originally from Xanthomonas holcicola . Besides being highly thermostable, the enzyme is produced in very large amounts by this bacterial strain . A single purification step renders it free of unspecific nucleases and suitable for performing restriction analysis and cloning experiments.

Nucleic Acids Res, 1985 Mar 25, 13(6), 1871 - 89
Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA; Price JV et al.; The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS) . The altered genes were transcribed and the RNA tested for self-splicing in vitro . A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity . Two of these insertions, located at a site 5 nucleotides preceding the 3'-end of the IVS, did not alter the choice of the 3' splice site . Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS . Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro . Three other regions were found to be necessary . The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences . The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing.

J Biol Chem, 1985 Mar 25, 260(6), 3251 - 4
Evidence for N coordination to Fe in the {2Fe-2S} clusters of Thermus Rieske protein and phthalate dioxygenase from Pseudomonas; Cline JF et al.; Rieske-type iron/sulfur proteins and several NADH-dependent oxygenases contain Fe/S clusters with similar spectral and magnetic properties . Purified Rieske iron/sulfur protein from Thermus thermophilus contains two apparently identical {2Fe-2S} clusters in a polypeptide having only four cysteine residues, and it has been proposed that each Fe/S cluster is coordinated to two cysteine S-atoms and to an unknown number of other non-sulfur atoms (Fee, J . A., Findling, K . L., Yoshida, T., Hille, R., Tarr, G . E., Hearshen, D . O., Dunham, W . R., Day, E . P., Kent, T . A., and Munck, E . (1984) J . Biol . Chem . 259, 124-133) . We have examined the Rieske protein from Thermus and the phthalate dioxygenase from Pseudomonas cepacia with electron nuclear double resonance (ENDOR) and pulsed EPR methods and report here evidence for the direct coordination of nitrogenous ligands to the Fe/S clusters in these proteins . The electron nuclear double resonance signals arising from 14N have been interpreted in terms of a strongly coupled ligand with AN = approximately 26-28 MHz and a weakly coupled ligand with AN = approximately 9 MHz . The pulsed EPR spectrum shows a rich pattern of lines in the Fourier transformed data having peaks in the range of 0.8 to 6.7 MHz . The lower frequency resonances are tentatively associated with coupling of the unpaired spin to the remote N-atoms of coordinated imidazole rings.

Eur J Biochem, 1985 Mar 15, 147(3), 503 - 10
Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA . An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA; Bohm S et al.; An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+ . These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups . T . thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure . Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs . Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.

Biochemistry, 1985 Mar 12, 24(6), 1476 - 83
Spectral properties and function of two lumazine proteins from Photobacterium; Lee J et al.; The spectral properties are compared for two 6,7-dimethyl-8-ribityllumazine proteins from marine bioluminescent bacteria, one from a psychrophile, Photobacterium phosphoreum, and the other from a thermophile, Photobacterium leiognathi . The visible spectral properties, which are the ones by which the protein performs its biological function of bioluminescence emission, are almost the same for the two proteins: at 2 degrees C and 50 mM Pi, pH 7, fluorescence quantum yield phi F = 0.59 and 0.54, respectively; fluorescence lifetime tau = 14.4 and 14.8 ns, respectively; fluorescence maxima, both 475 nm; absorption maximum, 417 and 420 nm, respectively; circular dichroism minima at around 420 nm, both -41 X 10(3) deg cm2 dmol-1 . The ligand binding sites therefore must provide very similar environments, and arguments are presented that the bound ligand is relatively exposed to solvent . The dissociation equilibrium was studied by steady-state fluorescence polarization . The thermophilic protein binds the ligand with Kd (20 degrees C) = 0.016 microM, 10 times more tightly than the other protein {Kd (20 degrees C) = 0.16 microM} . The origin of the binding difference probably resides in differences in secondary structure . The tryptophan fluorescence spectra of the two proteins are different, but more significant is an observation of the decay of the tryptophan emission anisotropy . For the psychrophilic lumazine protein this anisotropy decays to zero in 1 ns, implying that its single tryptophan residue lies in a very "floppy" region of the protein . For the other protein, the anisotropy exhibits both a fast component and a slow one corresponding to rotation of the protein as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Mar 12, 24(6), 1467 - 75
Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum; O'Kane DJ et al.; The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared . An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements . Neither protein contains metal cofactors, organic phosphorus, or carbohydrate . Both proteins are anionic and hydrophilic . They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P . phosphoreum and P . leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300 . In the P . phosphoreum protein both Cys residues are accessible, but in the P . leiognathi protein the single Cys is "buried" . Modification of this buried Cys and at least one Cys in the P . phosphoreum protein prevents binding of the ligand . The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe.

Nucleic Acids Res, 1985 Mar 11, 13(5), 1543 - 57
Topoisomerase I has a strong binding preference for a conserved hexadecameric sequence in the promoter region of the rRNA gene from Tetrahymena pyriformis; Andersen AH et al.; Topoisomerase I is in situ associated with DNaseI hypersensitive sites located in the promotor and terminator regions of the extrachromosomal rDNA in Tetrahymena thermophila at sites with sequences fitting the motif (sequence in text) Reconstitution experiments with purified topoisomerase I and cloned fragments of rDNA demonstrate that the enzyme exhibits the same binding and cleavage properties on naked DNA . These observations are striking as topoisomerase I previously has been found to exhibit low sequence specificity . The specific binding of the enzyme has an absolute requirement for divalent cations with a preference for Ca2+ . The strong binding to the hexadecamer has been characterized by competition experiments, and it has been used to determine the molecular weight of the enzyme.

J Biochem (Tokyo), 1985 Mar, 97(3), 899 - 909
Fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase from Thermus aquaticus YT-1, an extreme thermophile: activation by citrate and modification reagents and comparison with Thermus caldophilus GK24 L-lactate dehydrogenase; Machida M et al.; Heat-stable fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase {EC 1.1.1.27} was purified from an extremely thermophilic bacterium, Thermus aquaticus YT-1 . The amino acid composition and NH2-terminal 34 amino acid sequence of the enzyme were determined . Its NH2-terminal sequence shows high homology with those of Thermus caldophilus GK24 (82% identity) and some other bacterial L-lactate dehydrogenases (44-53% identity), indicating the close phylogenic relationship of the two Thermus species . At the same time, the two Thermus L-lactate dehydrogenases were found not to be identical not only chemically but also kinetically and immunologically . Citrate activated the T . aquaticus enzyme in the weak acidic pH region, while fructose 1,6-bisphosphate did in both acidic and neutral pH regions . The maximum activity obtained with citrate at pH 5.0 was about 2.5 times higher than that in the presence of fructose 1,6-bisphosphate at pH 6.7 . The enzymes modified with 2,3-butanedione, acetic anhydride and diethyl pyrocarbonate in the presence of both NADH and oxamate were desensitized to fructose 1,6-bisphosphate, and the modified enzymes were active even in the absence of fructose 1,6-bisphosphate . All of the modified enzymes examined were still activated by citrate similarly to the native enzyme . These results suggest that the mechanism of activation by citrate is different from that by fructose 1,6-bisphosphate, and that the citrate-binding site is different from the fructose 1,6-bisphosphate-binding site.

Appl Environ Microbiol, 1985 Mar, 49(3), 724 - 6
Effect of seeding during thermophilic composting of sewage sludge; Nakasaki K et al.; The effect of seeding on the thermophilic composting of sewage sludge was examined by measuring the changes in CO2 evolution rates and microbial numbers . Although the succession of thermophilic bacteria and thermophilic actinomycetes clearly reflected the effect of seeding, no clear difference was observed in the overall rate of composting or quality of the composted product.

J Bacteriol, 1985 Mar, 161(3), 981 - 8
Electron microscopy of the secondary structure in partially denatured rRNAs of Escherichia coli and Bacillus stearothermophilus; Klein BK et al.; Partially denatured 16S and 23S rRNAs from the thermophile Bacillus stearothermophilus show characteristic loop patterns when observed by electron microscopy . The patterns are very similar to those seen in rRNAs from Escherichia coli . At least 2 of 4 most stable interactions in 16S rRNA and 8 of 12 interactions in 23S rRNA are in common for the two species . These interactions correspond well to features of secondary structure in models inferred for rRNA from phylogenetic sequence comparisons and chemical modification studies . However, two additional large loops, enclosing large portions of the 23S rRNA, have been detected in B . stearothermophilus for the first time, and even though other loops are similar, their relative frequencies vary in the two species . Much of the variation is consistent with relative delta G degree values for putative base-paired stems at the base of different loops; but the 5'-terminal loops in 23S rRNA, for example, are unaccountably far less stable in B . stearothermophilus . Also, in general, structural features are not differentially stabilized in B . stearothermophilus; the relative stability of secondary structure in its ribosomes at elevated growth temperatures must involve interactions with ribosomal proteins or other cellular components.

Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Mar, 47(3), 291 - 7
Membrane damage and its repair in the thermophilic bacterium, Thermus thermophilus HB-8 exposed to u.v . radiation and 60Co gamma-rays; Suzuki S; When exposed to either u.v . radiation or 60Co gamma-rays, the thermophilic bacterium, Thermus thermophilus HB-8, which can grow at 49-85 degrees C, lost its ability to take up extracellular K+ in a dose-dependent manner . However, the loss was reduced by incubation at 37 degrees C after exposure to u.v . radiation or gamma-rays . Cell survival after exposure to 60Co gamma-rays, as measured by colony formation, was increased by incubation at 37 degrees C after exposure, whereas cell survival after u.v . radiation was not . These results, therefore, indicate that the loss of ability of cells to take up K+ after u.v . radiation was not due to cell death but some damage to the membrane itself, and that the membrane damage can be repaired . Lipid peroxidation is not responsible for the membrane damage, because HB-8 cells do not contain unsaturated fatty acids in their membranes.

J Inorg Biochem, 1985 Mar-Apr, 23(3-4), 279 - 88
Potentiometric study of cytochrome c1aa3 from Thermus thermophilus; Yoshida T et al.; We have examined the redox behavior of the cytochrome c1aa3 complex from Thermus thermophilus . In potentiometric titrations the cytochrome c behaves as an independent center having n = 1 and E = 205 mV (NHE) . Under the assumption that the individual centers equilibrate independently in this experiment, changes in the absorption band at 603 nm have been resolved into two components: cytochrome a (n = 1, Em = 270 mV, 60% spectral contribution) and cytochrome a3 (n = 2, Em = 360 mV, 40% spectral contribution) . The n = 2 process was attributed to strong chemical coupling between cytochrome a3 and CuB . The enzyme was also titrated with a mixture of NADH and PMS, and the results are shown not to conform to a model of intramolecular equilibrium according to the equilibrium constants obtained from the potentiometric titration . It is suggested that a conformational equilibrium within the complex may control electron transfer between cytochromes a and a3.

Biol Chem Hoppe Seyler, 1985 Mar, 366(3), 223 - 31
Purification, amino-acid sequence and some properties of the ferredoxin isolated from Bacillus acidocaldarius; Schlatter D et al.; Ferredoxin was isolated from the aerobic, thermophilic and acidophilic bacterium Bacillus acidocaldarius and its sequence of 78 amino acids completely determined by automated Edman degradation of the protein and of peptides derived from chemical cleavage between aspartic acid and proline and from enzymatic digestions . The optical spectrum of the oxidized protein has a broad maximum around 400 nm . The ferredoxin is thermostable: its absorbance begins to decrease only at incubation over 71 degrees C . The number of iron and inorganic sulphur atoms per molecule was determined to be 5.3 and 5.0, respectively . The calculated molar extinction coefficient was 23 000 M-1 X cm-1, the molecular mass of the apoferredoxin 8 872 Da . Contrary to all expectations, the sequence of B . acidocaldarius ferredoxin shows very little homology to that of B . stearothermophilus but closely resembles that of Thermus thermophilus.

Can J Biochem Cell Biol, 1985 Mar, 63(3), 153 - 61
Complexes of cytochrome caa3 from the thermophilic bacterium PS3 formed with ligands and during catalytic activity; Sone N et al.; PS3 (thermophilic bacterium) cytochrome caa3 reacts slowly with cyanide which forms a low-spin complex with the CuBa3 centre . Partial reduction under catalytic conditions increases the rate of cyanide binding, and the reaction constant is rather similar to that of the mammalian enzyme, but the partially reduced complex dissociates more rapidly than does the corresponding eukaryotic complex . A simple biphasic reaction can account for the results obtained . The azide complex of partially reduced PS3 cytochrome caa3 shows an alpha-peak blue shift similar to that of the mammalian enzyme . PS3 cytochrome caa3 forms an oxyferri ("oxygenated") species like the mammalian enzyme, but does not undergo high- to low-spin changes during the aerobic steady state with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine as substrates . Interactions seen in cytochrome oxidase-ligand reactions and its spin-state changes are therefore intrinsic to the enzyme's large catalytic subunits and do not require the presence of the small nuclear-encoded subunits found in eukaryotic systems.

Nucleic Acids Res, 1985 Feb 25, 13(4), 1399 - 412
DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine; Ehrlich M et al.; While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C) . Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy . Eight of the bacterial DNAs contained m4C . Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile . The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs . Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns . Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes . Instead, some of them have deamination-resistant m4C residues.

J Biol Chem, 1985 Feb 25, 260(4), 2064 - 8
Differential selectivity of cholinephosphotransferase and ethanolaminephosphotransferase of Tetrahymena for diacylglycerol and alkylacylglycerol; Smith JD; The glycerophospholipids of the ciliate protozoan Tetrahymena thermophila differ greatly in their content of alkylacylglycerol with phosphatidylcholine, phosphatidylethanolamine, and 2-aminoethylphosphonolipid containing 60, 4, and 53% glyceryl ether, respectively . This difference is achieved by differences in the selectivities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) for alkylacylglycerol and diacylglycerol . When the two enzymes are assayed in vitro using only endogenous diglyceride as substrate, the newly formed phosphatidylcholine contains 37% glyceryl ether, while the newly formed phosphatidylethanolamine contains 5% glyceryl ether . The ethanolaminephosphotransferase is stimulated equally well by addition of diolein and dipalmitin, but the diacylglycerols have no effect on the glyceryl ether content of phosphatidylethanolamine . In contrast, the glyceryl ether content of newly formed phosphatidylcholine decreases to 16% when the cholinephosphotransferase is exposed to diolein or dipalmitin . The ethanolaminephosphotransferase is not stimulated by addition of a 60:40 mixture of alkylacylglycerol/diacylglycerol . The cholinephosphotransferase is stimulated by the mixture to the same extent as it is by the diacylglycerols, with the glyceryl ether content of the newly formed phosphatidylcholine increasing to 52% . With the addition of alkylacylglycerol alone, the glyceryl ether content of the newly formed phosphatidylethanolamine increases to 10%, while that of the newly formed phosphatidylcholine increases almost to 60%.

Nature, 1985 Feb 28-Mar 6, 313(6005), 787 - 9
Two contrary modes of chemolithotrophy in the same archaebacterium; Segerer A et al.; Sulphur-dependent archaebacteria, which are found around nearly boiling continental solfataric springs and mud holes, can be assigned to two distinct branches: the aerobic, sulphur-oxidizing Sulfolobales and the strictly anaerobic sulphur-reducing Thermoproteales . Here, we report the isolation of a group of extremely thermophilic solfataric archaebacteria that are able to grow either strictly anaerobically by reduction, or fully aerobically by oxidation of molecular sulphur, depending on the oxygen supply . We have also established that the ability to grow in these two ways is shared by Sulfolobus brierleyi, a well-known less thermophilic sulphur-oxidizing archaebacterium capable of ore-leaching . The phenomenon may be dependent on a fundamental switch in genome expression . These organisms might represent the primitive fore-runners of sulphur-oxidizing archaebacteria, meeting their energy requirements either by oxidation or by reduction of the same element.

J Protozool, 1985 Feb, 32(1), 32 - 7
S-Adenosylhomocysteine hydrolase deficiency in cysteine auxotrophs of Tetrahymena thermophila; Murphy SE et al.; The biochemical lesion in two cysteine auxotrophs of Tetrahymena thermophila has been established as a defect in S-adenosylhomocysteine hydrolase, an enzyme of the transsulfuration pathway . As a result, these mutants require cysteine (or cystathionine or homocysteine) for growth in a defined medium . Cell-free extracts of the mutants contained less than 5% of the level of the enzyme seen in the wild type . One of the mutant strains accumulated intracellular levels of S-adenosylhomocysteine as high as 1380 microM, a level 200 times normal . When both mutant strains were maintained in defined medium without cysteine, growth occurred after a long lag; this phenomenon was termed "adaptation." Adaptation was a) reversed by passage through rich medium, b) was not a recovery of S-adenosylhomocysteine hydrolase, and c) was probably linked to induction of an alternate pathway for cysteine biosynthesis, involving a lysosomal S-adenosylhomocysteine nucleosidase activity.

J Protozool, 1985 Feb, 32(1), 126 - 39
Oral ultrastructure and oral development of the misaligned undulating membrane mutant of Tetrahymena thermophila; Lansing TJ et al.; The misaligned undulating membrane (mum) mutant of Tetrahymena thermophila is a non-conditional, single gene recessive mutation . The major effect of the mum mutation is the production of multiple undulating membrane (UM) fragments in the oral apparatus (OA) . The ultrastructure of the UM fragments of mum OAs is identical to that of the single UM of wild-type OAs . Analysis of OA development at midbody using a combination of light microscopy of protargol-stained cells and SEM of demembranated whole cells showed that the phenotypic effect of the mum mutation first becomes evident during mid to late stage 4 and is fully manifested in early stage 5 . The effect of the mutation involves a proliferation of excess basal bodies in the UM field . Subsequent events in the development of the mum OA from mid to late stage 5 are identical to those in wild-type OAs . This study suggests that the mum mutation establishes conditions that allow the production of multiple UMs and thus reveals that the UM field is competent for the complete and coordinated development of several adjacent UMs . This level of regional control is not clearly evident when a single UM is present . The comparison of development of wild-type and mum OAs required an extensive reanalysis of stages 4 and 5 of normal oral development . On the basis of current and previous observations, we propose a new and more subdivided staging system for oral development in Tetrahymena.

J Dairy Res, 1985 Feb, 52(1), 197 - 207
Nutritional value of yogurt; Hewitt D et al.; Yogurt, made from fortified skim milk by conventional methods using Streptococcus thermophilus and Lactobacillus bulgaricus, was used in studies of the effect of fermentation on nutritional value of milk . In all experiments, the product was compared with the uninoculated base milk . The concentration of most vitamins was less in yogurt than in milk and was most noticeably so for biotin which was 60% less . The effect on folic acid content was inconsistent . In nutritional experiments with rats, high values for true digestibility, biological value and net protein utilization were obtained for both yogurt and its base milk, only minor differences being apparent between the two materials . In growth tests with rats, yogurt was not found to be consistently superior to the base milk when the milk was subjected to a double heat treatment to reduce bacterial contamination . Yogurt did not confer a nutritional advantage on fresh milk in this respect.

J Clin Microbiol, 1985 Feb, 21(2), 222 - 5
Illness associated with Campylobacter laridis, a newly recognized Campylobacter species; Tauxe RV et al.; Campylobacter laridis, a recently described thermophilic Campylobacter species found principally in seagulls, has not previously been linked to illness in humans . Six clinical isolates of this species were referred to the national campylobacter reference laboratory in 1982 and 1983 . Each isolate was confirmed by biochemical characterization and by DNA relatedness studies . The six isolates were obtained during an illness: enteritis in four, severe crampy abdominal pain in one, and terminal bacteremia in an immunocompromised host in one . The infections occurred in persons 8 months to 71 years old . Neither the geographic distribution nor the reports of the patients suggest that seagulls played a direct role in the epidemiology of these infections . This potential human enteric pathogen appears to be clinically, epidemiologically, and microbiologically similar to Campylobacter jejuni and may be mistaken for it if nalidixic acid susceptibility screening is not routinely performed.

J Infect Dis, 1985 Feb, 151(2), 227 - 35
Susceptibility of Campylobacter isolates to the bactericidal activity of human serum; Blaser MJ et al.; Although Campylobacter jejuni and related thermophilic organisms are more common human pathogens than are Campylobacter fetus, most bloodstream or systemic isolates are C . fetus . To understand the pathophysiology related to this observation, the authors studied susceptibility to the bactericidal activity of normal human serum of Campylobacter coli, C . jejuni, and C . fetus isolates from feces and blood . In standardized assays, 10 of 15 C . jejuni and related isolates showed 90% kill (mean, 90.6% +/- 5.9); under more stringent conditions, the relatively resistant strains were completely killed . In contrast, all C . fetus strains were highly serum resistant under both standard and stringent conditions . Killing of C . jejuni was ablated by heating serum to 56 C but restored by addition of complement . Both classical and alternative complement pathways may contribute to killing, and adsorption studies demonstrated antibody dependence . Serum resistance may permit systemic infection by C . fetus, whereas complement- and antibody-mediated serum sensitivity of C . jejuni may account for the relative infrequency of systemic invasion.

Cell, 1985 Feb, 40(2), 371 - 80
The Tetrahymena rRNA intron self-splices in E . coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function; Waring RB et al.; We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E . coli as the host . A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor . Plaque phenotypes correlate well with levels of excised intron RNA . Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S . All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important . The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function.

J Protozool, 1985 Feb, 32(1), 6 - 9
Processing of digestive vacuoles in Tetrahymena and the effects of dichloroisoproterenol; Fok AK et al.; The digestive-lysosomal system in Tetrahymena has been extensively studied; however, the various vacuole stages and the existence of a required processing period prior to defecation have not been clearly defined . In this study the presence of such a required processing period and the rate of DV defecation in Tetrahymena thermophila were determined . Like the cycle in Paramecium, a digestive cycle in Tetrahymena consisted of two periods: the processing period was 45 min and the defecation period was approximately 2 h, making the complete cycle approximately 3 h . During the defecation period vacuole egestion followed the kinetics of a first-order rate reaction and had a rate constant of 0.0187/min and a t1/2 of 37 min (82 min into the cycle) . Using the naphthol AS-TR phosphate-hexazotized rosanilin method to visualize acid phosphatase activity at the light microscopic level, DVs became positive beginning at 10 min . The number of positive DVs increased to a maximum of 13% when DVs were 20-min old and declined to 5-7% beyond 30 min . Although dichloroisoproterenol (DCI) has been reported by others to stimulate vacuole defecation, we found it inhibited the defecation rate . The extent of inhibition depended on the age of the DVs when exposed to DCI . Vacuole formation was completely blocked in cells preexposed to 40 microM DCI for only 10 min; however, upon further exposure, cells could recover from this inhibition . The time required for complete recovery increased with increasing DCI concentrations . If DCI was given to cells simultaneously with latex beads, it was found to exert a dose-dependent inhibitory effect on DV formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1985 Feb 1, 236(2), 567 - 75
The varied responses of different F1-ATPases to chlorpromazine; Bullough DA et al.; The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined . While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature . Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C . Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C . At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature . Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5 . Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation . Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5 . Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA . The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison . Chlorpromazine protected MF1 and EF1 against cold inactivation . Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine . Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard . Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP.

Nucleic Acids Res, 1985 Jan 11, 13(1), 73 - 87
DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila; Harrison GS et al.; We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila . DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation . Anlagen DNA was first detectably methylated at GATC sites 3-5 hours after its synthesis . Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation . The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells . Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time . Upon pair separation, less than 20% of old macronuclear DNA remained . A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing anlagen.

