Biochim Biophys Acta, 1992 Nov 20, 1160(2), 206 - 12 Expression of a hyperthermophilic aspartate aminotransferase in Escherichia coli; Arnone MI et al.; The gene for an archaebacterial hyperthermophilic enzyme, aspartate aminotransferase from Sulfolobus solfataricus (AspATSs), was expressed in Escherichia coli and the enzyme purified to homogeneity . A suitable expression vector and host strain were selected and culture conditions were optimized so that 6-7 mg of pure enzyme per litre of culture were obtained repeatedly . The recombinant enzyme and the authentic AspATSs are indistinguishable: in fact, they have the same molecular weight, estimated by means of SDS-PAGE and gel filtration, the same Km values for 2-oxo-glutarate and cysteine sulphinate and the same UV-visible spectra . Moreover, recombinant AspATSs is thermophilic and thermostable just as the enzyme extracted from Sulfolobus solfataricus . The protocol described may be used to produce thermostable arachaebacterial enzymes in mesophilic hosts. J Biol Chem, 1992 Nov 15, 267(32), 22787 - 97 The organization and expression of essential transcription translation component genes in the extremely thermophilic eubacterium Thermotoga maritima; Liao D et al.; A 5789-nucleotide-long EcoRI fragment from the genome of Thermotoga maritima, identified by cross-hybridization to L11, L1, L10, and L12 ribosomal protein gene sequences from Escherichia coli, was cloned and sequenced . The fragment encodes five tRNAs (tRNA(met1), anticodon complementary to AUG; tRNA(met2), AUG; tRNA(thr), ACA; tRNA(tyr), UAC; tRNA(trp), UGG), the transcription termination-antitermination factor nusG, the four 50 S subunit ribosomal proteins L11, L1, L10, and L12, and the amino-terminal portion of the RNA polymerase beta subunit protein . The five tRNA genes, the nusG gene, and the L11, L1, L10, and L12 ribosomal protein genes form a complex transcription unit . Transcripts appear to be initiated from an upstream promoter, P1, located in front of the tRNA(met1) gene and from three internal promoters: P2 is located immediately in front of the tRNA(met2) gene; PL10 is near the beginning of the L1-L10 intergenic space, and PL12 is at the end of the L10 gene sequence . The tRNA sequences are excised from the leader regions of the P1- and P2-initiated transcripts . Three putative but potentially important regulatory sequences were identified within this operon: an L1 translational control site, a transcription attenuator, and a strong rho-independent terminator . The strong terminator located distal to the L12 gene overlaps a fifth promoter, P beta, which is used to initiate transcripts of the downstream RNA polymerase beta subunit gene . The T . maritima NusG protein exhibits 43% amino acid sequence identity when aligned to the E . coli protein; the alignment is interrupted by a large 171-amino acid-long insertion into the T . maritima protein after codon 45. J Protozool, 1992 Nov-Dec, 39(6), 719 - 23 Immobilization antigens from Tetrahymena thermophila are glycosyl-phosphatidylinositol-linked proteins; Ko YG et al.; We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class . Following exposure of the strains to {3H}ethanolamine or {3H}myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide electrophoresis gels . Furthermore, antibodies raised to the i-antigens of the T . thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain . The lipid moieties labeled by {3H}myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria . However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from {3H}myristate were rapidly cleaved from the putative i-antigens . On the basis of available data, it was concluded that T . thermophila i-antigens contain covalently-linked glycosyl-phosphatidylinositol anchors. Nucleic Acids Res, 1992 Nov 11, 20(21), 5607 - 15 Structure determination of two new amino acid-containing derivatives of adenosine from tRNA of thermophilic bacteria and archaea; Reddy DM et al.; Two new nucleosides have been identified in unfractionated transfer RNA of two thermophilic bacteria, Thermodesulfobacterium commune, and Thermotoga maritima, six hyperthermophilic archaea, including Pyrobaculum islandicum, Pyrococcus furiosus and Thermococcus sp . and two mesophilic archaea, Methanococcus vannielii and Methanolobus tindarius . Structures were determined primarily by mass spectrometry, as 3-hydroxy-N-{{(9-beta-D-ribofuranosyl-9H-purin-6- yl)amino}carbonyl}norvaline, (hn6A), structure 1, and 3-hydroxy-N-{{(9-beta-D-ribofuranosyl-9H-2-methylthiopurin-6- yl)amino}carbonyl}norvaline (ms2hn6A), 2 . The amino acid side chain was characterized as 3-hydroxynorvaline (3) by gas chromatography-mass spectrometry of the trimethylsilyl derivative after cleavage from 1 and 2 by alkaline hydrolysis . Evidence for the amino acid-purine carbamoyl linkage was obtained from the collision-induced dissociation mass spectrum of trimethylsilylated 1, and the total structure was confirmed by chemical synthesis of 1. J Biol Chem, 1992 Nov 5, 267(31), 22087 - 94 Lactose transport system of