Nucleic Acids Symp Ser, 1985, (16), 225 - 8
Two types of tRNA(Gm)methylase found in extreme thermophile, Thermus thermophilus strains HB 8 and HB 27; Hori H et al.; There are two distinct strains HB 8 and HB 27 in an extreme thermophile, Thermus thermophilus, and both strains have their own tRNA(Gm)methylases, which specifically methylates the 2'-OH of the ribose ring in the D loop of tRNA . The Gm-methylases are very similar with respect to the recognition mechanism of substrate tRNA and the molecular weight, but differ in the temperature dependency of the enzyme activity . Gm-methylase from strain HB 8 possesses its activity even at low temperature (40 degrees C), whereas that of strain HB 27 shows very low activity at the temperature and increases the activity as the incubation temperature is raised . Amino acid compositions of both the enzymes are very similar except for Glx and Asx, but the content of secondary structure is very different as judged by circular dichroism.

Antonie Van Leeuwenhoek, 1985, 51(2), 193 - 201
Thermostability of enzymes of the tricarboxylic acid cycle of Bacillus coagulans; Jones MV et al.; The thermostability of four enzymes of the tricarboxylic acid cycle has been studied in the facultative thermophile, Bacillus coagulans . Although isocitrate dehydrogenase appeared to be more temperature-sensitive in whole-cell extracts of cultures grown at 30 degrees C compared with that in cultures grown at 55 degrees C, this difference could be largely eliminated by the removal of cell-wall material . The specific activity of each of the enzymes examined was approximately threefold higher in cultures grown at 55 degrees C than in those grown at 30 degrees C . The maximum temperature, Arrhenius plot and effect of stabilizing agents for each enzyme were examined and found to be independent of growth temperature . Sodium chloride (10% w/v) was an effective protective agent for fumarase, aconitase and malate dehydrogenase . Protection from thermal denaturation of isocitrate dehydrogenase, aconitase and fumarase but not malate dehydrogenase was also given when the enzymes were heated in the presence of their substrates . These results are discussed in light of the generalized theories of facultative thermophily which have been proposed.

Antonie Van Leeuwenhoek, 1985, 51(2), 155 - 65
Temperature-dependent lipid content and fatty acid composition of three thermophilic bacteria; Aerts JM et al.; The lipid content and fatty acid composition of a strain of Bacillus caldolyticus and of two facultative thermophiles (B . flavothermus and strain NZ-2) were analysed after growth at different temperatures . In all three strains the amount of membrane, as a fraction of total cellular dry mass, was found to increase with temperature, however, in varying degrees . Changes of lipid content and protein/lipid ratio in B . caldolyticus between 60 degrees C and 100 degrees C and in strain NZ-2 between 45 degrees C and 70 degrees C were minor; in B . flavothermus the alterations in the 50 degrees C-70 degrees C range were more pronounced . The same was found for changes observed in the phospholipid/total lipid and phospholipid/membrane ratios, and also in the amounts of individual phospholipids . The alterations of the fatty acid composition were most significant in B . caldolyticus, especially between 80 degrees C and 95 degrees C . In contrast, the main changes in B . flavothermus and NZ-2 were found to occur between 30 degrees C and 50 degrees C, and between 45 degrees C and 60 degrees C, respectively . Based on these data, strain NZ-2 could be characterized as the least and B . flavothermus as the most versatile of the three organisms.

Int J Biochem, 1985, 17(5), 661 - 3
Artificial and natural thermostabilization of subunit enzymes . Do they have similar mechanism?
Trubetskoy VS, Torchilin VP.
Rabbit skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was stabilized by intramolecular intersubunit crosslinking with diimidoesters . Half-inactivation temperature for optimal cross-linker-treated enzyme preparation increased by 11 degrees C . Stabilization effect correlated with the content of crosslinked fractions in enzyme preparation, as proved by SDS gel-electrophoresis . It is proposed that artificial crosslinks stabilize the enzyme in a similar fashion to salt bridges in the thermophilic bacteria enzymes, i.e . preventing dissociation into inactive subunits.

Comp Biochem Physiol B, 1985, 80(3), 485 - 7
Electron microscopic mapping of Thermus thermophilus RNA polymerase binding sites on plasmid pBR322; Gonzalez B et al.; The binding of RNA polymerase from the extreme thermophile T . thermophilus HB8 to plasmid pBR322 was measured by electron microscopy . DNA-protein complexes were prepared at 35 and 60 degrees C . At both temperatures the enzyme binds strongly to sites which coincide with promoters P1, P2, P3 and P4 present in pBR322 . At 60 degrees C, an additional binding site appears, which is located between P3 and P4 . There is a high degree of correlation between RNA polymerase binding sites and the location of A-T rich regions on pBR322 DNA.

Comp Biochem Physiol B, 1985, 80(4), 827 - 9
Changes in ornithine decarboxylase activity and polyamine levels during the growth of Tetrahymena thermophila cultures; Yao KM et al.; Ornithine decarboxylase activity and polyamine levels were determined at various growth phases of Tetrahymena thermophila cultures . Enzyme activity and intracellular polyamines increased in exponentially growing cells and peaked just before the stationary phase . Putrescine was the predominant polyamine and spermidine and spermine concentrations were low throughout . The increase in putrescine level can be totally accounted for by the enzyme activity detected, provided that there is an ample supply of the precursor, L-ornithine.

Mol Cell Biol, 1985 Jan, 5(1), 93 - 8
Elimination of micronuclear specific DNA sequences early in anlagen development; Brunk CF et al.; After conjugation in Tetrahymena thermophila, the old macronuclei degenerate, and new macronuclei (anlagen) develop . During anlagen development a number of DNA sequences found in the micronuclear genome (micronuclear limited sequences) are eliminated from the anlagen . A cloned copy of a repetitive micronuclear limited sequence has been used to determine the developmental stage at which micronuclear limited sequences are eliminated . DNAs from anlagen of various developmental stages were examined by Southern analysis . It was found that micronuclear limited sequences are present in 4C anlagen and essentially absent in 8C and 16C anlagen . The precipitous loss of these sequences in the 8C anlagen rules out under-replication as the mechanism for the loss and suggests that these sequences are specifically degraded early during anlagen development.

J Appl Bacteriol, 1985 Jan, 58(1), 77 - 86
Menaquinone composition of mycolic acid-containing actinomycetes and some sporoactinomycetes; Collins MD et al.; The menaquinones of 141 actinomycetes representing the genera Caseobacter, Mycobacterium, Nocardia, Rhodococcus and some related taxa lacking mycolic acids were examined by mass spectrometry . The mycolic acid-containing strains were assigned to four groups on the basis of the predominant isoprenologue detected: Rhodococcus coprophilus, R . equi, R . erythropolis, R . globerulus, R . rhodnii, R . rhodochrous and R . ruber contained dihydrogenated menaquinones with eight isoprene units; Nocardia asteroides, N . brasiliensis, N . carnea, N . otitidis-caviarum and N . transvalensis contained tetrahydrogenated menaquinones with eight isoprene units; Caseobacter polymorphus, R . bronchialis, R . rubropertinctus and R . terrae and representatives of twenty-one approved species of Mycobacterium contained dihydrogenated menaquinones with nine isoprene units; a single strain of 'Mycobacterium album', contained unsaturated menaquinones with nine isoprene units . Actinomycetes containing meso-diaminopimelic acid, arabinose and galactose in the wall peptidoglycan but lacking mycolic acids were recovered in two groups: tetrahydrogenated menaquinones with eight isoprene units were the main components from 'Nocardia' autotrophica and Pseudonocardia thermophila whereas Saccharopolyspora hirsuta and Pseudonocardia spp . contained tetrahydrogenated menaquinones with nine isoprene units . Promicromonospora citrea and 'skin coryneforms' with LL-diaminopimelic acid and glycine in the wall peptidoglycan also contained tetrahydrogenated menaquinones with nine isoprene units as the major isoprenologue . In contrast, representatives of the genera Kitasatoa, Microellobosporia, Streptomyces and Streptoverticillium were characterized by the presence of complex mixtures of tetra-, hexa- and octa-hydrogenated menaquinones with nine isoprene units . The menaquinone data correlate well with other developments in actinomycete systematics and confirm earlier suggestions that menaquinone analyses are of value in both the classification and identification of actinomycetes . Indeed, the data suggest that minimal descriptions of wall chemotype IV taxa should ideally include information on menaquinone composition.

J Cell Physiol, 1985 Jan, 122(1), 155 - 8
Utilization of iron complexes in an animal cell; Rasmussen L et al.; The ciliate protozoan Tetrahymena thermophila was grown in synthetic nutrient medium in the absence of the iron chelator citrate . Utilization and toxicity of various iron compounds or complexes in iron-starved cells were assessed from the number of cell doublings obtained within a standard time . The compounds tested included complexes formed between ortho-phosphates and two forms of ferric hydroxides, native and cationized ferritin, and tris-acetylacetonato Fe(III) . The ferric hydroxo ortho-phosphate particles are toxic and can be removed from the medium by Millipore filtration . Uptake of ferritin and tris-acetylacetonato-Fe(III) is independent of food vacuole formation and seems to occur by micropinocytosis and by plasma membrane translocation, respectively.

J Mol Evol, 1985, 22(4), 351 - 60
Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen; Hamilton PT et al.; A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacterium Methanobacterium thermoautotrophicum delta H has been cloned and sequenced . This DNA complements mutations in both the purE1 and purE2 loci of Escherichia coli . The sequence of the M . thermoautotrophicum DNA predicts that complementation in E . coli results from the synthesis of a polypeptide with a molecular weight of 36,249 . A polypeptide apparently of this molecular weight is synthesized in E . coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA . We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogen Methanobrevibacter smithii . The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology . The genome of M . thermoautotrophicum has a molar G + C content of 49.7%, whereas the genome of M . smithii is 30.6% G + C . Conservation of encoded amino acids while accommodating the very different G + C contents is accomplished by use of different codons that encode the same amino acid . The majority of base changes occur at the third codon position . The intergenic regions of the cloned M . thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters . Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene from M . smithii maintains a codon usage that conforms to the RNY rule.

Eur Biophys J, 1985, 12(1), 19 - 24
A comparison of the translational diffusion of a normal and a membrane-spanning lipid in L alpha phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers; Vaz WL et al.; We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L alpha phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE) . The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus . The translational diffusion was examined between about 15 degrees and 45 degrees C . It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is approximately 2/3 that for NBD-POPE which spans only one monolayer of the bilayer . The result is interpreted in terms of existing models for translational diffusion in lipid membranes.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1985, (4), 84 - 8
{Distribution of microorganisms lysing Pseudomonas aeruginosa and Staphylococcus aureus in the system of sewage waters}; Seliavko AV et al.; The regularities of lytic microorganisms distribution in domestic sewage have been studied . A reproduction of mesophilic gram-negative bacteria producing lytic substances against Pseudomonas aeruginosa and Staphylococcus aureus has been shown to take place at mechanical cleaning stages . In the primary sediment trap the number and the relative content of microorganisms lysing P . aeruginosa at mean temperature and the number of microorganisms lysing S . aureus are maximum . The number of gram-positive sporogenous bacteria lysing P . aeruginosa under conditions close to thermophilic does not change considerably till the secondary sediment trap and remains comparatively high . Certain stages of purification can be regarded as a source of microorganisms producing lytic substances.

Comp Biochem Physiol B, 1985, 80(4), 817 - 9
Comparison of the immunochemical responses by anti-NADPH-cytochrome c reductase of Tetrahymena pyriformis (strain NT-1) in protozoan cells and mammalian liver microsomes; Fukushima H et al.; Comparison of the microsomal NADPH-cytochrome c reductase activities in the four Tetrahymena cells (pyriformis, strain GL and NT-1; thermophilia; ISO) and rat liver was studied . The reductase activity in strain NT-1 was lowest among four Tetrahymena cells grown at 24 degrees C . Rabbit antibody was prepared against the purified NADPH-cytochrome c reductase from Tetrahymena pyriformis (strain NT-1) microsomes . Microsomal NADPH-cytochrome c reductase activities in various Tetrahymena cells were inhibited in proportion to the amount of antibody added, in the order of GL greater than NT-1 greater than thermophilia greater than ISO . No inhibition of reductase activity by antibody was observed in rat liver microsomes.

Curr Genet, 1985, 9(6), 441 - 5
Formation and stability of linear plasmids in a recombination deficient strain of yeast; Zakian VA et al.; Natural termini from macronuclear DNA of the ciliated protozoans Tetrahymena thermophila and Oxytricha fallax can support telomere formation yeast . However, plasmids carrying these ciliate termini are modified by the addition of DNA which hybridizes to the synthetic oligonucleotide poly {d(C-A}), a sequence which also hybridizes to terminal restriction fragments from yeast chromosomes but not to Tetrahymena or Oxytricha macronuclear DNAs . Thus, in yeast, the creation of new telomeres on ciliate termini involves the acquisition of yeast-specific terminal sequences presumably by either recombination or non-templated DNA synthesis . The RAD52 gene is required for the majority of yeast mitotic and meiotic recombination events . Moreover, the absence of an active RAD52 gene product results in high rates of chromosome loss . Here we demonstrate that terminal restriction fragments from Tetrahymena macronuclear ribosomal DNA (rDNA) support the formation of modified telomeres in a yeast strain carrying a defect in the RAD52 gene . Moreover, linear plasmids bearing these modified ciliate termini are stably propagated in rad52- cells.

J Free Radic Biol Med, 1985, 1(3), 233 - 7
Paraquat toxicity and effect of hydrogen peroxide on thermophilic bacteria; Allgood GS et al.; Paraquat (PQ++) increased cyanide-resistant univalent respiration in cell suspensions of five strains of obligately thermophilic bacteria . PQ++ was reduced by an NADH: or NADPH:paraquat diaphorase and selectivity for NADH, NADPH, or both electron donors varied among the thermophiles . Superoxide anion production that was dependent on the presence of PQ++ was shown by following the superoxide dismutase-inhibitable reduction of cytochrome c . In addition, the PQ++-dependent formation of hydrogen peroxide from superoxide anion was evident in two of the thermophilic strains . Catalase synthesis was induced by adding hydrogen peroxide to the growth medium of the thermophiles . The induction of catalase to eliminate hydrogen peroxide appears to be an important response of these thermophilic bacteria to oxygen toxicity.

Adv Biophys, 1985, 20, 13 - 29
Effect of amino acid substitutions on conformational stability of a protein; Yutani K et al.; This paper reviews studies on thermostable proteins from thermophilic bacteria and on mutant proteins of human hemoglobin, tryptophan synthase alpha-subunit of E . coli, T4 phage lysozyme, and phage lambda repressor with respect to the role of the constituting amino acid residues in stabilization of conformation . The stability of a protein is easily affected by single amino acid substitutions, by which the protein undergoes change(s) of one or more of the following: a hydrogen bond, a salt bridge, a hydrophobic interaction, the volume of the residue, a disulfide bond, or the relative position of two aromatic rings.

Am J Ind Med, 1985, 8(3), 241 - 51
Health hazards of natural and introduced chemical components of boatbuilding woods; Jagels R; The major components of untreated wood--cellulose, hemicellulose, and lignin--have not been implicated as toxicants, but extractive substances, especially in heartwood, can be toxic . Decay-resistant woods are more likely to contain irritants or sensitizers than nondurable woods . Short-term exposures to certain wood dusts may result in asthma, conjunctivitis, rhinitis, or allergic dermatitis, but long-term effects may include nasal cancer and Hodgkin's disease . Some thermophilic microorganisms found in wood are human pathogens, and septic splinters (chromomycosis) and inhalation of ascomycete spores from stored wood chips have been implicated in human illnesses . Reconstituted wood can contain formaldehyde resins, which pose health risks in enclosed humid areas . Pentachlorophenol (PCP)-treated wood is particularly toxic--short-term exposures to PCP-treating solutions can lead to aplastic anemia and mortality, while diseases such as Hodgkin's disease are associated with long-term exposures . Since much commercial lumber is dipped in PCP, the separation of the chronic effects of wood dust from PCP exposure is difficult . Chromated copper arsenate (CCA)- and ammoniacal copper arsenite (ACA)-treated wood may leach arsenic . CCA-treated wood is potentially safer, since it contains the pentavalent arsenic, which is a common constituent in the environment . ACA contains the trivalent arsenic, which is more toxic.

Ciba Found Symp, 1985, 111, 40 - 56
Large-scale purification of enzymes; Scawen MD; For an enzyme to be employed as a reagent in any field, be it clinical chemistry or organic synthesis, it must first be purified to a degree that removes any other enzyme capable of catalysing undesirable side-reactions . This may or may not mean purification to homogeneity . For the enzyme to be commercially viable, purification must yield tens or hundreds of grams of protein . On this scale, the availability of sufficient starting material may pose a problem, particularly for animal tissues . For this and other reasons, bacteria may provide the most valuable source of enzymes . Bacteria are readily produced in enormous quantities and, by virtue of their diverse metabolism, contain many enzymes not present in other organisms . Furthermore, the enzymes from thermophilic species often show a desirable increase in stability under the arduous conditions which may be encountered in a reactor . The preparation of enzymes on this scale from bacteria, or any other source, can usually be accomplished by the application of the normal range of techniques available to the protein chemist . Thus precipitation methods, gel filtration, ion exchange and affinity chromatography are all valuable . A major difference between this and normal laboratory-scale scale purification is cost; large-scale enzyme purification requires a considerable investment in equipment, materials and manpower . This cost must be viewed in terms of the value of the product.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 436 - 9
Adolescence in Tetrahymena thermophila; Rogers MB et al.; The development of sexual maturity in Tetrahymena thermophila has been shown to include an intermediate stage, adolescence, during which cells are capable of mating with mature cells but not other adolescent cells . When the progeny of successfully mated cells are grown logarithmically and tested frequently for the ability to mate, they are unable to form mating pairs for about 65 generations . This period is known as immaturity . During the next stage, the progeny pair with mature cells but not with other adolescent cells despite the presence of complementary mating types . Adolescence persists for 20-25 generations before the cells attain maturity, which is defined as the ability to mate with any cell of different mating type . Once paired with mature cells, adolescents successfully complete conjugation . Cytological preparations show that both members of the pair undergo meiosis and form macronuclear anlagen . The proteins synthesized during a mating between adolescents and mature cells are similar to those synthesized during a mating between mature cells as determined by two-dimensional gel analysis . Both the adolescent cell and the mature partner contribute genetic markers to the progeny.

Mol Gen Genet, 1985, 201(1), 65 - 75
Eliminated sequences with different copy numbers clustered in the micronuclear genome of Tetrahymena thermophila; White TC et al.; As the ciliated protozoan Tetrahymena thermophila develops a new macronucleus (MAC) from products of its micronucleus (MIC), several repetitive sequences are eliminated from the MAC genome . Four MIC DNA clones containing repetitive sequences that are eliminated from the MAC were obtained . One clone contains a representative from each of three families of eliminated sequences . One, present in 200-300 copies in the MIC, is almost completely eliminated from the MAC . A second, present in approximately 50 copies in the MIC, is scattered throughout the genome, although up to half of the family members examined could be localized to chromosome 2 . Approximately one tenth of the members of this less repetitive family persist in the MAC while the rest are eliminated . The third type of eliminated sequence has three to four members, all of which are eliminated from the MAC . Three of the members are located on three of the five MIC chromosomes, and one could not be mapped . This sequence is clustered with the other two families of sequences in at least three of the four sites . All three types of eliminated sequences are found in similar arrangements in the MIC of several different inbred strains of T . thermophila.

Gene, 1985, 37(1-3), 131 - 8
Nucleotide sequence of the tetracycline resistance gene of pTHT15, a thermophilic Bacillus plasmid: comparison with staphylococcal TcR controls; Hoshino T et al.; The nucleotide (nt) sequence of the tetracycline resistance (TcR) region (1628 bp) of the Bacillus plasmid pTHT15 was determined . A single open reading frame (ORF), encoding a 458 amino acid (aa) 50-kDal protein (TET), is present after a GTG initiation codon preceded by a ribosome-binding site (RBS-2) . The transcriptional start point, at a position 120 nt upstream from the GTG codon was determined by S1 mapping . This upstream region contains a short ORF (30 aa) which is preceded by RBS-1 . The presence of three inverted repeats, which can form two different conformations of the mRNA very similar to those of the control region of the macrolide-lincosamide streptogramine B resistance gene of pE194 {Horinouchi and Weisblum, Proc . Natl . Acad . Sci . USA 77 (1980) 7079-7083; Gryczan et al., Nucl . Acids Res . 8 (1980) 6081-6097; Shivakumar et al., Proc . Natl . Acad . Sci . USA 77 (1980) 3903-3907}, suggests that the TcR gene is regulated by a translational attenuation mechanism . A Rho-independent transcriptional terminator structure is present immediately after the translational stop codon (TAA) of the TET protein . Comparison of the TET protein with the staphylococcal TcR proteins of pT181 revealed considerable homology.

J Biol Chem, 1984 Dec 25, 259(24), 15234 - 41
Structural elucidation of a unique macrocyclic membrane lipid from a new, extremely thermophilic, deep-sea hydrothermal vent archaebacterium, Methanococcus jannaschii; Comita PB et al.; The membrane lipid of a new deep-sea hydrothermal vent methanogen, Methanococcus jannaschii, was isolated, purified, and structurally characterized . The total lipid extract, amounting to 32.2 micrograms/mg, dry cell weight, was fractionated on silica gel into a neutral lipid and polar lipid fraction . The neutral lipid fraction consisted of a series of isoprenoid hydrocarbons and free (nonphospholipid) alkylglycerol ethers . The polar phospholipid and glycolipid fraction (8.44 micrograms/mg, dry cell weight) was hydrolyzed with methanolic HCl, and the resulting alkylglycerol ethers were analyzed by a combination of chemical and spectroscopic techniques . The hydrolyzed polar lipid was primarily (95%) a unique, macrocyclic glycerol diether, heretofore unknown . High-field (250 MHz) proton nuclear magnetic resonance and infrared spectra of this novel macrocyclic compound are nearly identical and overlapping those of the known bis-(phytanyl)glycerol diether and bis-(diphytanyl)diglycerol tetraether . A field desorption mass spectrum revealed a molecular weight of 650 for the macrocyclic glycerol diether, 2 mass units less than that of bis-(phytanyl)glycerol diether . Degradation of the macrocyclic ether with boron tribromide resulted in diphytanyl dibromide, and further reaction of this dibromide with lithium aluminium hydride resulted in diphytane as determined by gas chromatography-mass spectrometry . The significance of the predominance of this structure in M . jannaschii is discussed . A survey of selected methanogenic Archaebacteria, including three thermophiles, failed to indicate the presence of the macrocyclic glycerol diether in any other microorganism, including two species of order Methanococcales, one species of Methanobacteriales, and three strains belonging to the order Methanomicrobiales.

Eur J Biochem, 1984 Dec 3, 145(2), 283 - 90
L-Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium . Desensitization to fructose 1,6-bisphosphate in the activated state by arginine-specific chemical modification and the N-terminal amino acid sequence; Taguchi H et al.; Heat-stable and fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 {Taguchi, H., Yamashita, M., Matsuzawa, H . and Ohta, T . (1982) J . Biochem . (Tokyo) 91, 1343-1348} . N-terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N-terminal arm region (first 1-20 residues) known for the vertebrate L-lactate dehydrogenases is completely missing in the T . caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD-binding domain . The C-terminal amino acid of the enzyme was phenylalanine . Analysis of the amino acid composition showed that T . caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate L-lactate dehydrogenases . On modification reaction with 2,3-butanedione in the presence of NADH and oxamate, an enhanced activity of the T . caldophilus L-lactate dehydrogenase was obtained independently of fructose 1,6-bisphosphate, and the modified enzyme was desensitized to fructose 1,6-bisphosphate . Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit . Desensitization of the enzyme was inhibited in the presence of fructose 1,6-bisphosphate . A similar desensitization was observed using 1,2-cyclohexanedione instead of 2,3-butanedione . The enzyme was irreversibly modified with 2,3-butanedione and characterized . The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6-bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6-bisphosphate . Fructose 1,6-bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased . Amino acid analysis indicated that only the arginine content was decreased by the modification . These results show that arginine residue(s) exist in the binding site for fructose 1,6-bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.

Int J Pept Protein Res, 1984 Dec, 24(6), 557 - 62
Enzymatically active subunits of Bacillus stearothermophilus enolase bound to Sepharose; Veronese FM et al.; The octameric enolase from Bacillus stearothermophilus was immobilized onto Sepharose 4B activated by the cyanogen bromide reaction under conditions for achieving essentially a single-point attachment . The immobilized enzyme was dissociated with guanidine hydrochloride to yield bound monomeric enolase . The Sepharose-bound subunit regained activity upon removal of the denaturant . It was also possible to rehydribize immobilized monomers to native octamers . Of note, the thermal stability of the immobilized enolase subunit does not appreciably differ from that of the parent soluble octameric enzyme . Thus, these results indicate that single subunits of thermophilic enolase are active and that oligomerization is not a prerequisite for the enzymic activity as well as for thermal stability.

Genetics, 1984 Dec, 108(4), 1035 - 45
Recombination and assortment in the macronucleus of Tetrahymena thermophila: a theoretical study by computer simulation; Doerder FP et al.; The compound nature of the macronucleus of Tetrahymena thermophila presents multiple opportunities for recombination between genes on the same macronuclear chromosome . Such recombinants should be detectable through their assortment at subsequent amitotic macronuclear divisions . Thus, a macronucleus that is initially AB/ab should produce recombinant assortees of the genotypes Ab/aB . Computer simulation shows that, when the recombination frequency is two or fewer times per cell cycle, recombinant assortees are produced at experimentally measurable frequencies of less than 40% . At higher recombination frequencies, linked genes appear to assort independently . The simulations also show that recombination during macronuclear development can be distinguished from recombination in subsequent cell cycles only if the first appearance of recombinant assortees is 100 or more fissions after conjugation . The use of macronuclear recombination and assortment as a means of mapping macronuclear genes is severely constrained by the large variances in assortment outcomes; with experimentally small sample sizes, such mapping is impossible.

Biochem J, 1984 Dec 1, 224(2), 407 - 14
Glucose metabolism in the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus; De Rosa M et al.; Sulfolobus solfataricus is a thermophilic archaebacterium able to grow at 87 degrees C and pH 3.5 on glucose as sole carbon source . The organism metabolizes glucose by two main routes . The first route involves an ATP-dependent phosphorylation to give glucose 6-phosphate, which readily isomerizes to fructose 6-phosphate . In the second route, glucose is converted into gluconate by an NAD+-dependent dehydrogenation; gluconate is then dehydrated to 2-keto-3-deoxygluconate, which, in turn, is cleaved to pyruvate and glyceraldehyde . Each metabolic step has been tested in vitro at 70 degrees C on dialysed homogenates or partially purified fractions; minimal requirements of single enzymes have been evaluated . Identification of the intermediates is based on chromatographic, spectroscopic and/or synthetic evidence and on specific enzymic assays . The oxidative breakdown of glucose to pyruvate occurring in S . solfataricus differs from the Entner-Doudoroff pattern in that there is an absence of any phosphorylation step.

Proc Natl Acad Sci U S A, 1984 Dec, 81(23), 7383 - 7
Specific DNA rearrangements in synchronously developing nuclei of Tetrahymena; Austerberry CF et al.; Specific rearrangement of internal chromosomal regions occurs during development of the somatic macronucleus in Tetrahymena thermophila and results in elimination of germ-line (micronuclear) DNA sequences . The timing and mechanism of genome rearrangement within one particular 9.3-kilobase region, which contains three distinct eliminated sequences, were investigated . Portions of this cloned region were used as probes in Southern hybridization experiments to analyze DNA from developing macronuclei (anlagen) . All three deletions were found to occur predominantly within a 2-hr time period in which the nuclear DNA contents increased from 4C to 8C (1C represents the amount of DNA present in a haploid genome) . The three deletion events can occur independently because intermediate forms, having sustained one or two deletions, were detected . One of the deletions occurs in two alternative ways, resulting in two equally abundant products of different size . Because reciprocal products expected from unequal sister chromatid exchange were not detected, an intramolecular DNA splicing mechanism is suggested.

J Bacteriol, 1984 Dec, 160(3), 1047 - 54
Cyclic AMP levels during induction and repression of cellulase biosynthesis in Thermomonospora curvata; Wood WE et al.; Specific cellulase production rates (SCPR) were compared with intracellular cyclic AMP (cAMP) levels in the thermophilic actinomycete, Thermomonospora curvata, during growth on several carbon sources in a chemically defined medium . SCPR and cAMP levels were 0.03 U (endoglucanase {EG} units) and 2 pmol per mg of dry cells, respectively, during exponential growth on glucose . These values increased to about 6 and 25, respectively, during growth on cellulose . Detectable EG production ceased when cAMP levels dropped below 10 . Cellobiose (usually considered to be a cellulase inducer) caused a sharp decrease in cAMP levels and repressed EG production when added to cellulose-grown cultures . 2-deoxy-D-glucose, although nonmetabolizable in T . curvata, depressed cAMP to levels observed with glucose, but unlike glucose, the 2DG effect persisted until cells were washed and transferred to fresh medium . SCPR values and cAMP levels in cells grown in continuous culture under conditions of cellobiose limitation were markedly influenced by dilution rate (D) . The maxima for both occurred at D = 0.085 (culture generation time of 11.8 h) . When D was held constant and cellobiose concentration was increased over a 14-fold range to support higher steady state population levels, SCPR values decreased about fivefold, indicating that extracellular catabolite accumulation may be a factor in EG repression . The role of cAMP in the mechanism of this repression appears to be neither simple nor direct, since large changes (up to 200-fold) in SCPR accompany relatively small changes (10-fold) in cellular cAMP levels.

Nucleic Acids Res, 1984 Nov 26, 12(22), 8489 - 507
Changes in chromatin structure accompany modulation of the rate of transcription of 5S ribosomal genes in Tetrahymena; Pederson DS et al.; The chromatin structure of a single cluster of six tandemly repeated 5S ribosomal RNA genes (5S genes) in Tetrahymena thermophila has been characterized . Indirect end labeling experiments indicate that the actively transcribed 5S genes in macronuclei are rapidly cut by DNAse I near the putative internal promotor and just 5' to the transcribed region . When cells are starved to reduce 5S gene transcription rates, the DNAse I sensitivity of the intragenic site is reduced relative to the 5' site . In the nontranscribed 5S genes in micronuclei, neither of these sites is hypersensitive to DNAse I . Thus structural alterations accompany both the activation of transcription during macronuclear development and physiological changes in the rate of transcription of the 5S genes . These DNAse I data together with studies using Staphylococcal nuclease suggest that rapidly transcribed 5S genes may not be associated with histones as nucleosomes . In contrast, the genes in starved cell macronuclei appear to be associated with one nucleosome per 280 base pair tandem repeat.

Biochim Biophys Acta, 1984 Nov 26, 767(2), 240 - 7
Kinetics of cytochrome c and TMPD oxidation by cytochrome c oxidase from the thermophilic bacterium, PS3; Nicholls P et al.; Cytochrome caa3 (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes . It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes . Proteoliposomal cytochrome caa3 develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c . Reduction of the a3 centre is blocked in the presence of cyanide . During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms . During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated CuA atom are reduced next . It is concluded that, despite its size and hydrophobic association with the aa3 complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.

Nature, 1984 Nov 15-21, 312(5991), 286 - 8
Dinitrogen fixation by a thermophilic methanogenic bacterium; Belay N et al.; Methanogenic bacteria are known to use NH+4 as a nitrogen source for growth . Previous work with an impure methanogenic culture suggested that a methanogen might fix atmospheric dinitrogen as a nitrogen source, but no further work on this phenomenon has been documented . We have now examined the use of N2 by Methanococcus thermolithotrophicus and find that the organism can grow well, with multiple transfers, in medium having N2 as the source of nitrogen . Control cultures without N2 and containing less than 0.1 mM NH+4 do not grow . Growth yields with N2 are on the average one-third those with NH+4, suggesting that, as in other nitrogen-fixing organisms, this bacterium requires a large amount of ATP for the reduction to occur . After growing in NH+4-containing medium, a long lag is observed before growth begins with N2 as the nitrogen source; the NH+4 levels must be very low for growth to begin . Cells grown in N2-fixing conditions reduce acetylene to ethylene . The discovery of a nitrogen-fixing archaebacterium has important implications for studies on the evolution of nitrogenase, and the fact that M . thermolithotrophicus nitrogenase is active at 64 degrees C suggests that a novel enzyme is involved.

J Mol Biol, 1984 Nov 15, 179(4), 689 - 712
Higher-order structure in the 3'-terminal domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus; Garrett RA et al.; An experimental approach was used to determine, and compare, the higher-order structure within domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus . This domain, which encompasses approximately 300 nucleotides at the 3' end of the RNAs, consists of two large subdomains . The 5' subdomain has been conserved during evolution and appears to be functionally important for the binding of the EF-1 X GTP X aminoacyl-tRNA complex in eukaryotes . The 3' subdomain has diverged widely between eubacteria and eukaryotes, and has produced the 4.5 S RNA in the chloroplast ribosomes of flowering plants . The structure of domain VI within the eubacterial RNAs was probed with chemical reagents in order to establish the degree of stacking and/or accessibility of each adenosine, cytidine and guanosine residue; the double-helical segments were localized with the cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were detected with the single-strand-specific ribonucleases A, T1 and T2 . The data enabled the three secondary structural models, proposed for the E . coli 23 S RNAs, to be examined critically and it was concluded that many of their structural features are correct . Various differences between the models were considered and evidence is provided for additional structuring in the RNA including the stacking of juxtaposed purines into double helices . The 5' subdomain constitutes a compact and resistant structure whereas the 3' subdomain is relatively accessible and contains most of the potential protein binding sites . Moreover, comparison of our results with the published results on 4.5 S RNA suggests that the latter forms essentially the same structure as the 3' subdomain, in contrast to earlier conclusions . A high level of structural conservation has occurred throughout the RNA domain during the evolution of the Gram negative and Gram positive bacteria although the thermophile was generally more stable at base-pairs adjacent to the terminal loops.

J Mol Biol, 1984 Nov 5, 179(3), 559 - 63
ATP-dependent DNA topoisomerase from the archaebacterium Sulfolobus acidocaldarius . Relaxation of supercoiled DNA at high temperature; Mirambeau G et al.; A topoisomerase, able to relax negatively supercoiled DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius . Relaxation was fully efficient in vitro between 70 degrees C and 80 degrees C and was dependent on the presence of ATP and magnesium ions . The enzyme did not exhibit gyrase-like activity and was poorly sensitive to gyrase inhibitors . These properties are reminiscent of eukaryotic type II topoisomerases . However, the enzyme was unable to relax positively supercoiled DNA . This thermophilic enzyme may be used in a variety of ways to study the structure and stability of DNA at high temperature.

Eur J Biochem, 1984 Nov 2, 144(3), 563 - 9
Lipid specificity for the reconstitution of well-coupled ATPase proteoliposomes and a new method for lipid isolation from photosynthetic membranes; Van Walraven HS et al.; The lipid specificity for the enzymatic and proton-translocating functions of a reconstituted thermophilic ATPase complex has been investigated . The proteoliposomes were prepared from the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 and various lipids and lipid mixtures extracted from this organism and from a related mesophilic strain . Some commercial lipids were used as well . An improved method of lipid extraction from chlorophyll-containing membranes is presented . This method is based on acetone extraction and additional chlorophyll separation and results in higher yields, less chlorophyll contamination and a simpler procedure than the conventional methods based on chloroform/methanol extraction . The lipids of Synechococcus 6716 thus extracted were fractionated by thin-layer chromatography . The fatty acyl chain composition of the separated lipids was analyzed by gas chromatography . The coupling quality of the reconstituted ATPase proteoliposomes made of different lipids was tested by a membrane-bound fluorescent probe and uncoupler stimulation of ATP hydrolysis . None of the separated lipids alone was able to produce a well-coupled system . The best results were obtained with the native lipid mixture . The minimum requirement was the combination of a typical bilayer-forming lipid and the non-bilayer (hexagonal II structure)-forming monogalactosyldiacylglycerol . Lipids from the mesophilic Synechococcus 6301 and commercial lipids (also mesophilic) produced poorly coupled vesicles but significant improvement was obtained when thermophilic monogalactosyldiacylglycerol was included . Both the reconstituted and solubilized ATPase complex have a sharp temperature optimum at 50 degrees C . The effect of reconstitution and measurement temperatures on the yield of well-coupled vesicles from different lipid sources was also studied.

Eur J Biochem, 1984 Nov 2, 144(3), 555 - 61
Proton movements and electric potential generation in reconstituted ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716; Van Walraven HS et al.; ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes . The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 {Van Walraven et al . (1983) Eur . J . Biochem . 137, 101-106} . The native cartenoids and added oxonol VI served as probes for the electric membrane potential generated by the net charge separation (negative outside, positive inside) . Their responses, with similar half-times as 9-tetradecylamino-6-chloro-2-methoxyacridine, are sensitive to valinomycin and stimulated by nigericin, as expected . The proton concentrations of extraliposomal and intraliposomal aqueous spaces were monitored by neutral red and cresol red; for internal measurements these pH indicators were trapped inside the vesicles during detergent dialysis . Internal acidification and external alkalinization induced by ATP hydrolysis are inhibited by nigericin and enhanced by valinomycin; at the commonly used higher valinomycin concentrations the neutral red response becomes transient, while the much slower cresol red response is diminished right from its onset . At smaller preset pH gradients both ATP hydrolysis activity and neutral red response are diminished . At increasing MgCl2 concentrations the neutral red responses are slowed down and the cresol red responses are slightly enhanced; this is observed for both internal and external dye responses . Neutral red permeation through the membrane is insignificant under our experimental conditions but is enhanced at temperatures below the lipid-phase transition . In the case of externally added neutral red the non-permeant buffer Hepes is only effective at high MgCl2 concentration, whereas some external cresol red response is visible only at high MgCl2 concentration in the presence of Hepes . The kinetics of the pH indicator and electric potential probe responses clearly distinguish fast interfacial and intra-membrane proton displacements from slow bulk proton equilibration . The data are summarized in a model that supports the importance of localized proton displacements for the primary energy-transducing events.

J Biochem (Tokyo), 1984 Nov, 96(5), 1599 - 607
Purification and characterization of glutamyl-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8; Hara-Yokoyama M et al.; Glutamyl-tRNA synthetase has been isolated from an extreme thermophile, Thermus thermophilus HB8 . The enzyme has been purified to homogeneity by successive chromatography on columns of DEAE-cellulose, DEAE-Sephacel, phosphocellulose and hydroxyapatite . 11.7 mg of purified enzyme has been obtained from 2 kg of T . thermophilus cells, with a purification factor of 600 with an 11% yield . From gel permeation chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme is found to be a monomer protein with a molecular weight of 50,000 . The optimum temperature for the aminoacylation of T . thermophilus tRNAGlu is 65 degrees C, and the optimum pH range is 8.0-9.0, in the presence of 5 mM Mg2+ . The Km values for ATP, L-glutamate, and T . thermophilus tRNAGlu are 230 microM, 70 microM, and 0.65 microM, respectively, in the presence of 50 mM KCl and 10 mM MgCl2 at pH 8.0 at 65 degrees C . Escherichia coli tRNA2Glu is also a good substrate with a Km value of 0.60 microM at 65 degrees C . The mole fractions of Arg and Leu residues are higher and that of Asx residues is lower than those of E . coli glutamyl-tRNA synthetase . Glutamyl-tRNA synthetase from T . thermophilus is remarkably thermostable; even after incubation for 9 h at 65 degrees C, 70% of the enzyme activity is retained in the absence of any protecting factors . Such an extremely thermostable enzyme with a low molecular weight will be useful for detailed physiochemical analyses on the molecular mechanism of strict recognition by aminoacyl-tRNA synthetases.

J Cell Biol, 1984 Nov, 99(5), 1669 - 77
Proteolytic processing of h1-like histones in chromatin: a physiologically and developmentally regulated event in Tetrahymena micronuclei; Allis CD et al.; Micronuclei isolated from growing cells of Tetrahymena thermophila contain three H1-like polypeptides alpha, beta, and gamma . Micronuclei isolated from young conjugating cells (3-7 h) also contain a larger molecular weight polypeptide, X, which is being actively synthesized and deposited into these nuclei (Allis, C . D., and J . C . Wiggins, 1984, Dev . Biol., 101:282-294) . Pulse-chase experiments (with growing and conjugating cells) suggested that X is a precursor to alpha and that alpha is further processed to gamma and a previously undescribed and relatively minor species, delta . These precursor-product relationships were supported by cross-reactivity with polyclonal antibodies raised against alpha and peptide mapping . While beta consistently became labeled under chase conditions (both in growing and mating cells), it was not clear whether it is part of the vivo processing event(s) which interrelates X, alpha, gamma, and delta . Beta was not recognized by alpha antibodies . Despite this uncertainty, these results suggest that proteolytic processing serves to generate significant changes in the complement of H1-like histones present in this nucleus.

Biochem J, 1984 Nov 1, 223(3), 809 - 13
Pulsed cytochrome c oxidase from the thermophilic bacterium PS3; Sone N et al.; A caa3-type terminal cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3 containing three subunits showed conversion from resting into pulsed form . Upon pulsing (reduction and re-oxidation), the cytochrome c oxidase activity increased over 10-fold . This enhanced activity of the pulsed enzyme gradually decayed . Addition of phospholipids, necessary for the enzyme activity, did not affect this decay process . Small changes in the absorption spectrum were observed for the resting-into-pulsed transition and for H2O2 ligation to the pulsed enzyme . The e.p.r . spectrum of the resting enzyme was very similar to that of mitochondrial enzyme, but the transient g = 5, 1.78 and 1.69 set of e.p.r . signals, associated with the pulsed bovine heart oxidase, were not observed in the case of pulsed bacterium-PS3 enzyme.

J Biochem (Tokyo), 1984 Nov, 96(5), 1625 - 32
Selective utilization of 2-thioribothymidine- and ribothymidine-containing tRNAs by the protein synthetic systems of Thermus thermophilus HB 8 depending on the environmental temperature; Watanabe K et al.; An extreme thermophile, Thermus thermophilus HB 8, contains two types of tRNAs, T- and S2T-containing tRNAs . Their relative content changes depend on the growth temperature of the bacterial cells (1-3) . To elucidate the reason why the extreme thermophile possesses the two types of tRNAs, an attempt was made to clarify how these tRNAs are utilized in in vivo protein synthetic systems of the bacteria cultured at different temperatures . First, a method was developed to isolate active polysomes from the thermophile cells cultured at 55 degrees C, 65 degrees C, and 77 degrees C . Then, tRNAs were separated from the polysomes and the T- and S2T-contents of the tRNAs were determined by HPLC . The relative content of S2T-tRNAs in the polysomes from 77 degrees C cells was much higher than that in bulk tRNAs from whole cells cultured at the same temperature, but the situation was reversed in 50 degrees C cells . These results clearly show that the protein synthetic systems of the thermophile have some selection mechanism to utilize either T- or S2T-containing tRNAs preferentially depending on the environmental temperature.

Biochemistry, 1984 Oct 9, 23(21), 5004 - 9
Kinetics of ATP hydrolysis by F1-ATPase and the effects of anion activation, removal of tightly bound nucleotides, and partial inhibition of the ATPase by covalent modification; Wong SY et al.; Eadie-Hofstee plots (v/{S} vs . v) of the kinetics of ATP hydrolysis by purified bovine heart mitochondrial F1-ATPase (MF1) over a substrate (MgATP) concentration range of 1-5000 microM were curvilinear, indicating negative cooperativity with respect to {MgATP} as originally shown by Ebel & Lardy (1975) {Ebel, R . E., & Lardy, H . A . (1975) J . Biol . Chem . 250, 191-196} . The data were computer analyzed for the best fit of the least number of straight lines, each representing a different apparent Km and Vmax . The best fits for MF1 and TF1 from the thermophilic bacterium PS3 were three lines in each case . The upper limits of the apparent Km values for MF1 were of the order of 10(-6), 10(-4), and 10(-3) M, and the corresponding apparent Vmax values (per minute per milligram of protein) were in the range of micromoles or less for the lowest Km line and decamicromoles for the other two . The results for TF1 were very similar . The presence of an activating anion (10 mM KHCO3) in the MF1 assay medium increased the overall Vmax by about 50% and eliminated the high Km but had essentially no effect on the intermediate and low Km's, indicating retention of negative cooperativity in the corresponding substrate concentration range . Kinetic data for MgITP as substrate also yielded two Km values (in the absence of KHCO3) differing by about 10(4)-fold . The relationship between {14C}dicyclohexylcarbodiimide {( 14C}-DCCD) binding to MF1 and activity inhibition was linear up to approximately 1 mol of DCCD bound/mol of MF1 . At this point, the degree of inhibition was about 95%.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1984 Oct, 4(10), 2170 - 9
Starved Tetrahymena thermophila cells that are unable to mount an effective heat shock response selectively degrade their rRNA; Hallberg RL et al.; Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock . An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins . Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells . Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment . We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment . However, selective rRNA degradation occurred in the nonsurviving cells during the heat shock and this presumably accounted for their inviability . A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.

J Appl Bacteriol, 1984 Oct, 57(2), 263 - 71
Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products; Chopra AK et al.; Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products . After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified . Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B . coagulans, five were B . circulans and four were B . licheniformis . Skim milk powder contributed the maximum number of B . stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%) . When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65 degrees C for 30 min . Protease of B . stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70 degrees C for 30 min.

J Bacteriol, 1984 Oct, 160(1), 413 - 20
Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110; Matsumura M et al.; The product of a kanamycin resistance gene encoded by plasmid pTB913 isolated from a thermophilic bacillus was identified as a kanamycin nucleotidyltransferase which is similar to that encoded by plasmid pUB110 from a mesophile, Staphylococcus aureus . The enzyme encoded by pTB913 was more thermostable than that encoded by pUB110 . In view of a close resemblance of restriction endonuclease cleavage maps around the BglII site in the structural genes of both enzymes, ca . 1,200 base pairs were sequenced, followed by amino-terminal amino acid sequencing of the enzyme . The two nucleotide sequences were found to be identical to each other except for only one base in the midst of the structural gene . Each structural gene, initiating from a GUG codon as methionine, was composed of 759 base pairs and 253 amino acid residues (molecular weight, ca . 29,000) . The sole difference was transversion from a cytosine (pUB110) to an adenine (pTB913) at a position + 389, counting the first base of the initiation codon as + 1 . That is, a threonine at position 130 for the pUB110-coded kanamycin nucleotidyltransferase was replaced by a lysine for the pTB913-coded enzyme . The difference in thermostability between the two enzymes caused by a single amino acid replacement is discussed in light of electrostatic effects.