Streptococcus thermophilus . Functional reconstitution of the protein and characterization of the kinetic mechanism of transport; Foucaud C et al.; The kinetic mechanism of the lactose transport system of Streptococcus thermophilus was studied in membrane vesicles fused with cytochrome c oxidase containing liposomes and in proteoliposomes in which cytochrome c oxidase was coreconstituted with the lactose transport protein . Selective manipulation of the components of the proton (and sodium) motive force indicated that both a membrane potential and a pH gradient could drive transport . The galactoside/proton stoichiometry was close to unity . Experiments which discriminate between the effects of internal pH and delta pH as driving force on galactoside/proton symport showed that the carrier is highly activated at alkaline internal pH values, which biases the transport system kinetically toward the pH component of the proton motive force . Galactoside efflux increased with increasing pH with a pKa of about 8, whereas galactoside exchange (and counterflow) exhibited a pH optimum around 7 with pKa values of 6 and 8, respectively . Imposition of delta pH (interior alkaline) retarded the rate of efflux at any pH value tested, whereas the rate of exchange was stimulated by an imposed delta pH at pH 5.8, not affected at pH 7.0, and inhibited at pH 8.0 and 9.0 . The results have been evaluated in terms of random and ordered association/dissociation of galactoside and proton on the inner surface of the membrane . Imposition of delta psi (interior negative) decreased the rate of efflux but had no effect on the rate of exchange, indicating that the unloaded transport protein carries a net negative charge and that during exchange and counterflow the carrier recycles in the protonated form. J Biol Chem, 1992 Nov 5, 267(31), 22014 - 7 Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn; Kimura S et al.; From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S . (1992) J . Biol . Chem . 267, 21535-21542) . Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability . Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively . We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability. Appl Environ Microbiol, 1992 Nov, 58(11), 3455 - 65 Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli; Wright RM et al.; Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli . Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside . Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes . One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E . coli . It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively . Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C . BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose . The DNA sequence of the 2.2-kb insert carrying bglB has been determined . An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1. J Gen Microbiol, 1992 Nov, 138 ( Pt 11), 2293 - 303 Campylobacter helveticus sp . nov., a new thermophilic species from domestic animals: characterization, and cloning of a species-specific DNA probe; Stanley J et al.; An atypical group of thermophilic catalase-negative Campylobacter strains, the 'CH' (Swiss) group, can be recovered from faeces of domestic cats and dogs after selection by filtration, or with the antibiotic cefoperazone . This group of strains shows no relative DNA homology with any species in rRNA superfamily VI (Vandamme et al., 1991, International Journal of Systematic Bacteriology 41, 88-103) except with four thermophilic Campylobacter species, notably C . upsaliensis . The group is homogeneous and possesses a DNA base composition, cellular morphology at the electron microscope level and phenotypic properties characteristic of Campylobacter . Nonetheless it is distinct from known species of Campylobacter in terms of conventional bacteriological tests, total cellular protein profile, rRNA gene profile, and genomic DNA homology . On the basis of an integrated study of phenotype and genotype, we conclude that these bacteria constitute a previously undescribed species for which we propose the name Campylobacter helveticus sp . nov . A species-specific recombinant DNA probe was cloned from the designated type strain (NCTC 12470) for use in identification and further analysis of the epidemiology, pathogenicity and transmission of C . helveticus. J Biochem (Tokyo), 1992 Nov, 112(5), 714 - 8 Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis; Shiratsuchi A et al.; Anthranilate synthase I (ASI) of Bacillus caldotenax, a thermophilic bacterium, was purified from a plasmid-bearing Escherichia coli and characterized . The molecular weight determination under native and denaturing conditions revealed that it was a monomeric enzyme of M(r) = 54,000 . The N-terminal amino acid sequence is the same as expected from DNA sequence of trpE except that the N-terminal methionine is lacking . All four cysteines in the molecule could be titrated with 5,5'-dithiobis (2-nitrobenzoic acid) in more than 8 M urea . The purified enzyme retained its full enzymatic activity