Biochem Biophys Res Commun, 1984 Sep 28, 123(3), 1040 - 6
Identification of an essential lysine residue in the beta subunit of the F1-ATPase from the thermophilic bacterium, PS3, using 7-chloro-4-nitro{14C}benzofurazan; Andrews WW et al.; When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro{14C}benzofurazan ({14C}Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of {14C}Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme . After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs . at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared . Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of {14C}Nbf-N-Lys per mol . A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues . The amino acid sequence immediately around the lysine residue labeled with {14C}Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G.. . .

J Chromatogr, 1984 Sep 28, 301(1), 199 - 219
Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA; Gehrke CW et al.; Improved, highly accurate high-performance liquid chromatographic methods for the measurement of the major and modified nucleosides in enzymatic digests of DNA using a single column are described . Four high resolution separation protocols (isocratic, binary, ternary and high speed) with specifically improved selectivity for 5-methyldeoxycytidine (m5dCyd) from Ade, dIno and Guo are presented . From a detailed study of the various factors contributing to the precision and accuracy of the measurement, optimized conditions and quantitative protocols were established . The ternary buffer allows for the first time the determination of N6-methyldeoxyadenosine (m6dAdo) in the same chromatographic analysis with the other deoxyribonucleosides . The binary system allows quantitation of the absolute amounts of each ribo- and deoxyribonucleoside as well as the mole % of each as the second buffer elutes 5'dA and the internal standard 8-bromoguanosine . The isocratic system allows precise quantitation of the mole % of each ribo- and deoxyribonucleoside while eliminating the need for buffer change valves, buffer cycling and column re-equilibration . Also, a high-speed isocratic system is described which permits separation of the deoxyribonucleosides in 6 min . The quantitative, enzymatic hydrolysis of DNA was evaluated by comparing a 40-h, three-enzyme system with a 4-h, two-enzyme procedure . The latter protocol proved to be an excellent hydrolysis method . These high resolution liquid chromatography techniques provide the most precise, sensitive and accurate measurement of m5dCyd available, in a straightforward method using as little as 1 microgram of DNA, and have allowed us to demonstrate: the existence of tissue-specific differences in levels of m5dCyd in DNA of humans, monkeys, rats and mice; that m5dCyd levels in DNA change during fetal development; that genomic undermethylation of DNA is correlated with cancer and the presence of m6dAdo in DNA of thermophilic organisms.

Biochem J, 1984 Sep 15, 222(3), 679 - 84
Putrescine biosynthesis in Tetrahymena thermophila; Yao KM et al.; The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures . A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected . CO2 was also liberated from L-arginine, but analyses by t.l.c . and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected . We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.

Nucleic Acids Res, 1984 Sep 11, 12(17), 6763 - 78
RNA structure analysis using T2 ribonuclease: detection of pH and metal ion induced conformational changes in yeast tRNAPhe; Vary CP et al.; We describe the use of an enzymic probe of RNA structure, T2 ribonuclease, to detect alterations of RNA conformation induced by changes in Mg2+ ion concentration and pH . T2 RNase is shown to possess single-strand specificity similar to S1 nuclease . In contrast to S1 nuclease, T2 RNase does not require divalent cations for activity . We have used this enzyme to investigate the role of Mg2+ ions in the stabilization of RNA conformation . We find that, at neutral pH, drastic reduction of the available divalent metal ions results in a decrease in the ability of T2 RNase to cleave the anticodon loop of tRNAPhe . This change accompanies an increase in the cleavage of the molecule in the T psi C and in the dihydrouracil loops . Similar treatment of Tetrahymena thermophila 5S ribosomal RNA shows that changes in magnesium ion concentration does not have a pronounced effect on the cleavage pattern produced by T2 RNase . T2 RNase activity has a broader pH range than S1 nuclease and can be used to study pH induced conformational shifts in RNA structure . We find that upon lowering the pH from 7.0 to 4.5, nucleotide D16 in the dihydrouracil loop of tRNAPhe becomes highly sensitive to T2 RNase hydrolysis . This change accompanies a decrease in the relative sensitivity of the anticodon loop to the enzyme . The role of metal ion and proton concentrations in maintenance of the functional conformation of tRNAPhe is discussed.

J Biol Chem, 1984 Sep 10, 259(17), 10653 - 6
A purified alanine carrier composed of a single polypeptide from thermophilic bacterium PS3 driven by either proton or sodium ion gradient; Hirata H et al.; An alanine carrier protein was isolated from membranes of the thermophilic bacterium PS3 using ion exchange column chromatography followed by high performance liquid chromatography with a hydroxylapatite column . The final preparation consisted of a single polypeptide, with Mr = 42,500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A polarity index of 33% was calculated from the amino acid analysis . Proteoliposomes reconstituted with the purified alanine carrier carried out an active alanine transport driven by either an electrochemical potential difference of protons or that of sodium ions.

J Biol Chem, 1984 Sep 10, 259(17), 10695 - 9
Manganese and iron superoxide dismutases are structural homologs; Stallings WC et al.; The crystal structure of a tetrameric manganese superoxide dismutase from a thermophilic bacterium, Thermus thermophilus HB8, has been determined at 4.4-A resolution by local averaging of electron density maps calculated by isomorphous replacement . The spatial arrangement of the principal secondary structural features of iron superoxide dismutase is conserved in manganese dismutase . The structural homology is displayed by orienting the polypeptide chain of Escherichia coli Fe dismutase in the electron density map of Mn dismutase . Densities corresponding to bound Mn3+ occur at locations equivalent to the Fe3+ positions in iron dismutase, indicating one metal binding site per chain, or four sites per tetramer . The Mn tetramer, with 222 symmetry, is approximately rectangular in shape and appears to be constructed with only two unique interfaces . One set of interchain contacts closely resembles the dimer interface of Fe dismutase, but the other interface utilizes an inserted polypeptide segment that has no equivalent in Fe dismutase.

Ann Microbiol (Paris), 1984 Sep-Oct, 135B(2), 187 - 98
Isolation and characterization of a new thermophilic Methanosarcina strain (strain MP); Ollivier B et al.; A thermophilic Methanosarcina strain was isolated from a digester fed with water hyacinths and inoculated with ground termites from the Congo . Optimal growth temperature was 55 degrees C . Methane production was at its optimum between pH 6.5 and 7.0 . The bacterium grew on acetate, methanol and methylamines in the absence of growth factors, but could not use H2-CO2 or formate . H2-CO2 inhibited acetate utilisation . Yeast extract and vitamins stimulated growth.

Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1363 - 8
{tRNA(adenine-1-)-methyltransferase from Thermus thermophilus HB8}; Morozov IA et al.; tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) was isolated from the extreme thermophile Thermus thermophilus strain HB8 . The specific activity of the enzyme is about 50 000 and the yield of activity more than 20% . The method of isolation consists of five steps and is valid for isolation of mg quantities of the enzyme . The purified protein preparation is practically homogeneous in SDS-gel electrophoresis, the position of the protein band corresponds to a molecular weight of 25 000 . By gel filtration on Sephadex G-100 the molecular weight of the native protein was found to be 70 000 . These data allow to suggest a subunit structure of the enzyme . The enzyme is highly thermostable and is most active at 80 degrees C . The only activity of the enzyme is to methylate A58 in the T psi X loop of tRNA.

J Clin Microbiol, 1984 Sep, 20(3), 453 - 60
Differentiation of Campylobacter species by protein banding patterns in polyacrylamide slab gels; Ferguson DA Jr et al.; Soluble protein extracts of 37 catalase-positive strains of Campylobacter species were examined by polyacrylamide slab gel electrophoresis (PAGE) . Electrophoretic banding patterns showed good correlation with biochemical tests and with available DNA homology data in distinguishing species of Campylobacter but did not differentiate subspecies or biotypes . PAGE patterns indicated that Campylobacter coli is a distinct species . Furthermore, the PAGE patterns indicated that C . jejuni and nalidixic acid-resistant thermophilic Campylobacter species (C . laridis) are each distinct species . The protein banding patterns of C . fetus subsp . venerealis and C . fetus subsp . fetus strains were distinctly different from those of the three thermophilic species.

J Biochem (Tokyo), 1984 Sep, 96(3), 645 - 50
Chemotaxis in thermophilic bacterium PS-3; Hirota N; Depositing the thermophilic bacterium PS-3 on semi-solid agar plate containing rich medium, several chemotactic rings were formed as in the cases of Escherichia coli and Salmonella typhimurium, indicating that the bacterium is chemotactic . The cells were attracted to L-amino acids: L-alanine, tryptophan, L-aspartate, and glutamate; and to sugars: D-glucose, maltose, D-fructose, and sucrose . In order to find out some sort of methylatable proteins were present, which has been proven to be important in the case of Escherichia coli, the PS-3 cells were labeled with radioactive methionine under conditions in which the protein synthesis had been inhibited . The results showed that the cells contained methylatable proteins of 60,000 to 88,000 daltons . The banding pattern of these methylated proteins was very similar to that of Escherichia coli when the proteins were analyzed by SDS-polyacrylamide gel electrophoresis . In addition, the methyl groups attached onto the proteins were alkaline labile, indicating that the site of methylation was on carboxyl group . The methylation and demethylation reactions of these proteins were affected by the presence of attractants.

J Clin Pathol, 1984 Sep, 37(9), 1007 - 13
Detection of campylobacter by immunofluorescence in stools and rectal biopsies of patients with diarrhoea; Price AB et al.; Rabbit antiserum, elicited by the intravenous injection of a strain of Campylobacter jejuni heated to 100 degrees C, cross reacted strongly with all other thermophilic campylobacters tested as well as with "C pyloridis" and could be detected by indirect fluorescence with labelled anti-rabbit serum . Antisera to formalin killed cells did not do so . The correlation of positive stool culture with positive immunofluorescence of stools and rectal biopsies from patients with diarrhoea was 70-80% . Some inconsistent, weak reactions showing differently shaped organisms have been seen with some strains of Bacteroides fragilis . Wolinella spp reacted weakly, but one strain of Vibrio cholerae tested did not . Other intestinal organisms, commensals, and pathogens tested were negative.

Afr J Med Med Sci, 1984 Sep-Dec, 13(3-4), 111 - 5
A possible new pathogenic Aspergillus isolation and general mycological properties of the fungus; Williams B et al.; A species of Aspergillus was isolated from vomitus and scrapings of the tongue of a patient with a form of respiratory illness . The fungus has since been identified as Aspergillus aculeatus, Iizuka . The fungus grew over a wide range of temperatures, the spores appeared to be thermophilic . Many local foodstuffs supported the growth of the fungus in culture . Ultraviolet light inhibited mycelia growth and sporulation of A . aculeatus . The fungicides brestan, benlate, fundazole and kocide 101 inhibited the growth of the fungus more than daconil, demosen and dithane M-45 . Amphotericin B and sulfadiazine completely arrested the growth of the fungus while sulfamycin, nalidixic acid and kanamycin had no detectable effects.

J Bacteriol, 1984 Sep, 159(3), 1040 - 6
Characterization of cytochrome c3 from the thermophilic sulfate reducer Thermodesulfobacterium commune; Hatchikian EC et al.; A c3 type cytochrome has been purified from the thermophilic, non-spore-forming, sulfate-reducing bacterium Thermodesulfobacterium commune . The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing . A pI of 6.83 was observed . The molecular weight of the cytochrome was estimated to be ca . 13,000 from both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The hemoprotein exhibited absorption maxima at 530, 408.5, and 351 nm in the oxidized form and 551.5 (alpha band), 522.5 (beta band), and 418.5 nm (gamma band) in the reduced form . The extinction coefficients of T . commune cytochrome c3 were 130,000, 74,120, and 975,000 M-1 cm-1 at 551.5, 522.5, and 418.5 nm, respectively . It contains four hemes per molecule, on the basis of both the iron estimation and the extinction coefficient value of its pyridine hemochrome . The amino acid composition showed the presence of eight cysteine residues involved in heme binding . T . commune cytochrome c3 had low threonine, serine, and glycine contents and high glutamic acid and hydrophobic residue contents . The electrochemical study of T . commune cytochrome c3 by cyclic voltammetry and differential pulse polarography has shown that the cytochrome system behaves like a reversible system . Four redox potential values at Eh1 = -0.140 +/- 0.010 V, Eh2 = Eh3 = Eh4 = -0.280 +/- 0.010 V have been determined . T . commune cytochrome c3, which acts as the physiological electron carrier of hydrogenase, is similar in most respects to the multiheme low-potential cytochrome c3 which is characteristic of the genus Desulfovibrio.

Biochim Biophys Acta, 1984 Aug 31, 766(2), 438 - 45
Purification and partial characterization of two cytochrome oxidases (caa3 and o) from the thermophilic bacterium PS3; Baines BS et al.; Two cytochrome oxidases, cytochrome aa3 (EC 1.9.3.1) and cytochrome o, have been purified from the membranes of a thermophilic bacterium, PS3 . The enzymes were solubilized with Triton X-100 and purified to apparent homogeneity on anion-exchange columns . The properties of the three-subunit cytochrome oxidase complex caa3 obtained here are compared with the same enzyme isolated by Sone, N . and Yanagita, Y . (1982) (Biochim . Biophys . Acta 682, 216-226) . On storage, the purified caa3 enzyme undergoes denaturation; a shoulder at 432 nm seen in (CO-reduced)-minus-reduced difference spectra may be due in part to denaturation products of the enzyme . The purified cytochrome o is more stable . At room temperature, the reduced-minus-oxidized difference spectrum shows absorbance maxima at 427 and 559 nm; at 77 K, its alpha-band is split into 554 and 557 nm components . At room temperature, the CO-reduced-minus-reduced spectrum shows troughs at 430 nm and 560 nm . Dissociating polyacrylamide gel electrophoresis suggests that the purified cytochrome o is composed of one type of subunit with an apparent molecular mass of 47 000-48 000 . Metal analysis of the purified enzyme demonstrated the lack of copper . Both oxidases, purified in the presence of Triton X-100, exist in highly polydisperse forms.

J Biol Chem, 1984 Aug 25, 259(16), 10041 - 7
Thermophilic DNA ligase . Purification and properties of the enzyme from Thermus thermophilus HB8; Takahashi M et al.; Thermophilic and thermostable DNA ligase was purified to near homogeneity from the extract of Thermus thermophilus HB8 . The purified enzyme has an isoelectric point at pH 6.6 and consists of a single polypeptide of about 79,000 in molecular weight on the bases of sodium dodecyl sulfate-polyacrylamide gel electrophoresis data and an equilibrium sedimentation method . The enzyme requires divalent cations, Mg2+ or Mn2+, and the optimum concentration of these ions being 5-9 X 10(-3) M and 3-6 X 10(-3) M, respectively . The enzyme also requires NAD as a cofactor . The apparent Km for NAD is 1.85 X 10(-8) M and that of (dT)10 is 1.4 X 10(-4) M . The pH optimum is 7.4-7.6 in Tris-HCl and 8.0 in collidine/HCl buffer . The joining reaction is activated by K+ and NH+4 at a concentration of 2-100 mM and inhibited by Na+ above 25 mM . The optimum temperatures of the joining of thymidylate oligomers in the presence of poly(dA) as a template are 27.5 degrees C for p(dT)s, 34.5 degrees C for p(dT)10, and 37 degrees C for p(dT)12-18 and that of cohesive-end DNA restriction fragments is 24-37 degrees C . The nick-closing activity of the enzyme was observed over a wide range of the temperature from 15 to 85 degrees C and the optimum temperature is 65-72 degrees C . The temperature dependency of ligation with HB8 DNA ligase for various substrates was found to shift to a region of 7-10 degrees C higher than that of T4 DNA ligase and the activity of HB8 DNA ligase decreased remarkably below 4 degrees C . The enzyme was stable for 1 week at 37 degrees C, its activity dropped by 50% within 2 days at 65 degrees C.

FEBS Lett, 1984 Aug 20, 174(1), 20 - 3
Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases from an extreme thermophile Thermus thermophilus HB8: protein structure and Zn2+ binding; Kohda D et al.; Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases have been purified from an extreme thermophile, Thermus thermophilus HB8 . Valyl-tRNA and isoleucyl-tRNA synthetases are found to be monomer proteins (Mr 108000 and 129000, respectively), while methionyl-tRNA synthetase is a dimer protein (Mr 150000) . These enzymes are very similar with respect to amino acid compositions and alpha-helix contents as estimated by circular dichroism analyses . Furthermore, two Zn2+ are tightly bound to each of these synthetases . These data suggest that valyl-tRNA and isoleucyl-tRNA synthetases consist of two domains, each corresponding to the subunit of methionyl-tRNA synthetase.

Arch Biochem Biophys, 1984 Aug 15, 233(1), 299 - 309
Conformational studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU; McLinden JH et al.; Among the various proposals that have been made in attempting to explain the ability of thermophiles to reproduce at high temperatures, there is no doubt that obligate and extreme thermophiles synthesize proteins (and other molecules) that have sufficient intrinsic molecular stability to withstand increased thermal stress . In contrast, the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU has been shown to be quite thermolabile in vitro . Thermal inactivation is not due to loss of bound NAD+ . It has also been shown that the enzymatic activity can be thermostabilized in vitro by increased ionic strength . As previously reported {J . W . Crabb, A . L . Murdock, and R . E . Amelunxen (1975) Biochem . Biophys . Res . Commun . 62, 627; (1977) Biochemistry 16, 4840}, the enzyme loses 94-97% of enzymatic activity after heat treatment at 55 degrees C for 5 min in 0.05 M sodium phosphate buffer (pH 7.1); however, by increasing the ionic strength to 1.8, complete protection was conferred at this temperature . Gel-filtration chromatography has been used to study the initial dissociation and subsequent aggregation of the glyceraldehyde-3-phosphate dehydrogenase after thermal inactivation . Aggregation occurs when the enzyme is heated at 50 degrees or 55 degrees C . Loss of enzymatic activity is correlated with changes in the tertiary structure as measured by the near-uv CD spectrum of the enzyme following heat inactivation, with essential disappearance of the peaks at 263 and 296 nm, and a blue shift of the far-uv spectrum, which is a measure of secondary structure . Estimation of secondary structure of the unheated protein from the far-uv CD data showed the enzyme contains approximately 26% alpha-helix, approximately 21% beta-structure, and approximately 53% disordered structure . Heat treatment at various temperatures resulted in only slight changes of the estimated secondary structure . Increased ionic strength prevents thermal alteration of the CD spectrum in both near- and far-uv regions . The data support the previous proposal that thermolabile enzymes such as the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile B . coagulans are thermostabilized in vivo mainly by the intracellular charged macromolecular environment.

Exp Cell Res, 1984 Aug, 153(2), 287 - 98
Proteolytic processing of micronuclear H3 and histone phosphorylation during conjugation in Tetrahymena thermophila; Allis CD et al.; During vegetative growth, micronuclei of the ciliated protozoan Tetrahymena thermophila contain two electrophoretically distinct forms of H3, H3S and H3F {4, 5} . Of these two forms, H3F is unique to micronuclear chromatin and is derived from H3S by a physiologically regulated proteolytic processing event {5} . While the function of this processing event is not clear, several lines of evidence {2, 5} suggest that it may be related to chromatin condensation during mitosis . In this report pulse-chase experiments have been used to study the processing of H3S into H3F during the sexual phase of the life cycle, conjugation . Our results demonstrate that even though micronuclei divide mitotically (and meiotically) several times during the mating process, processing of H3S into H3F does not occur . Failure of H3S to be converted into H3F during these divisions causes a significant increase in the amount of H3S (relative to H3F) as conjugation proceeds . By 10 h of conjugation, essentially all of the micronuclear H3 is in the form of H3S (also see {3}) . As long as mating cells are maintained under starvation conditions, processing of H3S into H3F does not occur . However, if exconjugants are returned to food and allowed to proceed through the first true cell division following exconjugation, processing of H3S into H3F occurs . Thus, the return of the processing of H3(3) into H3F following conjugation seems to be tightly coupled to a division which is part of a cell division cycle (as appears to be the case with vegetatively growing cells) . The relevancy of these results to the differentiation of new macro- and micronuclei is discussed . H3F is specifically phosphorylated in growing cells, and it has been suggested that this phosphorylation event may be related to chromatin condensation during mitosis {2} . Since in mating cells H3S becomes the more predominant form of H3, the pattern of histone phosphorylation was examined during stages of conjugation where micronuclei are active in mitotic division (6-7 h) . While a low level of phosphate label is observed over H3S in mating cells, more phosphate label is associated with the small amount of H3F which remains in micronuclei at this stage of conjugation . We also observe significant amounts of phosphate label associated with micronuclear H2A, H2B, and H4 and each of the micronuclear H1-like molecules, alpha, beta and gamma.

J Embryol Exp Morphol, 1984 Aug, 82, 67 - 95
Mutational analysis of patterning of oral structures in Tetrahymena . II . A graded basis for the individuality of intracellular structural arrays; Frankel J et al.; The ciliary arrays of the oral apparatus of the ciliated protozoan Tetrahymena thermophila each have their own unique 'pattern signature', which varies little so long as the number of arrays remains the same . In this study, we analyse the consequence of increases in the number of these arrays (membranelles) brought about by certain mutations . In oral apparatuses of mutant cells, the addition of a membranelle is associated with specific alterations in at least one of the other membranelles . The features that are altered include the relative lengths of membranelles, the state of ciliation of basal bodies located at specific positions within these membranelles, and the spatial configurations resulting from displacement of ciliary units during late oral development . The final organization of each membranelle depends upon its relative position along the length of the oral apparatus . This indicates that the membranelles are not individually 'named' by the organism, and suggests that the unit of pattern organization is the membranelle field as a whole . In the Discussion, we consider means for testing whether the same underlying idea might also apply to multicellular systems, such as the vertebrate limb, in which spatially ordered differences appear to be superimposed upon a fundamental repeating pattern.

J Embryol Exp Morphol, 1984 Aug, 82, 41 - 66
Mutational analysis of patterning of oral structures in Tetrahymena . I . Effects of increased size on organization; Frankel J et al.; The oral apparatus (OA) of the ciliated protozoan Tetrahymena thermophila consists of four ordered arrays of ciliary units . In wild-type cells, these arrays are constant in spatial organization and vary little in size except during extreme starvation . Recessive mutations at five gene loci are known to increase the size of the OA . They do this by increasing the length of the ciliary arrays, without affecting their width and often without increasing their number beyond the usual four . Comparison of the oral arrays over a large range of sizes has revealed: (1) that the lengths of the anterior two of three parallel arrays (membranelles) are rather tightly coordinated; (2) that the specific basal body configurations resulting from remodelling of the membranelles are only slightly affected by large changes in lengths of membranelles; and (3) that the third membranelle is restricted to a nearly constant length, except in the very largest OAs in which the structure is lengthened but interrupted by a gap in the middle . This gap may reveal the spatial extent of a putative zone of basal body regression . These phenomena are not specific to any of the genotypes utilized in this investigation; the effect of the mutations is to loosen quantitative restrictions and thus reveal underlying associations and constraints.