after being heated at 60 degrees C . Six mutated genes for the ASI with histidine in place of each conserved arginine, Arg321, Arg353, Arg358, Arg416, Arg429, and Arg452, were prepared by site-directed mutagenesis . All the mutated genes except one, the gene encoding an ASI mutant with histidine in place of Arg452 (R452H ASI) complemented an E . coli (trpE) . The mutated ASIs were purified and compared with the wild type ASI . No distinctive differences in enzymatic properties were found between the wild type and the enzymatically active mutated ASIs . R452H ASI was enzymatically inactive, though its conformation seemed to be unchanged after the substitution based on CD spectra and the SH titration curve. Chem Pharm Bull (Tokyo), 1992 Nov, 40(11), 3017 - 20 Studies on thermophile products . V . Immunosuppressive profile in vitro of Bacillus stearothermophilus component, Fr.5-B; Kohama Y et al.; The immunosuppressive profile of Bacillus stearothermophilus UK563 component, Fr.5-B, is presented in in vitro studies . Fr.5-B (0.1-1000 ng/ml), provided it was added at the initiation of mixed leukocyte reaction (MLR), inhibited dose-dependently the incorporation of tritiated thymidine ({3H}TdR) into mouse spleen cells and human peripheral blood lymphocytes . Even the addition of Fr.5-B 48 h after the onset of culture suppressed mouse MLR, unlike cyclosporin A (CYA) . Fr.5-B significantly inhibited cytotoxic T lymphocyte generation determined by {3H}TdR-release micro-cytotoxicity assay by using mouse mastocytoma P815 as targets . Moreover, this component decreased dose-dependently the expression of class II major histocompatibility molecules (Ia) on mouse peritoneal macrophages induced by concanavalin A supernatant . The present results revealed the unique immunosuppressive property of Fr.5-B which was different from that of CYA. Isr J Med Sci, 1992 Nov, 28(11), 772 - 5 Mycobacterium xenopi, a potential human pathogen; Lavy A et al.; Mycobacterium xenopi is infrequently recognized as a cause of pulmonary disease . During a 12-year survey (1978-89), . 108 strains of this Mycobacterium were isolated from 90 persons and 6 hot water samples . From 87 patients 89 occasional strains of M . xenopi were isolated, and 3 patients were diagnosed as having pulmonary mycobacteriosis caused by it . The treatment and the response in these three cases were variable, depending on clinical conditions and sensitivity to drugs . Most of the strains isolated came from patients hospitalized at the Barzilai Hospital, Ashkelon, therefore a local environmental contamination was suspected . The suspicion was confirmed by the isolation of this thermophile organism from the hot water samples of the above hospital. Exp Lung Res, 1992 Nov-Dec, 18(6), 761 - 73 Mouse hypersensitivity pneumonitis: depletion of NK cells abrogates the spontaneous regression phase and leads to massive fibrosis; Denis M; The contribution of natural killer cells (NK) in the progression of mouse hypersensitivity pneumonitis induced by repeated intranasal instillations with the thermophilic actinomycete Faeni rectivirgula was examined . These instillations determined a very large increase in the lung index (ca . twofold at 3 weeks), used as a measure of inflammation . In addition, this instillation was associated with a tenfold increase in the number of cells recovered in the bronchoalveolar lavage (BAL) at 3 weeks and thereafter . Most of these cells were macrophages, whereas T lymphocyte numbers increased at 6 weeks and thereafter . The instillations were also associated with a substantial fibrotic response in the lungs, as seen by large increases in hydroxyproline levels in the lungs . This fibrotic response, however, diminished after 6 weeks of instillations . Similarly, examination of histological preparations of lungs of challenged mice showed that F . rectivirgula induced inflammatory infiltrates of macrophages, lymphocytes, and neutrophils . The severity of the lesions were reduced in mice given more than 6 weeks of the actinomycete challenge . The involvement of NK cells on the development of this pulmonary pathology was determined by infusing F . rectivirgula-challenged mice with anti-NK 1.1 antibody . The depletion protocol was validated by verifying that such treatments effectively blocked lung NK cell activity . Such NK cell-depleted mice responded to the F . rectivirgula challenge with an increased lung index at 9 and 12 weeks, compared to mice challenged with F . rectivirgula and given control antibody . NK cell-depleted mice also responded to the actinomycete with a superior cellular recruitment in the BAL, with this increase mostly mediated by macrophages . Similarly, NK cell-depleted mice developed a fibrotic response that was much higher than that seen in control challenged mice, at 6, 9, and 12 weeks after initiation of the transnasal instillations . This was corroborated by scoring the severity of the histopathological lesions, which showed that NK cell-depleted mice had more severe lesions than challenged control mice . The importance of NK