Biochem J, 1984 Jul 15, 221(2), 407 - 13
Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351; Khoo TC et al.; Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+ . A variety of metal ions were able to substitute for Ca2+ . Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h . Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme . The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities . Average binding constants of the two types of binding sites were 2.8 X 10(4)M-1 (for the low-affinity sites) and 7.5 X 10(5) M-1 (for the high-affinity sites) . The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase . It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.

Biochem J, 1984 Jul 15, 221(2), 529 - 33
Reaction of caa3-type terminal cytochrome oxidase from the thermophilic bacterium PS3 with oxygen and carbon monoxide at low temperatures; Sone N et al.; Reaction of O2 and CO with a caa3-type terminal cytochrome oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3 grown with high aeration was studied at low temperatures . The CO recombination at the temperature range studied (-50 degrees C to -80 degrees C) followed first-order kinetics with an activation energy of 29.3 kJ/mol (7.0 kcal/mol) . In the presence of O2 at -113 degrees C the photolysed reduced form binds O2 to form an 'oxy' intermediate similar to Compound A . At a higher temperature (-97 degrees C) another intermediate, similar to Compound B, is formed as a result of electron transfer from the enzyme to the liganded O2.

Exp Cell Res, 1984 Jul, 153(1), 236 - 9
Tetrahymena . Adaptation to high iron; Rasmussen L et al.; Fe-starved ciliates Tetrahymena thermophila cease to multiply at Fe(III) concentrations above 10 microM in a synthetic nutrient medium lacking a good iron chelator such as citrate . If, however, the Fe(III) concentration is gradually increased over a series of subcultivations the cells will tolerate up to 300 microM Fe(III) . Our experiments rule out the possibility of genetic selection of Fe-tolerant clones and suggest a physiological type of adaptation.

Ann Microbiol (Paris), 1984 Jul-Aug, 135B(1), 69 - 78
Identification and evolution of the cellulolytic microflora present during composting of cattle manure: on the role of Actinomycetes sp; Godden B et al.; Population changes in cellulolytic microflora were studied during composting of cattle manure . Six bacterial and nine actinomycetes species were isolated . Among the isolates, two actinomycetes, Micromonospora chalcae and Pseudonocardia thermophila, were cellulolytic strains which were numerically dominant and might therefore play a notable role in the degradation of cellulose in cattle manure . Fungi were practically absent during the process, but two cellulose-degrading bacterial species, Sporocytophaga myxococcoides and Cytophaga hutchinsonii, appeared to coexist in equilibrium with actinomycetes during the maturation phase.

Ann Microbiol (Paris), 1984 Jul-Aug, 135B(1), 79 - 89
Growth and cellulase production of Micromonospora chalcae and Pseudonocardia thermophila; Malfait M et al.; Beta(1,4)glucosidases and carboxymethylcellulases were demonstrated in both strains when using carboxymethylcellulose as a carbon source for growth . Beta(1,4)Glucosidases appeared mainly as cell-bound activities, whereas carboxymethylcellulases were evenly distributed between the incubation fluids and the cellular fractions . In both microorganisms, glucose appeared to repress biosynthesis of the enzymes, and cellobiose and carboxymethylcellulose acted as inducers of the cellulase complex.

Antimicrob Agents Chemother, 1984 Jul, 26(1), 53 - 60
Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis; Eustice DC et al.; Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics . Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 microM, respectively, inhibited growth by 50% . A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T . thermophila . Analysis of polysome profiles from paromomycin- and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo {14C} lysine incorporation . In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes . In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin . We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation . Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes . In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.

Ann Intern Med, 1984 Jul, 101(1), 55 - 7
Campylobacter laridis causing bacteremia in an immunosuppressed patient; Nachamkin I et al.; An unusual species, Campylobacter laridis , belonging to the group of nalidixic acid resistant thermophilic Campylobacter species, was isolated from the blood of a 71-year-old man with multiple myeloma, hyperviscosity syndrome, and renal failure . The organism was first recognized in the laboratory by gram-stain reaction and resistance to nalidixic acid . The organism differs from C . jejuni and C . coli by its resistance to nalidixic acid, whereas anaerobic growth in the presence of trimethylamine N-oxide hydrochloride differentiates this organism from other Campylobacter species . Biochemical characterization and DNA homology studies confirmed the identity of this species as being C . laridis . To our knowledge, this is the first recorded case of bacteremia due to C . laridis in humans.

Biochemistry, 1984 Jun 5, 23(12), 2826 - 31
Resonance Raman study of the aa3-type cytochrome oxidase of thermophilic bacterium PS3; Ogura T et al.; Resonance Raman spectra of the aa3-type cytochrome oxidase of thermophilic bacterium PS3, which has a simpler subunit composition than the mitochondrial enzymes but very similar enzymatic properties, are investigated under various conditions and compared with those of mitochondrial enzymes . The intensities of the two marker lines of reduced cytochrome a3 at 1667 and 213 cm-1 had different dependences on the incubation temperatures and pH . With regard to the incubation temperature dependence, the intensity of the 1667-cm-1 line, the peripheral CH = O stretching mode of the a3 heme, behaved in nearly the same way as that of the oxidase activity whereas the intensity of the 213-cm-1 line, the Fe-histidine stretching mode of the a3 heme, exhibited a similar dependence to that of the proton pumping activity . The 213-cm-1 line disappeared upon binding of carbon monoxide, upon raising the pH above 9.2, or after incubating above 55 degrees C . The Raman line at 1611 cm-1, which was recently suggested to probe the proton pump activity {Babcock, G.T., & Callahan, P.M . (1983) Biochemistry 22, 2314-2319}, remained unaltered after incubation at 60 degrees C for 20 min despite a reduction of proton pumping activity to one-third . This argues against the proposed mechanism . The frequencies of the Raman lines were the same for the intact membrane and the isolated enzyme in the reduced state . The Raman spectra of cytochrome oxidase isolated from bacterium, yeast, and bovine heart were different in the lower frequency region below 600 cm-1 but closely alike in the higher frequency region above 1200 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

J Cell Biol, 1984 Jun, 98(6), 2107 - 17
Timing of the appearance of macronuclear-specific histone variant hv1 and gene expression in developing new macronuclei of Tetrahymena thermophila; Wenkert D et al.; Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus . Earlier studies ( Allis , C . D., C . V . C . Glover , J . K . Bowen, and M . A . Gorovsky , 1980, Cell, 20:609-617; and Allis , C . D., Y . S . Ziegler , M . A . Gorovsky , and J . B . Olmsted, 1982, Cell, 31:131-136) demonstrated the existence of a macronuclear-specific histone variant, hv1 , which is enriched in small punctate regions in nucleoli of several mammalian cell lines . These observations suggest that this histone variant is highly conserved in evolution and may be associated with actively transcribed sequences . Despite large differences in structure and function during vegetative growth, macro- and micronuclei are related . During conjugation, the sexual phase of the life cycle in Tetrahymena, postzygotic division products of micronuclei give rise to new micro- and macronuclei, while the old macronucleus moves to the posterior of each cell and is eliminated . In this study using antiserum specific for hv1 , we determined by indirect immunofluorescence the time during conjugation at which hv1 first appears in the developing new macronuclei . In growing, starved, and young mating cells (2-5 h after mixing opposite mating types), only macronuclei are detected with affinity-purified antibodies against hv1 . Newly formed macronuclei are either not stained or only weakly stained in cells in which the old macronucleus is located in the center of the cell . However, new macronuclei are clearly observed in cells in which the old macronucleus has moved to the posterior of the cell (approximately 8 h) . During later stages of conjugation (10-16 h), the intensity of hv1 staining in new macronuclei increases with time corresponding to the increasing DNA content of these nuclei . Disappearance of detectable hv1 from old macronuclei begins nearly 1 h after these nuclei reach the posterior cytoplasm (approximately 9-10 h) and is sometimes complete before these nuclei are eliminated from the cells . Autoradiography of cells labeled for brief periods with {3H}uridine shows that new macronuclei begin to synthesize RNA very soon after the second postzygotic division (approximately 8 h) . During stages when hv1 is clearly detected in new macronuclei, anlagen are active in RNA synthesis . RNA synthesis in old macronuclei ceases very close to the time when RNA synthesis begins in new macronuclei . Thus, the addition of hv1 coincides closely with the transformation of a transcriptionally inactive germinal nucleus into that of a transcriptionally active somatic nucleus . We suspect that addition of hv1 plays a fundamental role in

J Gen Microbiol, 1984 Jun, 130 ( Pt 6), 1399 - 408
Two replication determinants of an antibiotic-resistance plasmid, pTB19, from a thermophilic bacillus; Imanaka T et al.; Two different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus . One replication determinant (designated RepA) was functional only in Bacillus subtilis, whereas the other (designated RepB) functioned in both B . subtilis and Bacillus stearothermophilus . A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1.0 MDal EcoRI fragment of a cryptic plasmid pBSO2 from B . stearothermophilus . The presence of this 1.0 MDal EcoRI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B . stearothermophilus 10(3) to 10(4) times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.

DNA, 1984 Jun, 3(3), 251 - 7
Electron microscopy mapping of Escherichia coli RNA polymerase-binding sites on plasmids from thermophilic bacteria; Gonzalez B et al.; The binding sites of Escherichia coli RNA polymerase to plasmid DNA from extremely thermophilic bacteria have been mapped by electron microscopy . Templates used in these studies included plasmids pTF62 (from Thermus flavus AT62) and pTT8 (from T . thermophilus HB8) and also hybrid molecules constructed by ligation of these plasmids to pBR322 . Although the affinity of the enzyme for heterologous DNA was about one-third of that for pBR322, it was possible to localize preferred binding sites on pTF62 and pTT8 . Six binding sites were identified in pTT8, mapping close to 7, 28, 47, 61, 65, and 81 map units (one unit being equal to 1% of the length of the DNA) . Seven such regions located at 3, 27, 48, 60, 67, 81, and 86 map units were found in pTF62 . RNA polymerase binding sites found in pBR322 coincided with promoters identified previously by electron microscopy analysis of transcriptional complexes prepared in vitro . These data indicate that E . coli RNA polymerase binds preferentially to specific sequences in plasmids from thermophilic bacteria, suggesting possible promoter locations in these plasmids.

J Biochem (Tokyo), 1984 May, 95(5), 1273 - 81
Reversible denaturation of thermophilic malate dehydrogenase by guanidine hydrochloride and acid; Iijima S et al.; Thermophilic malate dehydrogenase {L-malate:NAD+ oxidoreductase, EC 1.1.1.37} was denatured at pH 2.0 with complete loss of enzyme activity but without dissociation to monomers, suggesting the presence of strong intersubunit contact . On the other hand, the enzyme was completely denatured and dissociated to monomers in the presence of 5 M GdnHCl . Inactivation and denaturation of the enzyme by acid and GdnHCl were reversible . Upon dilution of the denaturants, the inactivated enzyme regained enzyme activity and the native structure with high yield (80-90%) . Kinetic analyses of reactivation of the enzyme denatured by GdnHCl and by acid revealed that the reaction obeyed first-order kinetics . The rate constant and Arrhenius activation energy of the reactivation of the acid-inactivated enzyme were almost the same as those of the enzyme inactivated by GdnHCl . These results suggest that the rate-limiting steps in the reactivation processes of the enzyme denatured by GdnHCl and by acid are the same and that a conformational change of the inactive dimer to active dimer is the rate-limiting step in the reactivation reaction.

Arch Microbiol, 1984 May, 138(1), 31 - 6
Thermophilic anaerobic bacteria which ferment hemicellulose: characterization of organisms and identification of plasmids; Weimer PJ et al.; Seven thermophilic anaerobic bacteria which ferment xylan were isolated from natural geothermal features in the western United States . Typically, these strains were Gram-negative non-sporeforming rods with an unusual double-layered cell wall which resembled that observed in Thermobacteroides acetoethylicus . The strains differed from known thermophilic anaerobes in their ability to utilize a very wide variety of carbohydrates and in their ability to grow in a chemically-defined medium and/or at pH 3.5 . Four of the strains contained cryptic plasmids of 1.2 or 1.5 X 10(6) daltons . The taxonomic characteristics of the strains are discussed in terms of their relatedness to those of Thermoanaerobium, Thermoanaerobacter, and Thermobacteroides species.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1984 May, 17(2), 86 - 91
{Spoilage microorganisms encountered in ultra-high temperature processed milk}; Lee CM; 40 strains of aerobic or facultative anaerobic microorganisms were isolated from a total of 37 spoiled ultra high temperature processed milk . 13 of them were identified as the genus Bacillus . They were 6 B . cereus, 5 B . licheniformis, 1 B . brevis and 1 B . pumilus . The other 27 strains were nonsporeforming microorganisms, which included 5 yeasts, 2 Pseudomonas sp., 3 Streptococcus sp., 12 Lactobacillus sp., 1 Shigella sp., 1 Aeromonas sp . and 3 Micrococcus sp . Results indicate that the spoilage of milk sample was caused mainly by the contamination during the filling operation . The Bacillus strains isolated were mesophilic or thermophilic, and some of them, were also psychrotrophic.

J Bacteriol, 1984 May, 158(2), 721 - 6
Formaldehyde oxidation and methanogenesis; Escalante-Semerena JC et al.; Formaldehyde oxidation by cell-free extracts of Methanobacterium thermoautotrophicum was shown to drive methanogenesis from CH3-S-coenzyme M or HCHO under a nonreductive atmosphere of N2 . Under N2 when HCHO was the sole source of carbon and reducing equivalents in the reaction, it underwent oxidation and reduction events (disproportionation), the sum of the reactions being 3 HCHO + H2O----CH4 + 2 HCOO - + 2H+ . This reaction predicts a CH4/HCHO ratio of 1/3, which is in agreement with the experimental finding of 1/2.9 . In extracts of the mesophilic methanogen Methanococcus voltae and the extreme thermophile Methanococcus jannaschii , which exhibited formate dehydrogenase activity, the CH4/HCHO ratio was 1/2 . NADPH stimulated methane formation from HCHO under N2 . An unidentified, oxygen-labile cofactor, the formaldehyde activation factor, present in boiled-cell extract was discovered . Methanopterin , an oxygen-stable molecule, also substituted for boiled-cell extract.

J Biochem Biophys Methods, 1984 May, 9(2), 163 - 9
Purification of a high molecular weight membrane protein by fast protein liquid chromatography: the ATPase complex of a thermophilic cyanobacterium; van Walraven HS et al.; Fast protein liquid chromatography (FPLC) with a strong anion-exchange (Mono Q) column is applied to the purification of a high molecular weight membrane protein . The ATPase complex of the thermophilic cyanobacterium Synechococcus 6716, partially purified by ammonium sulfate precipitation, was fractionated in the presence of the detergent octylglucoside . The ATPase complex containing fractions were eluted by a linear NaCl gradient at about 0.4 M and within 10 min . The FPLC fractions were analyzed for protein and pigment contents and by polypeptide composition . The purest fraction is essentially free of pigments and has a high specific ATP hydrolysis activity (about 1.6 mumol ATP X min-1 X mg protein-1) which is sensitive to N,N'-dicyclohexylcarbodiimide.

Appl Environ Microbiol, 1984 May, 47(5), 933 - 41
Biokinetic analyses of adaptation and succession: microbial activity in composting municipal sewage sludge; McKinley VL et al.; The interactions between temperature and the microbial communities in composting municipal sewage sludge were studied to determine the optimal temperature range for efficient decomposition (stabilization) of the sludge . Information concerning thermophilic successions in such communities was also obtained . Samples were taken from several different temperature areas in a production-scale composting pile throughout the 19-day processing run . Optimum temperatures for microbial activity, determined as the rate of {14C}acetate incorporation into microbial lipids, were determined for each sample . Biomass was determined from the lipid phosphate content of the sample . Maximal activities were generally found in samples coming from lower-temperature areas (25 to 45 degrees C), whereas samples from high temperatures (55 to 74 degrees C) usually had relatively little activity . The temperature giving the optimum activity in samples incubated at a variety of temperatures during the assay tended to increase as the composting time progressed, but never exceeded about 50 degrees C . Many of these temperature response curves were similar in nature to curves reported for purified enzyme systems and pure cultures of bacteria . Comparisons of the apparent energies of activation calculated for different temperature ranges over time also indicated that the overall community was better adapted to higher temperatures during the latter part of the composting run . It was also found that the relationship between the apparent energies of activation and the apparent energies of inactivation (apparent heats of denaturation) consistently changed with sample temperature throughout the composting run, suggesting that the microbial communities from hotter samples were better adapted to high temperatures than those from cooler samples, and vice versa.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1984 Apr 27, 786(1-2), 9 - 17
L-tryptophan 2,3-dioxygenase of a moderate thermophile, Bacillus brevis . Purification, properties and a substrate-mediated stabilization of the quaternary structure; Matsumura M et al.; L-Tryptophan 2,3-dioxygenase (L-tryptophan: oxygen 2,3-oxidoreductase ( decycling ), EC 1.13.11.11) from Bacillus brevis, a moderately thermophilic bacteria, was purified to apparent homogeneity . The enzyme had a molecular weight of 110 000 and consisted of four subunits of equal molecular size . The enzyme exhibited the typical absorption spectra of a protohemoprotein . The amino acid composition and catalytic properties of the thermophilic enzyme were almost similar to those of its mesophilic counterpart from Pseudomonas acidovorans . However, the stabilities of the enzyme differed markedly between the two . The thermophilic enzyme was more resistant to heat and several chemical denaturants . The addition of L-tryptophan protected the enzyme from heat- and SDS- denaturations , and the tryptophan-mediated stabilization was more evident for the thermophilic enzyme . The effect of L-tryptophan on the stabilization of the thermophilic enzyme was more effective in preventing the dissociation of the tetrameric form of the enzyme (i.e . stabilizing it) in the case of the native, as compared to the mesophilic enzyme.

Nucleic Acids Res, 1984 Apr 11, 12(7), 3115 - 26
Comparative structural analysis of eubacterial 5S rRNA by oxidation of adenines in the N-1 position; Pieler T et al.; Adenines in free 5S rRNA from Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus have been oxidized at their N-1 position using monoperphthalic acid . The determination of the number of adenine 1-N-oxides was on the basis of UV spectroscopic data of the intact molecule . Identification of the most readily accessible nucleotides by sequencing gel analysis reveals that they are located in conserved positions within loops, exposed hairpin loops and single-base bulge loops . Implications for the structure and function of 5S rRNA will be discussed on the basis of this comparative analysis.

Exp Cell Res, 1984 Apr, 151(2), 374 - 83
Stage-specific changes in protein synthesis during conjugation in Tetrahymena thermophila; Suhr-Jessen PB; Conjugation in the free-living ciliate Tetrahymena thermophila is an inducible developmental system which results in a synchronized reorganization of the genetic material in both mates of a pair . The cytological events were followed by Feulgen stainings of simultaneously mating cells and protein synthesis was revealed using {35S}methionine pulse labelling and two-dimensional gel electrophoresis . At least 33 proteins, including 24 conjugation-specific proteins, with apparent molecular weights (Mr) between 61 and 200 X 10(3) are stimulated during conjugation . Two slightly acidic proteins (Mr 89 and 73 X 10(3), respectively) are stimulated shortly after mixing of mating-competent cells and mainly before tight pairs are formed . Ten proteins are stimulated during meiosis, and two of these (Mr 90 and 78 X 10(3), respectively) are particularly interesting, since they are highly stimulated and more basic (pI values around 8.5) than most other proteins detected . Twelve proteins are stimulated essentially between pairing and early macronuclear development, three are stimulated from shortly before zygote formation and during the post-zygotic divisions, and six are stimulated during late conjugation, at various parts of macronuclear development . The functions of the conjugation-stimulated proteins are discussed.

J Appl Bacteriol, 1984 Apr, 56(2), 237 - 45
The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat; Garvie EI et al.; Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk . Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously . Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined . Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet . Streptococcus thermophilus but not Lact . bulgaricus grew in the rat diet when tested in vitro . Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates . Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction . The bacterial origin of all this increased activity is discussed . The other lactose-containing diets did not affect enzyme activity to the same degree . Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact . reuteri ) to one which was predominantly homofermentative (Lact . salivarius).

Scand J Work Environ Health, 1984 Apr, 10(2), 115 - 9
Airborne molds and actinomycetes in the work environment of farmer's lung patients in Finland; Kotimaa MH et al.; Occurrence of molds and actinomycetes in the breathing zone of farmers during the handling of hay, straw, or grain was studied with the use of an Andersen sampler on 35 farms in Finland . On 24 farms there was a person with recently diagnosed farmer's lung disease, and on 11 farms people were free of the disease . The total spore concentration and the concentrations of the spores of Thermoactinomyces (T) vulgaris, Micropolyspora (M) faeni, and Aspergillus (A) umbrosus were statistically significantly higher on the farms of patients with farmer's lung than on the disease-free farms . The mean proportions of the spores of thermotolerant and thermophilic microbes were greater on the farms of farmer's lung patients than on the reference farms . T vulgaris was the predominant actinomycete species . Both T vulgaris and A umbrosus were found on all farms of farmer's lung patients, but M faeni on only about half of such farms . The findings match the results of previous microbiological analyses of Finnish moldy hay and serological analyses of Finnish farmer's lung patients . It seems that T vulgaris, not M faeni, may be the main causative agent of farmer's lung in Finland . The possible etiologic role of A umbrosus requires further investigation . Because the farmers often failed to identify the moldiness of the plant material in contrast to researchers, it might be possible, through training, to improve farmers' ability to identify moldiness.

Cell, 1984 Apr, 36(4), 933 - 42
Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena; Palen TE et al.; The chromatin structure of regulatory regions of the extrachromosomal rRNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei . The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II . These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication . The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA . The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region . Comparison of the chromatin structures of the central spacer of T . thermophila and T . pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp . This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome.

J Cell Sci, 1984 Apr, 67, 203 - 15
Isolation and phenotypic characterization of Tetrahymena thermophila size mutants: the relationship between cell size and regulation of DNA content; Seyfert HM et al.; Temperature-sensitive size mutants of the ciliate Tetrahymena thermophila were selected following chemical mutagenesis . Phenotypical characteristics are given for seven cell lines, which have a range of average cell volumes from 8000 microns 3 to more than 100 000 microns 3 . wild-type Tetrahymena cells have an average cell volume of 15 000 microns 3 . Two of the mutagenized cell lines have comparatively small cells at 29 degrees C but normal cells at 37 degrees C; whereas the other five lines are normal at 29 degrees C but large at 37 degrees C . While the small cells are poor growers, the large cells grow excellently at 37 degrees C . Measurements of DNA, RNA and protein contents indicate a significant correlation between all parameters and cell size . However, since the cells tolerate considerably different concentrations of each class of macromolecules, the amount of any of these macromolecules cannot be tightly controlled by cell size.