cells was confirmed by demonstrating that mice given anti-NK 1.1, challenged with F . rectivirgula and reconstituted with Percoll gradient-enriched lung NK cells had hydroxyproline levels at 9 and 12 weeks that were comparable to that seen in intact mice, as well as a histopathological score similar to control challenged mice . Overall, this suggests that in the course of a pulmonary inflammatory response, NK cells exert a suppressive effect on cellular recruitment in the BAL, contribute to down-regulating the inflammatory response, and are involved in blocking the appearance of fibrosis. Br Vet J, 1992 Nov-Dec, 148(6), 547 - 56 Campylobacter infections in calves, piglets, lambs and kids in Trinidad; Adesiyun AA et al.; Faeces or rectal swabs from 689 diarrhoeic and non-diarrhoeic animals were cultured for thermophilic campylobacters and their antibiograms were determined . Three hundred and fifteen (45.7%) samples were positive for Campylobacter . Piglets had the highest prevalence, 79.3% (233/294) and lambs, the lowest with 17.9% (15/84) being positive . The difference was statistically significant (P < or = 0.01; chi 2) . In calves, 20.5% (60/293) and in kids 38.9% (7/18) were positive for campylobacters . The prevalence of infection was not significantly (P > or = 0.05; chi 2) different between diarrhoeic (46.1%) and non-diarrhoeic (45.1%) animals nor between male (47.5%) and female (43.8%) . The frequency of isolation of campylobacters harvested from semi-intensively managed animals (75.4%) was, however, significantly higher (P < or = 0.001; chi 2) than from intensively or extensively managed animals . Overall, C . coli strains (32.8%) were more frequently isolated than C . jejuni strains (12.9%) and the difference was significant (P < or = 0.001; chi 2) . Biotype I accounted for 67.3% (152/226) of C . coli and 64.0% (57/89) of C . jejuni strains isolated . A total of 245 (77.8%) strains of Campylobacter exhibited resistance to one or more antibiotics and was highest to streptomycin (76.5%), kanamycin (28.6%) and neomycin (26.7%) . It was concluded that Campylobacter infections were widespread in livestock in Trinidad, particularly C . coli in piglets. J Protozool, 1992 Nov-Dec, 39(6), 655 - 62 An assessment of proteolytic enzymes in Tetrahymena thermophila; Straus JW et al.; Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity . Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate . Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity . Activity was also strongly diminished by increasing concentrations of divalent cations . Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity . Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity . Phenylmethanesulfonyl fluoride had no apparent effect . Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000 . Five of these apparent proteases were detected in 70% ammonium sulfate precipitates . Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation . Cellular extracts of T . thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents . Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases. J Clin Microbiol, 1992 Nov, 30(11), 3019 - 23 Fatal disseminated infection caused by Myceliophthora thermophila, a new agent of mycosis: case history and laboratory characteristics; Bourbeau P et al.; We report a case of human infection caused by the hyphomycete Myceliophthora thermophila . A 7-year-old male with neurofibromatosis (type 1) was diagnosed in 1987 with acute myeloblastic leukemia associated with the chromosomal abnormality monosomy 7 . The patient experienced multiple serious infections over a three-year period before expiring in 1990 while in the end stage of leukemia . Autopsy findings included fungal vegetations of the left atrium, ascending aorta, and pulmonary arteries and fungal invasion of both lungs . Cultures yielded M . thermophila . We believe that this is the first reported fatality caused by M . thermophila. J Bacteriol, 1992 Nov, 174(22), 7458 - 62 Sequence of the S-layer gene of Thermus thermophilus HB8 and functionality of its promoter in Escherichia coli; Faraldo MM et al.; The nucleotide sequence of the slpA gene, which is responsible for the synthesis of the S-layer protein of Thermus thermophilus HB8, is described . This gene is transcribed as a unit in which the coding region is preceded by a 127-base-long leader mRNA sequence . The promoter region is also recognized by the RNA polymerase of Escherichia coli because of the presence of homologous -35 and -10 boxes . Homologies with other promoters from Thermus spp . are also presented. J Bacteriol, 1992 Nov, 174(22), 7227 - 34 Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome; Stettler R et al.; A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI . The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests . The derived map is circular, and the genome size was estimated to be 1,623 kb . Several cloned genes were hybridized to restriction fragments to locate their positions on the map . Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome . In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis . The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed. J Bacteriol, 1992 Nov, 174(21), 6840 - 3 Chemolithoautotrophic assimilation of dinitrogen by Streptomyces thermoautotrophicus UBT1: identification of an unusual N2-fixing system; Gadkari D et al.; Streptomyces thermoautotrophicus UBT1, which was isolated previously from a burning charcoal pile, was shown to utilize N2 as a sole nitrogen source when growing chemolithoautotrophically with CO or H2 plus CO2 under aerobic conditions at 65 degrees C . Doubling times under diazotrophic conditions were 10 h . S . thermoautotrophicus is a new CO- or H2-oxidizing, obligately chemolithoautotrophic, thermophilic, free-living, aerobic, N2-fixing streptomycete . Its ability to fix N2 was also evident from (i) the incorporation of substantial amounts of 15N2 (about 13%) into cell material, (ii) the formation of H2 during diazotrophic growth, (iii) the repression of 15N2 assimilation and H2 formation by ammonia, and (iv) culture growth yields with N2 as a nitrogen source that were significantly higher than those without any added nitrogen compounds (ca . 2.4 versus < 0.1 mg {dry weight}) . The N2-fixing system of S . thermoautotrophicus exhibited several properties not apparent in the diazotrophic bacteria studied so far, since it was (i) incapable of reducing acetylene to ethylene or ethane and (ii) resistant to inhibition by acetylene or ethylene (5% {vol/vol} each), CO (40 to 70% {vol/vol}), or H2 (40% {vol/vol}) . Under stringent conditions, nifH and nifDK gene probes from Klebsiella pneumoniae did not hybridize with total DNA from S . thermoautotrophicus. Mikrobiol Zh, 1992 Nov-Dec, 54(6), 32 - 40 Screening for restriction endonucleases in methane-oxidizing bacteria; Romanovskaya VA et al.; 51 methane-oxidizing bacteria strains such as Methylomonas methanica, M . rubra, Methylococcus capsulatus, M . thermophilus, M . luteus, M . ucrainicus, M . whittenburyi, Methylosinus trichosporium, M . sporium, Methylocystis parvus isolated from various ecological niches and geographical regions of the Ukraine and also the strains received from R . Whittenbury and Y . Heyer were screened for restriction endonucleases . Type II restriction endonucleases were detected in IMV B-3112 (= 12 b), IMV B-3027 (= 26), IMV B-3019 (= 9 c), IMV B-3017 (= 17 c), IMV B-3226 (= 26 v), IMV B-3033 (= Y), IMV B-3100 (= 100) and IMV B-3494 (= 1E494) . The results obtained were indicative of relatively high frequency of restriction enzymes occurrence in methane-oxidizing bacteria . There were Kpn I (Asp 7181) restriction endonuclease isoschizomers in crude extracts of IMV B-3112, B-3017, B-3019, B-3027 isolated from fresh-water silt as well as in IMV B-3226 strain isolated from waste-water silt . Although these isolates had bee previously considered as untypical strains of M . ucrainicus, more detailed study of their properties allowed placing them with Methylovarius luteus (= Methylococcus luteus) . IMV B-3494 strain was identified as Methylococcus capsulatus . Strain IMV B-3033 had earlier been allocated to Methylovarius whittenburyi (= Methylococcus whittenburyi) . Specificity of restriction endonucleases of this strain was not tested . Therefore, for the first time restriction endonucleases were detected in methane-oxidizing bacteria . 8 strains (3 species) among 51 strains (13 species) were found to produce restriction endonucleases displaying three different types of specificity in the least . Producers of restriction endonucleases having Kpn I (Asp 7181) specificity were isolated from different water and silt samples of the Dnieper flood-land more than 20 years ago. Eur J Biochem, 1992 Nov 1, 209(3), 1013 - 8 A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei; Schmitz RA et al.; We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II . Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-iron-sulfur protein . We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is a molybdenum-iron-sulfur protein . The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten (< 0.01 mol) . The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide . The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour . The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the alpha and beta subunits were identical up to residue 23, indicating that the two proteins are isoenzymes . The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution. FEBS Lett, 1992 Oct 26, 311(3), 192 - 4 Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA(Phe); Stepanov VG et al.; Phenylalanyl-tRNA synthetase from the extreme thermophilic bacterium Thermus thermophilus can incorporate more than one molecule of phenylalanine into the tRNA(Phe) . It is shown that the 'hyperaminoacylated' tRNA(Phe) is the bis-2',3'-O-phenylalanyl-tRNA(Phe), and its formation is typical for the thermophilic enzyme but does not occur for E . coli phenylalanyl-tRNA synthetase under the same conditions. Nucleic Acids Res, 1992 Oct 25, 20(20), 5389 - 96 Molecular characterisation of a DNA ligase gene of the extremely thermophilic archaeon Desulfurolobus ambivalens shows close phylogenetic relationship to eukaryotic ligases; Kletzin A; A 3382 bp fragment containing a gene for a DNA ligase from the extremely thermophilic, acidophilic, and facultatively anaerobic archaeon (archaebacterium) Desulfurolobus ambivalens was cloned and sequenced . The deduced amino acid sequence (600 amino acids, 67619 molecular weight) showed 30-34% sequence identity with the ATP-dependent eucaryal (eukaryotic) DNA ligases of Schizosaccharomyces pombe, Saccharomyces cerevisiae, the human DNA ligase I, and with the Vaccinia DNA ligase . Distant similarity to the DNA ligases from the bacteriophages T3, T4, T6, T7 and the African swine fever virus was found, whereas no similarities were detectable to the NAD-dependent DNA ligases from the bacteria (eubacteria) Escherichia coli and Thermus thermophilus, to the ATP-dependent RNA-ligase of bacteriophage T4, and to the tRNA-Ligase from S.cerevisiae . A detailed comparison of the phylogenetic relationship of the amino acid sequences of all known DNA and RNA ligases is presented including a complete alignment of the ATP-dependent DNA ligases . The in vivo-transcription initiation and termination sites of the D.ambivalens gene were mapped . The calculated transcript length was 1904-1911 nt. J Biol Chem, 1992 Oct 25, 267(30), 21650 - 5 Primary structure of the alanine carrier protein of thermophilic bacterium PS3; Kamata H et al.; Purified alanine carrier proteins were cleaved into peptides either chemically after solubilization in 1,1,1,3,3,3-hexafluoro-2-propanol or proteolytically with lysylendopeptidase . From the amino acid sequence analyses of these peptides, we synthesized a DNA probe and utilized it for successful cloning of a gene encoding the alanine carrier protein (acp gene) . The 5'-flanking region was determined by an inverse polymerase chain reaction, and an open reading frame consisting of 1,335 nucleotides was found . The amino acid sequence deduced from the open reading frame consists of 445 amino acids, and all the partial amino acid sequences determined are included in the sequence . Although the calculated M(r) of 47,803 is significantly larger than the apparent M(r) of 42,500 as reported previously (Hirata, H., Kambe, T., and Kagawa, Y . (1984) J . Biol . Chem . 259, 10653-10656), an in vitro translation experiment revealed that the product of the acp gene migrates at a position coinciding with that of the purified alanine carrier . Hydropathy analysis suggests that the protein contains at least 8 hydrophobic segments presumably spanning membrane . A homology search on a database reveals relatively high scores of homology with either the Escherichia coli melibiose carrier or the human Na+/glucose symporter, particularly in the region from Leu246 to Glu286 . Furthermore, the region also reveals low but significant similarities to other Na(+)-coupled symporters. J Biol Chem, 1992 Oct 25, 267(30), 21535 - 42 Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart; Kimura S et al.; Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M . (1992) J . Biol . Chem . 267, 10184-10192) . The identity in the amino acid sequences of these enzymes is 52% . As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability . A variety of mutant proteins of E . coli RNase HI were constructed and analyzed for protein stability . In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T . thermophilus RNase H . Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability . Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative . Replacement of all four regions (R4-R7) gave the most stable mutant protein . The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E . coli RNase HI . These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner. Biochem J, 1992 Oct 15, 287 ( Pt 2), 367 - 74 The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1; Coolbear T et al.; The proteinase from the extremely thermophilic Bacillus strain EA.1 exhibits maximum stability at a pH of approx . 6.5 . In the presence of calcium ions the half-life at 95 degrees C of the enzyme at this pH was 17 min, and loss of activity followed first-order decay kinetics . The role of metal ions in the activity and stability of the enzyme was studied using the holoenzyme, the metal-depleted apoenzyme, and a zinc-enriched apoenzyme preparation . Zinc and calcium ions were the preferred bivalent cations for the active site and stabilization site(s) respectively . Stabilization by metal ions was not in itself a highly stringent process, but ions other than calcium which stabilized the enzyme generally had a concomitant inhibitory effect on activity . Inhibition and stabilization of the enzyme by cations were concentration-dependent effects and certain ions activated the apoenzyme but not the holoenzyme . Manganese(II) ions conferred some stability and also activated the enzyme, but in the latter case were not as effective as zinc ions . The results are discussed with reference to the ionic radii, co-ordination