Nucleic Acids Res, 1984 Mar 26, 12(6), 3003 - 21
Sequence organization within and flanking clusters of 5S ribosomal RNA genes in Tetrahymena; Pederson DS et al.; Macro- and micronuclei of Tetrahymena thermophila each contain approximately 30 clusters of 5S genes per haploid genome . Structural changes in DNA sequences associated with some of these clusters occur during the development of the transcriptionally active macronucleus from the transcriptionally inert micronucleus . Exonuclease digestion indicates that DNA fragmentation is not responsible for these changes, making it likely that sequence rearrangements occur near some 5S genes during macronuclear development . These rearrangements appear to be random in location with respect to the 5S genes and do not alter either the tandem repeat organization of the genes, the average number (five) or the range in number (one to about 16) of genes per cluster . The 5S gene clusters are not closely linked and are not flanked by common repeating elements large enough to cross-hybridize . Sequence analysis of one tandem repeat suggests that Tetrahymena 5S genes have intragenic promoters . These observations indicate that features other than DNA rearrangements or DNA sequence per se are responsible for the transcriptional activation of 5S genes during macronuclear development.

J Biol Chem, 1984 Mar 10, 259(5), 2956 - 60
High guanine plus cytosine content in the third letter of codons of an extreme thermophile . DNA sequence of the isopropylmalate dehydrogenase of Thermus thermophilus; Kagawa Y et al.; In studies on the cause of the extreme stability of the macromolecules of Thermus thermophilus HB8, the leuB gene coding for 3-isopropylmalate dehydrogenase of the leucine synthesis pathway and its flanking regions were cloned and sequenced . The leuB gene of T . thermophilus was expressed in a leuB-less mutant of Escherichia coli, and thermostable dehydrogenase was purified from an extract of the cells . The primary structure of the thermophilic isopropylmalate dehydrogenase was deduced from the nucleotide sequence leuB gene (1017 base pairs) and the amino acid sequence of the peptides isolated from the purified dehydrogenase . The thermophilic dehydrogenase has Mr = 35,968, and the value was close to that determined for the monomer of dehydrogenase (36,000) by gel electrophoresis . The molecular weight of active dimeric dehydrogenase was found to be 73,000 by high speed liquid chromatography . The primary structure of dehydrogenase was consistent with the amino acid composition of the dehydrogenase . In contrast to the isopropylmalate dehydrogenase of E . coli which contains 8 cysteine residues, there was no cysteine in thermophilic isopropylmalate dehydrogenase . The 5'-noncoding region contained a typical Shine-Dalgarno sequence . The guanine plus cytosine content of the coding region was 70.1%, and that of the third letter of the codons was extremely high (89.4%).

Sem Hop, 1984 Mar 8, 60(11), 776 - 9
{Value of co-immunoelectrodiffusion on cellulose acetate for the demonstration of remarkable precipitating systems in the diagnosis of farmer's lung}; Gari M et al.; Farmer's Lung is an extrinsic allergic alveolitis due to the inhalation of actinomycetes thermophiles . Micropolyspora faeni is the principal allergen implicated in this disease which is very difficult to diagnose . The authors discuss the value of immuno-electro-diffusion on cellulose acetate which is a rapid and sensitive technique for the demonstration of remarkable precipitating systems: polysaccharide and chymotrypsin arcs . This investigation enables subjects who really have the disease to be distinguished from "contact" subjects.

J Cell Sci, 1984 Mar, 66, 167 - 73
Attachment of the cap to the central microtubules of Tetrahymena cilia; Dentler WL; The central microtubule cap is bound to the ends of the two central microtubules in Tetrahymena thermophila cilia by plug-shaped structures similar in appearance to the distal filament plugs attached to the ends of the A-microtubules . The caps have been separated from the microtubules and are composed of a bead, two plates, and two peg-like plugs to which the microtubules are attached . The structure of the cap is discussed in relation to the directionality of microtubule assembly in vivo.

Vopr Pitan, 1984 Mar-Apr, (2), 63 - 5
{Objective evaluation of the aroma and taste of Bulgarian sour milk}; Kondratenko M et al.; The purpose of the study was to discover the correlation between a subjective organoleptic evaluation of the samples of Bulgarian sour milk and the results of microbiological assays of the amount of and correlations among lactic bacteria, of yeast, and other foreign microflora . A total of 135 samples of milk supplied by different milk factories for tasting were examined . The mean arithmetic value of the percentage of S . thermophilus was found to be different for the samples of sour milk classified with different groups according to the aroma and taste . The data obtained were treated by the passive experimental-statistical method with the use of the REGR program for multiple regression analysis . Based on the theoretical conceptions several experimental models were suggested . The mathematical-statistical treatment allowed the most significant model to be selected for practical use . Control of the adequacy of the suggested models has shown that the model selected may be used for the characteristics of the aroma and taste of Bulgarian sour milk, enabling the tasters to objectively confirm the results of their assessment.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1984 Mar, 179(1), 56 - 72
{Acanthamoebae, Naegleria and other free-living Amoebae in cooling and rinsing water of dental treatment units}; Michel R et al.; 215 water samples were taken from 49 dental treatment units and investigated for the existence of free-living amoebae . In all water-carrying systems of the dental treatment units it was possible to verify the incidence of one or more amoeba species . In 8.2 per cent of the units Naegleria species was found and in 12.2 per cent Acanthamoeba species was present . Seven Naegleria and six Acanthamoeba strains (2 A . castellanii and 4 A . polyphaga) were isolated . From samples originating from 12 dental treatment units (DTU) another 42 amoeba strains were isolated which consisted of 14 different species within 9 classes . Among them Vannella mira (in 19 per cent of samples) and Hartmannella vermiformis (10.6 per cent) were found to be the most frequent species, followed by H . cantabrigensis (9.5 per cent), V . platypodia, Platyamoeba stenopodia and V . simplex (7.1 per cent each) . In 10 per cent of samples monotrichous and bitrichous flagellates such as the Bodo species were found, whereas two samples contained ova, larvae and adult free-living nematodes . Among the isolated Naegleria strains no thermophilic strain was present . Consequently they belong to the N . gruberi complex . Among the Acanthamoebae five of the six strains were thermophilic . All strains were investigated for pathogenic properties by means of the mice inoculation test . Two strains proved pathogenic - it was possible to isolate them from the brain and lung of dead mice . Another two strains proved to have invasive properties because they were isolated from the brain of infected animals; however, they did not give rise to disease or death of the respective animals . Supplementary microbiological tests demonstrated the existence of bacteria and fungi in 84 per cent of dental treatment units . Pseudomonas spec . were detected in 75% of dental units, Serratia marcescens in 2% and fungi in nearly 3% . 58.3% of all water samples contained total germ counts of more than 100/ml.

Genetics, 1984 Mar, 106(3), 387 - 401
Chromosome-designated mutation selection in Tetrahymena thermophila; Altschuler MI et al.; Two protocols are presented that allow the selection of mutations mapping to micronuclear chromosome 5 in Tetrahymena thermophilia . One protocol involves crossing mutagenized diploid cells directly to a strain nullisomic for chromosome 5 and screening the monosomic progeny for a mutant phenotype . The second protocol first takes the mutagenized diploid cells through round I of genomic exclusion to create useful and reusable mutant heterokaryons, which are then assayed for the presence of mutations on chromosome 5 by crossing to the nullisomic 5 strain . Of 14 putative chromosome 5 mutations obtained by these two methods, seven are shown by genetic analysis to be recessive mutations on chromosome 5; one mapped elsewhere in the genome; six were infertile or failed to yield progeny in some of the diagnostic crosses and, thus, their genetic nature could not be determined with certainty.

Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1768 - 70
Are DNA spacers relics of gene amplification events?
Wong WM, Abrahamson JL, Nazar RN.
The genome of a thermophilic fungus, Thermomyces lanuginosus, contains a tandemly arranged cluster of sequences that are approximately equal to 50% homologous with the cytoplasmic 5S RNA and that are selectable by hybridization techniques . Unlike typical pseudogenes, these sequences are not truncated; rather, they bear a limited sequence homology with the entire length of the 5S RNA and are oriented end to end without significant intervening sequences . We suggest that these are gene relics that were duplicated by a rolling circle-like mechanism and that have evolutionarily drifted to become gene spacers . Accordingly, we raise the possibility that this offers a fortuitous glimpse at the origins for many of the gene spacers in the eukaryotic genome.

Quad Sclavo Diagn, 1984 Mar, 20(1), 55 - 62
{Diagnostic methods in the isolation of Campylobacter in animals, animal foods and man}; Crotti D et al.; We report some techniques for the enrichment-broths and for the isolation-media in the research of thermophilic Campylobacter, over all in animal's foods; moreover we have compared four commercial media for direct isolation of this bacterium . We think that it is useful, as in alimentary as in animal and human microbiology, the research of Campylobacter utilizing selective and specific enrichments; for the isolation of Campylobacter we prefer the temperature of 42 degrees C in respect of 37 degrees C, always in microaerophilic systems . We haven't observed significant differences using the four commercial plates, but we think that in would be better prepare it laboratory the selective media for the goal.

Mikrobiologiia, 1984 Mar-Apr, 53(2), 242 - 5
{Selection of a culture forming alkaline phosphatase from thermophilic bacteria in the genus Thermus}; Egorova LA et al.; An obligate thermophilic Thermus ruber 132- 4K bacterial culture was selected . The culture has the activity of alkaline phosphatase . Its optimal growth temperature is 55 degrees C . The culture is recommended to be used for the production of alkaline phosphatase.

Nucleic Acids Res, 1984 Feb 24, 12(4), 2055 - 60
The number of ribosomal RNA genes in Thermus thermophilus HB8; Ulbrich N et al.; We have examined the number of rRNA genes in Thermus thermophilus HB8 by hybridization of Bam HI -, Hind III - and Pst I - digests of DNA to 3'- (3 2p) 23S, 16S and 5S rRNAs according to the Southern procedure . The restriction gels gave two radioactive bands with 23S and 5S rRNA . Furthermore, band positions were indistinguishable from one another when 23S and 5S rRNAs were used as probes to Bam HI and Hind III digests, indicating that each band contains sequences corresponding to the 3'-end of 23S and 5S rRNAs . The Pst I digest also gave two radioactive bands with 23S and 5S rRNAs as probes, where one band position was identical, but the other different . The 16S rRNA did hybridize with two fragments, using a Bam HI, as well as a Bam HI - Hind III double digest . The Hind III digest gave one band using 16S rRNA as a probe . It is concluded that the Thermus thermophilus HB8 chromosome carries at least two sets of genes for 23S, 16S and 5S rRNAs.

Nucleic Acids Res, 1984 Feb 24, 12(4), 1961 - 75
Tetrahymena H4 genes: structure, evolution and organization in macro- and micronuclei; Bannon GA et al.; The ciliated protozoan Tetrahymena thermophila contains two types of H4 histone genes (H4-I and H4-II) . Southern blotting and analysis of DNA from nullisomic strains indicate that H4-I and H4-II are on different chromosomes and that only H4-II is closely linked to an H3 gene . No DNA sequence rearrangements are observed for either of the H4 genes when the transcriptionally inert, germ line, micronucleus is compared to the transcriptionally active, somatic macronucleus . Comparison of the H4-I gene and its flanking sequences to H4 gene sequences of other organisms indicates that there are evolutionary constraints on coding nucleotides that are unrelated to their protein coding function and that these evolutionary pressures operate at the level of translation.

Nature, 1984 Feb 23-29, 307(5953), 737 - 40
Possible artefactual basis for apparent bacterial growth at 250 degrees C; Trent JD et al.; Baross and Deming reported that thermophilic marine bacteria isolated from the vicinity of a submarine hot-spring grow at temperatures up to at least 250 degrees C . They did not, however, conduct the appropriate control experiments to eliminate the possibilities of chemical artefacts or contamination . Here, in experiments using the same growth medium, the same temperature and pressure apparatus and the same sampling and analytical procedures, we report results nearly identical to theirs . We conclude that their evidence indicating bacterial growth at 250 degrees C may be due to artefacts produced in the medium and to contaminants introduced during sample processing.

J Biol Chem, 1984 Feb 10, 259(3), 1405 - 8
High vectorial proton stoichiometry by cytochrome c oxidase from the thermophilic bacterium PS3 reconstituted in liposomes; Sone N et al.; The stoichiometry of vectorial H+ ejection, coupled to ferrocytochrome c oxidation by a three-subunit bacterial cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3, was measured . Three methods of measuring the H+/e- ratio were applied to proteoliposomes containing a relatively small amount of PS3 cytochrome oxidase, which showed a relatively low oxidation rate and a very low H+ leakage, as follows: (a) simultaneous measurements of H+ ejection and cytochrome c oxidation upon addition of a yeast ferrocytochrome c pulse, which enable us to calculate the H+/e- ratio as H+ ejected per cytochrome c oxidized; (b) computer simulations to find out the fit for the pH meter trace by changing the H+/e- ratio and the velocity constant of leakage; and (c) two successive measurements of initial rates of H+ movement in the absence and presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone . The H+/e- ratios obtained were 1.39, the 10-s value after ferrocytochrome c addition in (a), 1.35 in (b), and 1.33 in (c) . This high H+/e- stoichiometry observed, exceeding 1 and as high as 1.4, is discussed with respect to the controversy of the H+/e- ratio at the cytochrome oxidase site.

J Protozool, 1984 Feb, 31(1), 112 - 6
Membrane protein differences correlated with the development of mating competence in Tetrahymena thermophila; Van Bell CT et al.; Initiation is the contact-independent phase of sexual conjugation which occurs when mature cells of Tetrahymena thermophila are shifted from growth medium to a low-salt starvation buffer . Immaturity, like high-salt starvation, restricts the ability of cells to conjugate; immature cells do not conjugate in either low- or high-salt buffers . Comparisons between sexually mature cells starved in initiation-restrictive and initiation-permissive buffers, and between immature and mature cells starved in an initiation-permissive buffer permitted the analysis of membrane protein expression correlated with mating competence . No polypeptides identified by lactoperoxidase-catalyzed iodination were found to be specific to mating-competent cells; however, several polypeptides not present in initiated cells were found to be common to the cell surfaces of immature and non-initiated cells which suggests that (1) initiation involves the removal of specific proteins from the cell surface, and (2) immaturity may be due to an inability to initiate.

J Gen Microbiol, 1984 Feb, 130 ( Pt 2), 357 - 62
DNA base composition, DNA-DNA homology and long-chain fatty acid studies on streptococcus thermophilus and Streptococcus salivarius; Farrow JA et al.; DNA base composition, DNA-DNA homology and long-chain fatty acid studies were performed on Streptococcus thermophilus and Streptococcus salivarius . These species possess similar mol % G + C values (about 37 to 41), long-chain fatty acid profiles and belong to a single DNA homology group . On the basis of the present and earlier studies it is proposed that Streptococcus thermophilus (Orla-Jensen) be reclassified as Streptococcus salivarius subsp . thermophilus comb . nov.

J Appl Bacteriol, 1984 Feb, 56(1), 151 - 7
The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide; Bolton FJ et al.; Nutrient agar plates stored in light and air for 48 h became inhibitory for Campylobacter jejuni, C . coli and nalidixic acid-resistant, thermophilic campylobacter (NARTC) strains . All five campylobacter test strains showed greater than 5 log reduction in counts on media which had been stored in light and air . Media stored in the dark and/or in a reduced atmosphere did not become inhibitory and supported the growth of campylobacters . Ferrous sulphate, sodium pyruvate, blood, charcoal or sodium metabisulphite, compounds frequently used as supplements in campylobacter media, were added to nutrient agar prior to storage of media in light and air . All additives except sodium metabisulphite prevented the accumulation of photochemically generated toxic oxygen derivatives and allowed growth of test strains . In qualitative tests to determine the ability of supplements to neutralize hydrogen peroxide, blood was the most active, charcoal and sodium pyruvate slightly less active and ferrous sulphate and sodium metabisulphite the least active . The results of this study confirm that supplements in campylobacter media act as quenching or detoxifying agents and not as enrichment factors.

J Clin Microbiol, 1984 Feb, 19(2), 157 - 60
Serotyping and biotyping of Campylobacter jejuni and Campylobacter coli from sporadic cases and outbreaks in Norway; Kapperud G et al.; Of 172 thermophilic campylobacters isolated from human cases of gastroenteritis in Norway, 149 (86.6%) were classified as Campylobacter jejuni, whereas 23 isolates (13.4%) belonged to Campylobacter coli . C . jejuni biotype 1 comprised 66.3% and C . jejuni biotype 2 comprised 20.3% of the total number . Using 50 unabsorbed antisera, we were able to serotype 109 (80.1%) of 136 campylobacters on the basis of heat-stable antigens identified by means of passive hemagglutination . The typable strains fell into 36 different serotypes . A large proportion of the strains were isolated from travellers returning from abroad, a state of affairs which may have influenced the serotype and biotype distribution . Two family outbreaks were found to be caused by a bio-serotype common to all diseased members of the particular families . A third family outbreak and an outbreak among employees at a poultry processing plant each involved two distinct strains.

J Clin Microbiol, 1984 Feb, 19(2), 153 - 6
Epidemiological aspects of enteritis due to Campylobacter spp . in Norway; Lassen J et al.; Data pertaining to 249 patients with stool cultures positive for thermophilic campylobacters are presented . Campylobacters were isolated from about 3% of all cases of acute enteritis and occupied second place in the bacterial etiology of this syndrome following Salmonella spp . Concomitant isolation of salmonellae or shigellae or both was achieved in 40 (16.1%) of the patients infected with campylobacters . The results suggest a bimodal age distribution with highest rates in young adults aged 20 to 29 years and children below 10 years of age . A majority of the campylobacters were isolated from travellers returning from abroad, and, to a lesser extent, from immigrants, particularly from Asia . Immigrants accounted for 45.2% of the patients below 10 years of age . The number of cases increased during the warmer months of the year . Travelling habits could, at least in part, explain the observed seasonality, age distribution, and geographical origin of infection . Eight outbreaks of Campylobacter enteritis were detected, five of which were family outbreaks, whereas three involved people from different families.

J Appl Biochem, 1984 Feb-Apr, 6(1-2), 29 - 38
Construction of a system for the regeneration of adenosine 5'-triphosphate, which supplies energy to bioreactor; Kondo H et al.; An engineering model was successfully developed for an ATP regeneration system by using two enzymes, acetate kinase (AK) and adenylate kinase (AdK), both obtained from the thermophile Bacillus stearothermophilus . This model is composed of five units: a substrate unit consisting of substrate solutions--AMP, ATP, and acetyl phosphate (AcOP)--an enzymatic reactor unit consisting of AK and AdK immobilized to Sepharose 4B, an auto sampler unit, an analytical unit made up of high-performance liquid chromatography, and a control unit made up of a microcomputer . Operation of the four units could be systematically controlled by the microcomputer . Fundamental, operational conditions were examined using this engineering model . The conversion of AMP to ATP concentration and space velocity (SV) . The minimum amount of ATP, which is required to obtain the 100% conversion of AMP to ATP, was determined to be about 4% of AMP concentration . The conversion of AMP to ATP was controlled effectively by changing the SV value . Based on the above experimental data, the continuous operation of an ATP regeneration system was tested at pH 7.5 and 30 degrees C under the conditions of 1.59 mM AMP, 0.084 mM ATP, and 5.0 mM AcOP . It was found that the conversion of AMP to ATP was more than 99% over a period of 6 days without changing SV.

J Appl Biochem, 1984 Feb-Apr, 6(1-2), 39 - 47
General stability of thermophilic enzymes: studies on 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus and yeast; Veronese FM et al.; The thermophilic enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase, decarboxylating, EC 1.1.1.44) from Bacillus stearothermophilus was much more resistant to inactivation under different conditions of temperature, pH, guanidine-hydrochloride, and organic solvents (dioxane, dimethylformamide, acetone) than its mesophilic counterpart from yeast . In addition, the thermophilic enzyme largely withstands proteolysis with trypsin, chymotrypsin, and elastase when compared with the yeast enzyme . It is proposed that thermophilic enzymes are not only thermostable, but also generally more stable to most common protein denaturants than their mesophilic counterparts . Because of their remarkable stability, enzymes isolated from thermophilic microorganisms may be ideally suited for technological applications.

J Hyg (Lond), 1984 Feb, 92(1), 45 - 51
The use of Preston enrichment broth for the isolation of 'thermophilic' campylobacters from water; Ribeiro CD et al.; The application of Preston enrichment broth to isolation of 'thermophilic' campylobacters from water has been investigated . Enrichment substantially increased the yield of such organisms . The optimum timing for subculturing the broth was after 48 h incubation . Despite the addition of aerotolerant supplement to the broth the number and variety of isolates was greater when the broth was incubated microaerically than when it was incubated in air.

Cell, 1984 Feb, 36(2), 441 - 5
Developmental rearrangements associated with a single type of expressed alpha-tubulin gene in Tetrahymena; Callahan RC et al.; Southern blotting analyses with diverse heterologous probes indicate that there is only a single type of alpha-tubulin gene in the ciliated protozoan Tetrahymena thermophila . Comparisons of the germ-line configuration of alpha-tubulin sequences in the transcriptionally inert micronucleus with the somatic configuration in the transcriptionally active macronucleus indicate that rearrangements involving breakage and rejoining of sequences flanking both sides of the alpha-tubulin gene accompany macronuclear differentiation.

Cell, 1984 Feb, 36(2), 433 - 40
DNA elimination in Tetrahymena: a developmental process involving extensive breakage and rejoining of DNA at defined sites; Yao MC et al.; Elimination of specific DNA sequences occurs during macronuclear development in the ciliate Tetrahymena thermophila . Recombinant DNA clones containing a segment of micronuclear (germinal) DNA involved in elimination and the corresponding segment of macronuclear (somatic) DNA produced after elimination were isolated . Detailed comparisons of the cloned DNAs, as well as the genomic DNAs, by hybridization indicated that DNA elimination is accompanied by specific DNA rearrangements . In this 9.5 kb region three defined DNA segments are deleted and the remaining sequences are linked together as one contiguous piece in the macronucleus . Specific DNA rearrangement of this kind occurs widely in the genome . Analysis of 20 randomly selected DNA clones suggests that there are more than 5000 such rearrangement sites in the genome . Thus specific breakage and rejoining of DNA occurs extensively during development, and might play an essential role in nuclear differentiation.

J Dairy Res, 1984 Feb, 51(1), 17 - 22
Stimulation of Streptococcus thermophilus growth in mastitic milk; Marshall VM et al.; Growth of Streptococcus thermophilus was faster in mastitic milk, aseptically collected following intramammary infusion of Escherichia coli endotoxin, than in normal aseptically collected milk . The contribution of polymorphonuclear leucocytes (PMN), of plasma and plasmin to this stimulation has been investigated . Addition of plasmin to low cell count milk stimulated growth of the streptococcus to the same degree as mastitic milk . However, addition of plasma or PMN from blood or from milk were less stimulatory . The part played by these components in providing casein breakdown products for the weakly proteolytic Str . thermophilus in mastitic milk is discussed.

Nucleic Acids Res, 1984 Jan 25, 12(2), 873 - 86
Studies on transcription termination and splicing of the rRNA precursor in vivo in the presence of proflavine; Nielsen OF et al.; In isolated nucleoli from Tetrahymena thermophila, low concentrations of the intercalating agent proflavine inhibit both transcription termination and splicing of the rRNA precursor . Proflavine also exerts an in vivo effect on the process of transcription termination under conditions, where the growth rate is only slightly reduced . Thus, approximately 40% of the rRNA precursor molecules, accumulated in nucleoli during 60 min of treatment with the drug, are longer than the normal 35S rRNA precursor . R-Loop mapping of these longer precursor molecules isolated after 30 and 60 min of incubation demonstrates that the RNA polymerases have a 50 fold lower elongation rate in the spacer region than in the coding region . Proflavine in the given concentration is found to have no significant effect on the splicing of properly terminated precursor molecules . In contrast, none of the longer non-terminated molecules are found to be spliced . These results indicate that proflavine primarily affects the process of transcription termination and that the splicing event is inhibited due to the improper termination of the precursor molecule.

Nucleic Acids Res, 1984 Jan 25, 12(2), 959 - 72
Nucleotide sequence of the 5'-terminal coding region for pre-rRNA and mature 17S rRNA in Tetrahymena thermophila rDNA; Engberg J et al.; The 5'-terminus of 35S pre-rRNA and mature 17S rRNA of Tetrahymena thermophila was mapped on cloned rDNA fragments by S1 nuclease protection experiments . A single site for transcription initiation was observed when pre-rRNA prepared by three different methods was used as RNA probe . These mapping results were unambiguously confirmed by sequencing the 5'-terminal region of in vitro capped 35S pre-rRNA . DNA sequence analysis of about 520 nucleotides upstream of the transcription initiation site revealed several distinct sets of highly conserved repeat sequences . In addition, the 840 nucleotides downstream of the transcription initiation site (+ 1) was determined and shown to include the 5'-terminus of the 17S rRNA coding region at position + 647 . A region surrounding the position + 195 contains an inverted repeat sequence which could be the structural basis for the recently described premature transcription termination event in this organism (Kister et al . (1983) Nucl . Acids Res . 11, 3487-3502).

J Biol Chem, 1984 Jan 25, 259(2), 1031 - 6
Studies on cytochrome c oxidase activity of the cytochrome c1aa3 complex from Thermus thermophilus; Yoshida T et al.; Cytochrome oxidase from T . thermophilus is isolated as a noncovalent complex of cytochromes c1 and aa3 in which the four redox components of aa3 appear to be associated with a single approximately 55,000-D subunit while the heme C is associated with a approximately 33,000-D peptide (Yoshida, T., Lorence, R . M., Choc, M . G., Tarr, G . E., Findling, K . L., and Fee, J . A . (1983) J . Biol . Chem . 258, 112-123) . We have examined the steady state transfer of electrons from ascorbate to oxygen by cytochrome c1aa3 as mediated by horse heart, Candida krusei, and T . thermophilus (c552) cytochromes c as well as tetramethylphenylenediamine (TMPD) . These mediators exhibit simple Michaelis-Menten kinetic behavior yielding Vmax and KM values characteristic of the experimental conditions . Three classes of kinetic behavior were observed and are qualitatively discussed in terms of a reaction scheme . The data show that tetramethylphenyldiamine and cytochromes c react with the enzyme at independent sites; it is suggested that cytochrome c1 may efficiently transfer electrons to cytochrome aa3 . When incorporated into phospholipid vesicles, the highly purified cytochrome c1aa3 was found to translocate one proton into the exterior medium for each molecule of cytochrome c552 oxidized . The combined results suggest that this bacterial enzyme functions in a manner generally identical with the more complex eucaryotic enzyme.

J Biol Chem, 1984 Jan 10, 259(1), 124 - 33
Purification and characterization of the Rieske iron-sulfur protein from Thermus thermophilus . Evidence for a {2Fe-2S} cluster having non-cysteine ligands; Fee JA et al.; We have purified the Rieske iron-sulfur protein from Thermus thermophilus . Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000 . The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mossbauer spectroscopies . Our data suggest the presence of a unique iron-sulfur center . Mossbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with {2Fe-2S} cores . The iron analyses and the Mossbauer data, taken together, suggest that the protein has two {2Fe-2S} clusters . This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe . Within the excellent resolution of the Mossbauer and EPR data, the two clusters are identical . Our results thus suggest that each {2Fe-2S} cluster is coordinated by at most two cysteine residues . The Mossbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian . The hyperfine parameters obtained are very similar to those reported for putidaredoxin . The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction . A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type {2Fe-2S} center.

J Biol Chem, 1984 Jan 10, 259(1), 112 - 23
Respiratory proteins from the extremely thermophilic aerobic bacterium, Thermus thermophilus . Purification procedures for cytochromes c552, c555,549, and c1aa3 and chemical evidence for a single subunit cytochrome aa3; Yoshida T et al.; We have developed a chemically defined, minimal growth medium for Thermus thermophilus which is suitable for nutritional studies, isotopic enrichment, and genetic manipulation of the organism . Reliable procedures are described for the large scale purification of cytochrome c552 from the periplasm and for cytochrome c555,549 and cytochrome c1 aa3 from the plasma membrane . In contrast to a previous report (Fee, J . A., Choc, M . G., Findling, K . L., Lorence, R., and Yoshida, T . (1980) Proc . Natl . Acad . Sci . U . S . A . 77, 147-151) which suggested a molecular weight near 200,000, the cytochrome c1aa3 complex was shown by protein and amino acid analyses to have Mr approximately 93,000 . Sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography, combined with amino acid analyses, revealed the presence of only two proteins in a 1:1 ratio: C-protein has Mr approximately 33,000, binds heme C, and is thought to correspond to cytochrome c1 . A-protein has Mr approximately 55,000 and is thought to bind the four redox components (2 heme A and 2 Cu) of cytochrome aa3.

Mol Gen Genet, 1984, 197(2), 244 - 53
The 5S ribosomal RNA gene clusters in Tetrahymena thermophila: strain differences, chromosomal localization, and loss during micronuclear ageing; Allen SL et al.; The organization of the 5S genes in the genome of Tetrahymena thermophila was examined in various strains, with germinal ageing, and the 5S gene clusters were mapped to the MIC chromosomes . When MIC or MAC DNA is cut with the restriction enzyme EcoRI, electrophoresed, blotted, and probed with a 5S rDNA probe, the banding patterns represent the clusters of the 5S rRNA genes as well as flanking regions . The use of long gels and 60 h of electrophoresis at 10 mA permitted resolution of some 30-35 5S gene clusters on fragments ranging in size from 30-2 kb (bottom of gel) . The majority of the 5S gene clusters were found in both MIC and MAC genomes, a few being MIC limited and a few MAC limited . The relative copy number of 5S genes in each cluster was determined by integrating densitometric tracings made from autoradiograms . The total number of copies in the MAC was found to be 33% greater than in the MIC . When different inbred strains were examined, the majority of the 5S gene clusters were found to be conserved, with a few strain-specific clusters observed . Nine nullisomic strains missing both copies of one or more MIC chromosomes were used to map the 5S gene clusters . The clusters were distributed non-randomly to four of the five MIC chromosomes, with 17 of them localized to chromosome 1 . A deletion map of chromosome 1 was constructed using various deletion strains . Some of these deletion strains included B strain clones which had been in continuous culture for 15 years . Losses of 5S gene clusters in these ageing MIC could be attributed to deletions of particular chromosomes . The chromosomal distribution of the 5S gene clusters in Tetrahymena is unlike that found for the well-studied eukaryotes, Drosophila and Xenopus.

Nucleic Acids Symp Ser, 1984, (15), 143 - 6
Various types of 5s rRNA crystals as studied by X-ray diffraction and electron microscopy; Morikawa K et al.; Six different types of 5 S rRNA crystals were obtained using the specimen purified from highly thermophilic bacteria, Thermus thermophilus HB8 . The best crystal available diffracts X-rays up to 13A Bragg spacing . One type of crystals was sufficiently thin to be subjected to electron microscopy . Optically filtered images of two different projections exhibit molecular regions distinctly from the medium . Preliminary investigation of packing of the molecules into the unit cell gives some implication of their overall shape.

Nucleic Acids Symp Ser, 1984, (15), 131 - 4
Recognition mechanism of tRNA with tRNA(guanosine-2')methyltransferase from Thermus thermophilus HB 27; Matsumoto T et al.; rRNA(Gm)methyltransferase from an extreme thermophile, Thermus thermophilus HB 27 specifically methylates the 2'-OH of the ribose ring of G18 in the invariant G18-G19 sequence in the D loop of tRNA . The interaction site on tRNA was presumed to be the D loop and stem structure . Destruction of tertiary structure of tRNA caused by heat resulted in a great decrease in the acceptor activity of methyl group . It was suggested by CD measurement that a conformational change of tRNA occurs when it forms an equimolar complex with Gm-methylase.

Radiat Environ Biophys, 1984, 23(3), 223 - 33
Effect of mercaptoethylamine on DNA degradation in thermophilic bacteria bac . Stearothermophilus exposed to gamma-, UV-radiation or methylnitrosourea; Fomenko LA et al.; The effect of mercaptoethylamine (MEA) on degradation of DNA in thermophilic bacteria Bac . stear . exposed to gamma-, UV-rays or methylnitrosourea (MNU) was studied . Using centrifugation on alkaline and neutral sucrose gradients, it was shown that MEA inhibits the accumulation of breaks in the DNA of Bac . stear . It also lowers the level of DNA degradation in toluene-treated cells of Bac . stear . under the action of the intrinsic nuclease, reduces the activity of the endonuclease specific for apurinic DNA, as well as that of S1-nuclease and DNase-I in vitro . The inhibition in the accumulation of DNA breaks is assumed to be due to a decrease of the endonuclease activity in the cells of thermophilic bacteria.

Anal Biochem, 1984 Jan, 136(1), 161 - 7
Isolation of tRNA isoacceptors by affinity chromatography on immobilized bacterial elongation factor Tu; Derwenskus KH et al.; Elongation factor Tu from Escherichia coli or Thermus thermophilus was immobilized on cyanogen bromide-activated Sepharose . Immobilized elongation factor Tu X GDP could be converted to Tu X GTP, which is able to bind aminoacyl-tRNA . When bulk tRNA from E . coli, baker's yeast, or bovine liver was aminoacylated by one amino acid only, the resulting aminoacyl-tRNA could be separated in one step from the rest of the tRNA using an affinity column of immobilized elongation factor Tu X GTP . Specific tRNA isoacceptors can be isolated in amounts sufficient for gel electrophorectic analysis, sequence determinations, and hybridization experiments.

Appl Environ Microbiol, 1984 Jan, 47(1), 201 - 4
Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata; Fennington G et al.; A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light . Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type . Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.

Mol Gen Genet, 1984, 193(1), 58 - 63
Cloning and expression of a thermophilic alpha-amylase gene from Bacillus stearothermophilus in Escherichia coli; Tsukagoshi N et al.; A 6.4 Kb HindIII fragment of Bacillus stearothermophilus DY-5 DNA cloned in Escherichia coli using pBR322 as a vector was shown to direct the synthesis of a thermophilic alpha-amylase . In attempts to reduce the size of the insert, the alpha-amylase gene was shown to be contained in a 3.1 Kb HindIII - BamHI fragment of the donor strain DNA . The alpha-amylase gene was stably maintained and expressed efficiently in E . coli . The enzymic properties of alpha-amylase produced in E . coli closely resembled those of the donor strain alpha-amylase and the temperature range for the maximal activity was from 65 degrees C to 80 degrees C . Nearly 100% of the activity remained after heating at 80 degrees C for 15 min . The alpha-amylase was shown to be accumulated in the periplasmic space . It was purified to a nearly homogenous protein with a molecular weight of 61,000, which was very similar in size to that produced by B . stearothermophilus DY-5.

Acta Microbiol Pol, 1984, 33(1), 57 - 66
Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp . AK-2; Srivastava RA et al.; Intracellular thermostable amylases from a thermophilic Baccilus sp . AK-2 have been isolated and purified . The crude enzyme, having pH optimum at 6.5 . and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography . Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0 . Electrophoretic mobility of these fractions showed a single band . Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions . The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively . The -SH group in the amylase molecule was essential for enzyme activity . Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities . EDTA showed dose dependent non-competitive inhibition . Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type . The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.

Mol Gen Genet, 1984, 195(1-2), 314 - 20
Nucleotide sequence of the triose phosphate isomerase gene of Escherichia coli; Pichersky E et al.; We report here the complete nucleotide sequence of the E . coli triose phosphate isomerase gene . The gene encodes a polypeptide of 255 amino acids which is approximately 46% homologous to eukaryotic triose phosphate isomerases, and approximately 38% homologous to the enzyme from a thermophilic bacterium, Bacillus stearothermophilus . The nucleotide sequence is 55% homologous to that of the corresponding gene in the yeast Saccharomyces cerevisiae . To our knowledge, this is the first report of the sequence of a gene coding a glycolytic enzyme from a prokaryotic organism.

J Biol Chem, 1983 Dec 25, 258(24), 14784 - 9
Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes . Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12; Guranowski A et al.; Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum . Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold . The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase . The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000 . Activity maximum is at pH 8.3 . The Km value computed for Ap4A is 25 +/- 3 microM . The sulfhydryl group(s) is essential for enzyme activity . Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively . Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis . Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively . Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations . Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations . Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold . The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine . Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.

J Biol Chem, 1983 Dec 10, 258(23), 14407 - 12
Modulation by ADP and Mg2+ of the inactivation of the F1-ATPase from the thermophilic bacterium, PS3, with dicyclohexylcarbodiimide; Yoshida M et al.; The soluble F1-ATPase from the thermophilic bacterium PS3 (TF1) contains no endogenous adenine nucleotides and contains about 0.2 g ions of Mg2+/mol which resists removal by repeated centrifugation-elution on columns of Sephadex G-50 . The isolated enzyme will not bind additional Mg2+ added in the absence of adenine nucleotides nor is the rate of inactivation of the isolated enzyme by dicyclohexylcarbodiimide (DCCD) affected by the addition of Mg2+ . When ADP is added to isolated TF1, a 1:1 TF1 X ADP complex is formed which is stable to repeated gel permeation on columns of Sephadex G-50 subjected to centrifugation-elution . On formation of the 1:1 TF1 X ADP complex, the rate of inactivation of the enzyme by DCCD is accelerated 6-fold . The rate of inactivation of the 1:1 TF1 X ADP complex by DCCD is not further stimulated in the presence of 2 mM ADP which indicates that the binding of ADP to a single site in the enzyme is sufficient to promote maximal stimulation of the inactivation . Addition of Mg2+ to the 1:1 TF1 X ADP complex results in the binding of about 1 g ion of Mg2+/mol of enzyme . The 1:1:1 TF1 X ADP X Mg2+ complex thus formed is sluggishly inactivated by DCCD . When the Mg2+ is removed from the TF1 X ADP X Mg2+ complex by treatment with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, the rate of inactivation of the enzyme by DCCD is accelerated 4-fold . Other divalent metal ions protect the 1:1 TF1 X ADP complex against inactivation by DCCD . Of these, Mn2+, Zn2+, Co2+, and Cd2+, which are about as equally effective as Mg2+ as cofactors for the hydrolytic reaction when present at 0.2 mM, offer about equal protection of the complex against inactivation by DCCD also when present at 0.2 mM . These results indicate that the binding site for ADP in the 1:1 TF1 X ADP complex is a catalytic site . TF1, inactivated by 92% with DCCD, has the same capacity to bind ADP as the active enzyme, forming a tight 1:1 TF1 X ADP complex which is stable to repeated centrifugation-elution on columns of Sephadex G-50 . The 1:1 TF1 X ADP complex retains its capacity to bind Mg2+ to form the 1:1:1 TF1 X ADP X Mg2+ complex after it is inactivated by 88% with DCCD.

Anal Biochem, 1983 Dec, 135(2), 466 - 9
A quantitative assay for ciliate chemotaxis; Leick V et al.; A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism . The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible . The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.

J Bacteriol, 1983 Dec, 156(3), 1332 - 7
Ultrastructure and extreme heat resistance of spores from thermophilic Clostridium species; Hyun HH et al.; The heat resistance and ultrastructural features of spore suspensions prepared from Clostridium thermocellum LQRI, Clostridium thermosulfurogenes 4B, and Clostridium thermohydrosulfuricum 39E were compared as a function of decimal reduction time . The decimal reduction times at 121 degrees C for strains LQRI, 4B, and 39E were 0.5, 2.5, and 11 min . The higher degree of spore heat resistance was associated with a spore architecture displaying a thicker cortex layer . Heat resistance of these spores was proportional to the ratio of spore cortex volume to cytoplasmic volume . These ratios for spores of strains LQRI, 4B, and 39E were 1.4, 1.6, and 6.6, respectively . The extreme heat resistance and autoclavable nature of C . thermohydrosulfuricum spores under routine sterilization procedures is suggested as a common cause of laboratory contamination with pure cultures of thermophilic, saccharide-fermenting anaerobes.

J Biomol Struct Dyn, 1983 Dec, 1(3), 657 - 67
Dynamics of ribosomal RNA structure; Erdmann VA et al.; The structural dynamics of ribosomal 5S RNAs have been investigated by probing single strandedness through enzymatic cleavage and chemical modification . This comparative study includes 5S rRNAs from E . coli, B . stearothermophilus, T . thermophilus, H . cutirubrum, spinach chloroplast, spinach cytomplasm, and Artemia salina . The structural studies support a unique tertiary interaction in eubacterial 5S rRNAs, involving nucleotides around positions 43 and 75 . In addition long range structural effects are demonstrated in E . coli 5S rRNA due to the conversion of C to U at position 92.

Eur J Biochem, 1983 Dec 1, 137(1-2), 95 - 9
Isolation, purification and characterization of the ATPase complex from the thermophilic cyanobacterium Synechococcus 6716; Lubberding HJ et al.; The ATPase complex is isolated and purified from membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 by octyl glucoside and cholic acid by a modification of the procedure for its extraction from spinach chloroplasts . The complex is purified by differential centrifugation and ammonium sulfate precipitation and by gel filtration on Sepharose 6B . The purified fraction, without any phycocyanin contamination, shows ATP hydrolysis activity and Pi/ATP exchange activity of 1564 and 350 nmol X min-1 X mg protein-1, respectively . N,N'-Dicyclohexylcarbodiimide inhibits the ATP hydrolysis activity of this purified fraction . On polyacrylamide gels most typical F1 ATPase polypeptides are identified, but the low-molecular weight polypeptides visible cannot be ascribed to the F0 part of the complex with certainty; non-identified bands around 30 kDa are also present.

Eur J Biochem, 1983 Dec 1, 137(1-2), 101 - 6
Characterization of reconstituted ATPase complex proteoliposomes prepared from the thermophilic cyanobacterium Synechococcus 6716; van Walraven HS et al.; The preparation and some properties are described of proteoliposomes consisting of the ATPase complex and lipids from the thermophilic cyanobacterium Synechococcus 6716 . In the proteoliposomes (about 200 nm in diameter) only a low amount of protein can be incorporated (protein/lipid ratio of 0.01 w/w) and they show very few protein particles on freeze-fracture replicas . The octyl glucoside and cholate dialysis method of reconstitution yielded stable proteoliposomes with a relatively low proton permeability . ATP hydrolysis and 32Pi/ATP exchange activities were about 400 and 120 nmol X min-1 X mg protein-1, respectively; the former was strongly stimulated by an uncoupler . ATP hydrolysis induces membrane energization as monitored by membrane-potential- and surface-potential-indicating probes and by different pH indicators trapped inside the vesicles . The probes used were a membrane-bound fluorescent aminoacridine, which monitors surface charge-density changes, the native carotenoids and added oxonol VI for monitoring electrical potential in the membrane and the pH indicators neutral red and cresol red . The different rise kinetics of these probes indicate that proton accumulation upon ATP hydrolysis involves at least two steps: a membrane-localized potential charge and proton transfer followed by a much slower acidification of the bulk intravesicular space . Internal neutral red and cresol red seem to discriminate between proton translocation to the internal interface and bulk space, respectively.

Biochem Biophys Res Commun, 1983 Nov 30, 117(1), 190 - 5
The temperature-dependent emission of low frequency sound by motile cultures of the ciliate Tetrahymena thermophilia; Hill RJ; When a sensitive condenser microphone is appropriately sealed and immersed into a motile suspension of Tetrahymena, a characteristic sound spectrum can be recorded . The spectrum of cells cultured at 25 degrees C and measured at 20 degrees contains three main components, centred around 40, 55 and 80 Hz . Both the intensity and distribution of the sound emission are altered when the cells are cooled to 12 degrees or warmed to 33 degrees C . No such sound emission is detectable from suspensions of sessile organisms.

Wien Klin Wochenschr, 1983 Nov 25, 95(22), 789 - 91
{Extrinsic allergic alveolitis: comparison of 2 methods (Ouchterlony gel precipitation and ELISA) for antibody detection in routine diagnosis}; Obexer G et al.; Sera from 58 patients with suspected extrinsic allergic alveolitis (EAA) were tested in parallel by the double diffusion test and the "enzyme-linked immunosorbent assay (ELISA)" using 10 different antigen solutions (extracts from thermophilic actinomycetes, various mycetes, pigeon serum, pigeon droppings and the wheat weevil sitophilus granarius) . The ELISA technique was more sensitive than the double diffusion test in detecting antibodies to the panel of antigens used . However, by using several antigen dilutions the number of sera positive sera with the double diffusion test increased and an overall correlation of 93.1% with the ELISA was achieved . The results of the study are discussed with regard to the advantages and disadvantages of both test systems for the in vitro diagnosis of EAA.

Nucleic Acids Res, 1983 Nov 25, 11(22), 7661 - 78
A site and strand specific nuclease activity with analogies to topoisomerase I frames the rRNA gene of Tetrahymena; Gocke E et al.; Exposure of macronuclear chromatin from Tetrahymena thermophila to sodium dodecyl sulfate causes an endogenous nuclease to cleave the extra-chromosomal rDNA at specific sites . All cuts are single-strand cleavages specific to the non-coding strand . Three cleavages map in the central non-transcribed spacer of the palindromic molecule at positions -1000, -600 and -150 bp with respect to the transcription initiation point . A fourth site is located close to the transcription termination point, while no cleavage is observed in the coding region . The position of each cleavage is in the immediate neighbourhood of DNAse I hypersensitive sites . Additionally, certain DNA sequence motifs are repeated in the region around the cleavages . Upon cleavage induction a protein becomes attached to the rDNA . Our results indicate covalent binding to the generated 3' end, in analogy to the aborted reaction of topoisomerase I.

Biochim Biophys Acta, 1983 Nov 17, 741(2), 251 - 7
Histone-like protein in the Archaebacterium Sulfolobus acidocaldarius; Green GR et al.; The Archaebacterium Thermoplasma acidophilum contains a basic chromosomal protein remarkably similar to the histones of eukaryotes . Therefore, it was of interest to examine a different Archaebacterium for similar proteins . We chose to examine Sulfolobus acidocaldarius because it is thermophilic, like T . acidophilum, but nevertheless the two organisms are not particularly closely related . Two major chromosomal proteins were found in S . acidocaldarius . The smaller of these was soluble in 0.2 M H2SO4 and had a molecular weight of 14500 . The larger was acid-insoluble and had a molecular weight of about 36000 . Together, the proteins protected about 5% of the DNA against nuclease digestion and stabilized about 50% against thermal denaturation . Overall, the properties of these proteins were intermediate between those of the Escherichia coli protein HU and T . acidophilum protein HTa.

J Biol Chem, 1983 Nov 10, 258(21), 13008 - 13
Studies of the ferredoxin from Thermus thermophilus; Hille R et al.; The soluble ferredoxin from Thermus thermophilus was examined by Mossbauer and EPR spectroscopies and by reductive titrations . These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one {4Fe-4S} center which is responsible for the rhombic EPR spectrum of the fully reduced protein . These assignments should replace those made by Ohnishi et al . (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T . (1980) J . Biol . Chem . 255, 345-348) prior to the discovery of the 3Fe clusters . The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.

J Cell Sci, 1983 Nov, 64, 49 - 67
Isolation and ultrastructural characterization of secretory mutants of Tetrahymena thermophila; Orias E et al.; Isolation of 14-secretory mutants (exo-) of Tetrahymena thermophila and ultrastructural characterization (freeze-fracture and thin-section) of two of these (SB255 and SB258) are described . The site of secretion is marked by an intramembrane particle array, the rosette, beneath which the secretory organelle rests . Using Alcian Blue (8GS) as a secretagogue, a screening procedure for exo- cells was developed . Of the resulting 14 clones isolated, 10 are stable and have a tight mutant phenotype . Two of these, SB255 and SB258, lack assembled rosettes . Electron microscopy shows that SB255 has a reduced total number of mucocysts, whereas SB258 appears to have the normal number . This study demonstrates a useful eukaryotic model with which to study by genetic dissection the regulatory mechanisms involved in membrane events in secretion.

Mol Cell Biol, 1983 Nov, 3(11), 1909 - 19
Germ line-specific DNA sequences are present on all five micronuclear chromosomes in Tetrahymena thermophila; Karrer KM; The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp . involves the elimination of specific DNA sequences (M . C . Yao and M . Gorovsky, Chromosoma 48:1-18 1974) . The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes . Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322 . A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe . Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific . They were all members of families of repeated sequences . Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T . thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes . One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes.

J Bacteriol, 1983 Nov, 156(2), 818 - 27
Adherence of Clostridium thermocellum to cellulose; Bayer EA et al.; The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied . The adherence phenomenon was determined to be selective for cellulose . The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars . A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS . Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells . By using these CBF-specific antibodies, crossed immunoelectrophoresis of cell extracts revealed a single discrete precipitation peak in the parent strain which was absent in the mutant . This difference was accompanied by an alteration in the polypeptide profile whereby sonicates of strain YS contained a 210,000-molecular-weight band which was missing in strain AD2 . The CBF antigen could be removed from cell extracts by adsorption to cellulose . A combined gel-overlay--immunoelectrophoretic technique demonstrated that the cellulose-binding properties of the CBF were accompanied by carboxymethylcellulase activity . During the exponential phase of growth, a large part of the CBF antigen and related carboxymethylcellulase activity was associated with the cells of wild-type strain YS . However, the amounts decreased in stationary-phase cells . Cellobiose-grown mutant AD2 cells lacked the cell-associated CBF, but the latter was detected in the extracellular fluid . Increased levels of CBF were observed when cells were grown on cellulose . In addition, mutant AD2 regained cell-associated CBF together with the property of cellulose adherence . The presence of the CBF antigen and related adherence characteristics appeared to be a phenomenon common to other naturally occurring strains of this species.

J Bacteriol, 1983 Nov, 156(2), 493 - 7
Eucaryote thermophily: role of lipids in the growth of Talaromyces thermophilus; Wright C et al.; The effects of growth temperature on the fatty acid composition of the phospholipids of the fungus Talaromyces thermophilus were investigated . This thermophilic organism was unable to increase the degree of unsaturation of its fatty acids when shifted from high to low growth temperatures . Inhibition of fatty acid synthesis by the antibiotic cerulenin was reversed by the addition of a mixture of palmitic, stearic, and oleic acids and ergosterol . The data obtained were consistent with the hypothesis that the thermophilic character of T . thermophilus is due to metabolic limitations that restrict its ability to regulate membrane fluidity.

Appl Environ Microbiol, 1983 Nov, 46(5), 1073 - 9
Alternative prey: a mechanism for elimination of bacterial species by protozoa; Mallory LM et al.; Antibiotic-resistant strains of Salmonella typhimurium and Klebsiella pneumoniae died readily after their addition to raw sewage, but they grew in sterilized sewage . The decline was not a result of abiotic stresses, and because the bacteria were able to survive in large numbers for at least 15 days in solutions containing no organic nutrients, it was not a result of competition . Toxin production, bacteriophages, and Bdellovibrio sp . did not cause the disappearance of the two bacterial species . A decline was also evident if the sewage was first passed through a 3-micron (pore size) filter or treated with cycloheximide or cycloheximide plus nystatin, but protozoa developed under these conditions . Little or no decline occurred if the sewage was filtered and treated with the eucaryotic inhibitors before the addition of S . typhimurium or K . pneumoniae, and protozoa were not detected . S . typhimurium increased in abundance if cycloheximide, streptomycin, and erythromycin or large amounts of glucose were added to sewage . Tetrahymena thermophilus did not significantly reduce the population of S . typhimurium in buffer when the density of the bacterium was about 10(4)/ml . However, when more than 10(8) Enterobacter agglomerans cells per ml were added to the buffer, T . thermophilus reduced the abundance of E . agglomerans and S . typhimurium to 10(6) and 10/ml, respectively . The density of S . typhimurium was further decreased by a second increment of E . agglomerans cells . The disappearance of S . typhimurium and K . pneumoniae from sewage thus is the result of predation by protozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1983 Oct 15, 170(1), 137 - 53
Small-angle X-ray scattering study of adenosine triphosphatase from thermophilic bacterium PS3; Furuno T et al.; Adenosine triphosphatase from the thermophilic bacterium PS3(TF1) has been studied by solution X-ray scattering . A structural change in TF1 caused by the binding of ADP was observed by examining the difference between the radii of gyration of the unligated and ligated forms . The radius of gyration of the unligated TF1 was found to be 49.5 +/- 0.3 A, and it decreased by approximately 3% after ligation with ADP . The positions and the amplitudes of a subsidiary maximum and a shoulder in the scattering profile showed subtle change on nucleotide binding . The lower limit of the maximum length of TF1 was determined to be 165 A for the unligated form and 150 A for the ligated form . The shape analysis of TF1 was performed by model calculations for simple triaxial bodies or their complexes . Among the various models tested, the one that gave the best fit with the experimental data consisted of seven ellipsoids of revolution; six identical ellipsoids with semi-axes: a = b = 18.5 A and c = 74 A . arranged hexagonally, and the other with a = b = 28 A and c = 45 A, located below the other six on the 6-fold axis . On the basis of this model it was suggested that there is a structural change on ligation with nucleotides, consisting of a shrinkage of the six long ellipsoids by 6% along their major axes.

J Mol Biol, 1983 Oct 5, 169(4), 963 - 74
Molecular symmetry of glyceraldehyde-3-phosphate dehydrogenase from Bacillus coagulans; Griffith JP et al.; The thermolabile glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans has a crystallographically exact 2-fold rotation axis of symmetry in one of its orthorhombic crystal forms (Lee et al., 1982) . Using various crystallographic techniques, we have now identified this axis to be the molecular R-axis, which is the symmetry axis that relates the two subunits that form each active site of the tetrameric enzyme . This is in contrast to the symmetry of the human skeletal muscle enzyme wherein the crystallographically exact axis was found to be the Q-axis (Buehner et al., 1974) . This finding could have important implications for the possible mechanism for the allosteric behavior of this molecule.

Eur J Biochem, 1983 Oct 3, 135(3), 425 - 34
Ribosomal subunits and ribosomal proteins of Tetrahymena thermophila . Effect of the presence of iodoacetamide during ribosome extraction on the properties of the subunits; Petridou B et al.; Proteolytic degradation of ribosomal proteins occurs during the preparation of subunits of the cytoplasmic ribosomes of the protozoa Tetrahymena thermophila and the isolated subunits are inactive . Addition of 5 mM iodoacetamide to cell suspensions before extraction inhibits proteolytic activity and permits isolation of active subunits . The protein complements of these subunits have been characterized in two different two-dimensional electrophoretic systems, and their molecular weights have been determined.

Mol Cell Biol, 1983 Oct, 3(10), 1857 - 65
Cloning of abundant mRNA species present during conjugation of Tetrahymena thermophila: identification of mRNA species present exclusively during meiosis; Martindale DW et al.; A cDNA library was constructed by using as a template the RNA present during the meiotic prophase of Tetrahymena thermophila . Clones containing cDNA sequences homologous to moderately abundant to abundant transcripts were detected by colony hybridization and confirmed by hybridizing purified cDNA plasmids on filters with labeled RNA probes . Eighteen clones were isolated, and the sizes of their cDNA inserts were determined . Cross-hybridization of individual cDNA plasmid pairs showed that each of these clones contained cDNA that was homologous to one of eight different RNA transcripts . The sizes of the eight RNA transcripts and the stages of the T . thermophila life cycle during which they were present were determined by hybridizing nick-translated cDNA probes against denatured, electrophoresed RNA from various stages . Clones were identified that contained sequences homologous to RNAs present only in early conjugation (meiosis); other clones contained sequences homologous to RNAs which were abundant during conjugation but present at other stages as well . One clone contained a sequence homologous to an RNA that was abundantly present only in nongrowing cells.

Environ Res, 1983 Oct, 32(1), 205 - 11
A survey of pathogenic and free-living amoebae inhabiting swimming pool water in Mexico City; Rivera F et al.; A survey of pathogenic and free-living amoebae in swimming pool waters of Mexico City was performed . Among the organisms isolated those which have public health importance were Naegleria fowleri Carter and Acanthamoeba castellanii Douglas . Amoebae of the genera Acanthamoeba, Naegleria, and Vahlkampfia were recovered in their cystic stage while those specimens of the genera Amoeba, Entamoeba, Thecamoeba, and Vanella were recovered only in their trophic stage during this study . Amoebae were concentrated through filtration procedures and subsequently cultured in different culture media . Nonpathogenic amoebae also isolated by culture included: Amoeba proteus (Pallas) Leidy, Amoeba striata Penard, Paratetramitus jugosus Page, Acanthamoeba astronyxis Ray and Hayes, Vahlkampfia avara Page, Vahlkampfia inornata Page, Thecamoeba verrucosa Ehrenberg, and Vanella mira Schaeffer . Trophozoites of Entamoeba gingivalis Gros, were also recovered, both directly and by culture . Most commonly found were amoebae of the species Naegleria gruberi Schardinger (59.02%), N . fowleri (16.77%), and A . castellanii (7.64%) . Least-frequently found amoebae belonged to the species Thecamoeba verrucosa (0.12%) . All isolated strains of N . fowleri and A . castellanii were thermophilic at 45 and 40 degrees C, respectively, and also pathogenic when inoculated into white mice . More populated by amoebae were those swimming pools of the indoor type with an inner side garden . It was also shown that the free residual chloride values of 0.50 to 1.5 mg/liter, ordinarily used in pool waters, are not adequate for elimination of amoebae.

Int J Pept Protein Res, 1983 Oct, 22(4), 469 - 75
Structural basis for the thermal stability of glyceraldehyde-3-phosphate dehydrogenases; Olsen KW; The amino acid sequences of two thermophilic and five mesophilic glyceraldehyde-3-phosphate dehydrogenases have been compared with the known three-dimensional structure of this enzyme to determine the factors responsible for thermal stability . The changes are greatest in the S-loop regions at the center of the tetramer, which show a quantitative increase in hydrophobicity and polarity that can strengthen subunit interactions in a complementary manner . The S-loops also show increases in residue volume and bulk that may indicate a tighter packing at the molecular center . In addition, there are changes in the secondary structural parameters indicating that the helices, in particular, may be more stable in the thermophilic proteins . Increases in the hydrophobicity of domain and subunit contacts for the Thermus aquaticus glyceraldehyde-3-phosphate dehydrogenase may explain why it is the most thermostable protein in this series.

Cryobiology, 1983 Oct, 20(5), 560 - 6
Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying; Font de Valdez G et al.; Cultures of 14 lactic acid bacteria species were freeze-dried in 10 or 20% non-fat skim milk and in distilled water containing one of the following additives: bovine albumin, glycogen, dextran, polyethylene glycol (PEG) 1000, PEG 4000, PEG 6000, glycerol, beta-glycerophosphate, sodium glutamate, asparagine, or cysteine . Each of the potential protective agents tested exhibited marked variations in the protection afforded to different species, none of them was effective for the preservation of viability of thermophilic lactobacilli . However, glycerol provided effective protection for L . leichmannii ATCC 4797 (90% survival), while L . bulgaricus ATCC 11842 reached a viability of 78% with 0.04 M cysteine.

Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5975 - 9
Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase; Mielenz JR; The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli . Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA . B . stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb . Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA . Cloning experiments with the four plasmids yielded alpha-amylase-producing E . coli that contained the same 9.7-kb chimeric plasmid . Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase . A spontaneous mutant of B . stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid . A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase . A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B . stearothermophilus . These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B . stearothermophilus with no genetic counterpart present on the chromosome.

Biochim Biophys Acta, 1983 Sep 7, 733(2), 274 - 82
Interaction of Escherichia coli F1-ATPase with dicyclohexylcarbodiimide-binding polypeptide; Loo TW et al.; Antibody raised against the N,N'-dicyclohexylcarbodiimide (DCCD)-binding polypeptide of Escherichia coli bound to the cytoplasmic surface of the cell membrane . A weak reaction was seen with everted vesicles of the thermophile PS3 . Rat-liver mitochondrial membranes did not react with the antibody . Reaction of the isolated DCCD-binding polypeptide with the antibody was prevented by oxidation of methionine residues or cleavage of the polypeptide with cyanogen bromide . Modification of the arginine residues of the DCCD-binding polypeptide did not affect interaction with the antibody . Purified F1-ATPase of E . coli bound to the isolated DCCD-binding polypeptide as shown by solid-phase radioimmune assay . Binding involved the alpha and/or beta subunits of F1 and the arginine residues of the polar central region of the DCCD-binding polypeptide . Our results are consistent with a looped arrangement of the DCCD-binding polypeptide in the membrane in which the carboxyl- and amino-terminal regions of the molecule are at the periplasmic surface and the polar central region, interacting with F1, is at the cytoplasmic surface of the cell membrane.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1983 Sep, 178(1-2), 155 - 7
{Bacteriological control of various methods of sewage sludge hygienization}; Breer C; As a result of extensive parallel investigations in a water treatment plant it was found that the fresh sludge pasteurization or prepasteurization with ensuing sludge digestion gives a product which is unobjectionable from an epidemiological hygienic point of view . The result were confirmed by investigations in a second plant . Similarly satisfactory results were obtained with the composting of previously desiccated sludge, with the aerobic-thermophilic fermentation of liquid sludge or with the drying of sewage sludge . An alternative to these thermal processes is the application of gamma rays or accelerated electrons.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1983 Sep, 178(1-2), 111 - 41
{Technology of sewage sludge hygienization}; Keller U; That the use of modern technology against the laws of Nature must fail, has been clearly demonstrated again some years ago when sewage sludge postpasteurization was rashly introduced . Although many attempts were made to improve this procedure, it had to be abandoned because of unavoidable massive regrowth of pathogens which invaded the germ-free postpasteurized sludge . In contrast of postpasteurization, long-term large-scale tests with the pasteurization of fresh sludge (prepasteurization) have demonstrated that this procedure where methane digestion with its pathogen displacing effect constitutes the final stage, is basically able to function . With respect to the Swiss Sewage Sludge Decree which came into force in May 1981, and which imposes sludge hygienization for most applications throughout the year, various thermal prepasteurization methods have been offered on the market ready for application to meet the legally prescribed requirements . However, some of them still need selective improvements in order to ensure the desired hygienisation effect permanently . For some time now, attention has been focussed on a novel biological 2-stage procedure based on partial aerobic thermophilic fermentation followed by anaerobic sludge digestion which in addition to good hygienisation promises improved sludge thickening, reduced digestion time, more favourable energy consumption and added process stability etc . Although it has already been offered on the market, this interesting process is being thouroughly tested and optimized in parallel pilot tests plant at the WWTP Altenrhein . Finally, reference is made to further sludge treatment processes such as sludge drying and sludge composting which mostly comprise efficent sludge hygienisation although they may not entirely prevent pathogenic regrowth . Moreover, some unconventional and less popular processes such as liquid sludge irradiation and chemical methods are also mentioned.

Arch Biochem Biophys, 1983 Sep, 225(2), 836 - 46
Two chlorophyll-binding subunits of the photosystem 2 reaction center complex isolated from the thermophilic cyanobacterium Synechococcus sp; Yamagishi A et al.; The reaction center of photosystem 2 has been highly purified from digitonin-solubilized thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp . by means of sucrose density gradient centrifugation and electrophoresis on polyacrylamide gels containing digitonin . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated reaction center complex yielded four chlorophyll a proteins named CP2-a, CP2-b, CP2-c, and CP2-d . When reelectrophoresed, CP2-a was transformed to CP2-d, and CP2-b was converted to CP2-a and CP2-d . The reaction center complex consisted of two major polypeptides of 47,000 and 40,000 Da and several minor polypeptides . CP2-b contained a 47,000-Da polypeptide together with 66,000- and 31,000-Da polypeptides, while CP2-a and CP2-d had only a 47,000-Da polypeptide . The apoprotein of CP2-c was a 40,000-Da polypeptide . Absorption spectra of CP2-a, -b, and -d were similar to each other but distinctly different from those of CP2-c at liquid nitrogen temperature . The reaction center complex showed two fluorescence emission bands at 686 and 694 nm at 77 degrees K . CP2-a, -b, and -d emitted the band at 694 nm, whereas the fluorescence peak at 686 nm was associated with CP2-c . It is concluded that the photosystem 2 reaction center complex contains two chlorophyll-binding subunits, CP2-d (or CP2-a) which may be the site of the primary photochemistry of photosystem 2 and CP2-c which may function as the antenna of the reaction center of photosystem 2.

Antonie Van Leeuwenhoek, 1983 Sep, 49(3), 313 - 26
Use of thermophilic lactic starters in the dairy industry; Auclair J et al.; The use of thermophilic lactic starters in the dairy industry is discussed . The functions of the thermophilic lactic starters in cooked cheese production and its ripening, the bacteria of the starter cultures and various types of starters are described.

Biochem Biophys Res Commun, 1983 Aug 12, 114(3), 907 - 12
The synthesis of enzyme-bound ATP by the F1-ATPase from the thermophilic bacterium PS3 in 50% dimethylsulfoxide; Yoshida M; Purified TF1 (F1-ATPase from a thermophilic bacterium PS3) synthesizes enzyme-bound ATP from medium Pi and enzyme-bound ADP in the presence of 50% dimethylsulfoxide (DMSO) . Once ATP was formed on the enzyme, it was not released even after removal of DMSO and Pi from the solution . The half maximal concentration of medium Pi for ATP synthesis was 1mM . The pH optimum for enzyme-bound ATP formation was about 6.5 . Under the optimum conditions, a yield of up to 0.8 mol of ATP/mol of TF1 was obtained.

Nucleic Acids Res, 1983 Aug 11, 11(15), 5131 - 45
Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila; Blackburn EH et al.; We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila . It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine . Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3' . A specific pattern of doubly methylated GATC sequences was found; hemimethylated sites were not detected . The patterns and levels of methylation of these sites did not change significantly in different physiological states . A molecular form of the rDNA found in the newly developing macronucleus and for several generations following the sexual process, conjugation, contained no detectably methylated GATC sites . However, both the bulk macronuclear DNA and palindromic rDNA from the same macronuclei were methylated . Possible roles for N-6-methyladenine in macronuclear DNA are discussed in light of these findings.

Biochim Biophys Acta, 1983 Aug 2, 740(3), 300 - 12
Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria; Cammarano P et al.; Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max . growth temp., 70 degrees C) and Escherichia coli (max . growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions . The principal results are as follows . Subunits of C . acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B . acidocaldarius (Tm = 77 degrees C) and E . coli counterparts (Tm = 72 degrees C) . Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species . The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C . acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B . acidocaldarius and 55% G . C for E . coli . The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s) . The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C . acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E . coli ribosomes . Compared to E . coli the C . acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.

J Protozool, 1983 Aug, 30(3), 592 - 8
Nucleosome phasing in Tetrahymena macronuclei; Pratt K et al.; Core-protected DNA can drive only 60% of the Tetrahymena thermophila macronuclear genome into duplexes in hybridization experiments . This core-protected DNA therefore contains only a subset of the genome complexity . We interpret this to mean that a large fraction, if not all, of the genome is phased with respect to nucleosome placement . Among the sequences present in total DNA and absent from core-protected DNA are most of the sequences containing N6-methyladenine (MeAde) residues, consistent with our previous demonstration that most of these residues lie in linker DNA . We show that these results are not due to artifacts resulting from the small size of the DNA driver, nor are they due to any sequence preferences exhibited by staphylococcal (staph) nuclease . This is the first evidence that nucleosome phasing may be a bulk genome characteristic.

Eur J Clin Microbiol, 1983 Aug, 2(4), 367 - 77
Nucleic acids in the classification of Campylobacters; Owen RJ; The importance of campylobacters in human disease has stimulated improvements in the methods for identification of strains from hospitals and the environment . Reliable and accurate identification depends on a sound classification for which nucleic acid analyses provide fundamental information about species relationships . Studies on the genus Campylobacter show that the genome DNA of species have base compositions of 29 to 38 mol% G + C and molecular weights of 1.54 X 10(9) to 2.31 X 10(9) . Campylobacter fetus, the type species, has a mean G + C content of 35.7 mol% whereas those of the thermophilic species Campylobacter jejuni, Campylobacter coli and the NARTC group (Campylobacter laridis) are between 31.5 and 32.6 mol% . DNA-DNA hybridizations, which are useful in clarifying relationships at the species level, show clear differences between most Campylobacter taxa . Distinct DNA sequence relatedness differences confirmed Campylobacter jejuni, Campylobacter coli and the NARTC group were separate species . The correlations between nucleic acid data and conventional biochemical test results, serology and fatty acid profiles are discussed . Further work is needed on the nucleic acids of Campylobacter sputorum and the various allied campylobacters.

Nucleic Acids Res, 1983 Jul 25, 11(14), 4667 - 76
Sequences of the 5S rRNAs of the thermo-acidophilic archaebacterium Sulfolobus solfataricus (Caldariella acidophila) and the thermophilic eubacteria Bacillus acidocaldarius and Thermus aquaticus; Dams E et al.; We have determined the nucleotide sequences of the 5 S rRNAs of three thermophilic bacteria: the archaebacterium Sulfolobus solfataricus, also named Caldariella acidophila, and the eubacteria Bacillus acidocaldarius and Thermus aquaticus . A 5 S RNA sequence for the latter species had already been published, but it looked suspect on the basis of its alignment with other 5 S RNA sequences and its base-pairing pattern . The corrected sequence aligns much better and fits in the universal five helix secondary structure model, as do the sequences for the two other examined species . The sequence found for Sulfolobus solfataricus is identical to that determined by others for Sulfolobus acidocaldarius . The secondary structure of its 5 S RNA shows a number of exceptional features which distinguish it not only from eubacterial and eukaryotic 5 S RNAs, but also from the limited number of archaebacterial 5 S RNA structures hitherto published . The free energy change of secondary structure formation is large in the three examined 5 S RNAs.

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