number and preferred ligand donors of the ions . Mercury(II) ions severely compromised enzyme activity and stability, and the effects of thiol-reactive agents suggest that thiol groups also have a role in enzyme integrity. FEBS Lett, 1992 Oct 12, 311(1), 22 - 4 Crystallization of the cpn60/cpn10 complex ('holo-chaperonin') from Thermus thermophilus; Lissin NM et al.; A stable complex of the chaperonins, cpn60 and cpn10 (Escherichia coli GroEL and GroES homologues), from the extremely thermophilic bacterium Thermus thermophilus has been isolated and crystallized . The crystals have dimensions up to 30 x 200 x 200 microns . Ultra-thin sections of the crystals estimated by electron microscopy showed a rectangular lattice with unit cell parameters of a = 17 nm, b = 27 nm, gamma = 90 degrees. Nucleic Acids Res, 1992 Oct 11, 20(19), 5047 - 52 Identification of the CTAG-recognizing restriction-modification systems MthZI and MthFI from Methanobacterium thermoformicicum and characterization of the plasmid-encoded mthZIM gene; Nolling J et al.; Two CTAG-recognizing restriction and modification (R/M) systems, designated MthZI and MthFI, were identified in the thermophilic archaeon Methanobacterium thermoformicicum strains Z-245 and FTF, respectively . Further analysis revealed that the methyltransferase (MTase) genes are plasmid-located in both strains . The plasmid pFZ1-encoded mthZIM gene of strain Z-245 was further characterized by subcloning and expression studies in Escherichia coli followed by nucleotide sequence analysis . The mthZIM gene is 1065 bp in size and may code for a protein of 355 amino acids (M(r) 42,476 Da) . The deduced amino acid sequence of the M.MthZI enzyme shares substantial similarity with four distinct regions from several m4C- and m6A-MTases, and contains the TSPPY motif that is so far only found in m4C-MTases . Partially overlapping with the mthZIM gene and in reverse orientation, an additional ORF was identified with a size of 606 bp potentially coding for a protein of 202 amino acids (M(r) 23.710 Da) . This ORF is suggested to encode the corresponding endonuclease R.MthZI. Biochemistry, 1992 Oct 6, 31(39), 9376 - 87 Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates; Goldbeck RA et al.; Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented . The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified . TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex . There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase {Goldbeck, R . A., Dawes, T . D., Einarsdottir, O., Woodruff, W . H., & Kliger, D . S . (1991) Biophys . J . 60, 125-134} . Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later . Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein . The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3 . Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3 . The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin. J Cell Sci, 1992 Oct, 103 ( Pt 2), 565 - 70 Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: studies with Tetrahymena thermophila; Leick V; Receptor-mediated binding of leukocyte chemotactic peptide, N-formylMet-Leu-Phe (fMLP), occurs in the ciliated protozoon Tetrahymena thermophila . In vivo labelling of the cells with N-formylMet-Leu-{3H}Phe ({3H}fMLP) shows that the cells bind the ligand with high affinity (KD = 4 x 10(-9) M to 1 x 10(-8) M) . Moreover, Scatchard transformations of the binding data show that there are about 5 x 10(5) binding sites per cell on the cell surface . Two fluorescent derivatives of leukocyte chemotactic peptide, N-dansylMet-Leu-Phe (dansMLP) and N-formylMet-Leu-Phe-(N-dansyl-)Lys (fMLPdanLys) compete for the N-formylMet-Leu-Phe (fMLP) binding sites on the cell surface . Moreover, both derivatives have retained significant chemoattracting potentials . Fluorescence from dansMLP, but not from fMLPdansLys and dansyl-beta-endorphin, is internalized preferentially into small vesicles . The differences may, however, reflect that the fluorescence from the dansyl group is strongly quenched by a hydrophilic microenvironment when using the two latter peptide derivatives . In contrast, the dansyl group from dansMLP must be assumed to be embedded in a hydrophobic microenvironment in the vesicular membrane or membrane protein . Rhodamine-labelled bovine serum albumin, egg albumin and cytochrome c as well as dansylated bovine serum albumin, which are poor chemoattractants, are preferentially seen to be internalized into large vesicles (food vacuoles). J Bioenerg Biomembr, 1992 Oct, 24(5), 441 - 5 The alpha beta complexes of ATP synthase: the alpha 3 beta 3 oligomer and alpha 1 beta 1 protomer; Kagawa Y et al.; The basic structures of the catalytic portion (F1, alpha 3 beta 3 gamma delta epsilon) of ATP synthase are the alpha 3 beta 3 hexamer (oligomer with cooperativity) and alpha 1 beta 1 heterodimer (protomer) . These were reconstituted from the alpha and beta subunits of thermophilic F1 (TF1), and the alpha 3 beta 3 hexamer was crystallized . On electrophoresis, both the dimer and hexamer showed bands with ATPase activity . Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics . The formation of the hexamer required neither nucleotide nor Mg . The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP . The hexamer, like mitochondrial F1 and TF1, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer. J Anim Sci, 1992 Oct, 70(10), 3178 - 87 Effects of bacterial inoculation of high-moisture ear corn on its aerobic stability, digestion, and utilization for growth by beef steers; Phillip LE et al.; High-moisture ear corn (HMEC) was treated with specific bacterial inoculants and evaluated for its aerobic stability and utilization for growth by beef steers . Immediately after harvest, HMEC (65% DM) was ensiled in tower silos after being either untreated (control) or treated with the following inoculants: 1) Ecosyl (E); 2) Ecosyl plus Serratia rubidaea (E + SR); and 3) Ecosyl plus Streptococcus thermophilus (E + ST) . A portion of HMEC was frozen immediately (-20 degrees C) and subsequently treated with eight bacterial inoculants before ensiling in laboratory silos; the fermented material was then exposed to air for 7 d for assessment of aerobic deterioration . The eight inoculants included the three used in the tower silos and four additional ones: Streptococcus thermophilus, Bacillus subtilis (BS), Serratia rubidaea, and a mixture of Ecosyl + B . subtilis (E + BS) . The growth trial was conducted for 112 d with 32 crossbred steers (average BW 296 kg) . A digestion trial was conducted, according to a 4 x 4 Latin square design, using an additional four steers (average BW 367 kg) . In both trials, steers were fed the same four diets containing inoculated (E, E + SR, and E + ST) or control HMEC . Upon exposure to air, Ecosyl-treated ensiled HMEC had the least increase in pH compared with other single inoculants; all inoculant treatments lessened (P less than .05) the increase in sample temperature compared with control . During aerobic exposure, treatment of HMEC with BS seemed to reduce the disappearance of water-soluble carbohydrates, whereas Ecosyl seemed to reduce lactic acid . Despite evidence of improved aerobic stability with Ecosyl and BS, inoculation of HMEC did not (P greater than .10) improve BW gain or feed efficiency; however, all inoculants reduced (P less than .05) digestibility of ADF. Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9196 - 200 Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants; Gaertig J et al.; Conjugating cells of the ciliate Tetrahymena thermophila were electroporated in the presence of plasmid DNA containing a paromomycin-resistant ribosomal RNA gene (rDNA) . Cells were selected with paromomycin following 12-24 hr of growth on nonselective medium . Resistant cells appeared after 2-3 days . Processing vectors containing the micronuclear rDNA and somatic vectors containing the macronuclear gene transformed the cells, with the former yielding frequencies up to 900 transformants per microgram of plasmid DNA . A ribosomal protein gene (rpL29) conferring cycloheximide resistance also transformed conjugating cells . The transformation efficiency of the plasmid containing only the rpL29 gene was increased by insertion of an rDNA replication origin and by cotransformation and preselection with an rDNA vector . These results indicate that electroporation can be used for the production of large numbers of transformed Tetrahymena. J Bacteriol, 1992 Oct, 174(20), 6424 - 31 Development of Thermus-Escherichia shuttle vectors and their use for expression of the Clostridium thermocellum celA gene in Thermus thermophilus; Lasa I et al.; We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VII-51B (ATCC 25105) and a Thermus sp . strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge . Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19 . The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermus-Escherichia cloning . Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained . As a general rule, those from T . aquaticus (pLU1 to pLU4) were more stable than those from the Thermus sp . (pMY1 to pMY3) . To probe their usefulness, we used one of the plasmids (pMY1) to clone in E . coli a modified form of the cellulase gene (celA) from Clostridium thermocellum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T . thermophilus HB8 . The hybrid product was expressed and exported by E . coli . When the gene was transferred by transformation into T . thermophilus, the cellulase protein was also expressed and secreted at 70 degrees C. Am J Respir Cell Mol Biol, 1992 Oct, 7(4), 441 - 6 Murine hypersensitivity pneumonitis: production and importance of colony-stimulating factors in the course of a lung inflammatory reaction; Denis M et al.; The release of colony-stimulating factors (CSFs) and their contribution to the inflammatory response in the lungs of mice exposed by the intranasal route to the actinomycete Faeni rectivirgula (150 micrograms/day, 3 days/wk), an important thermophilic actinomycete that determines farmer's lung in humans, was examined . Bronchoalveolar lavages (BAL) and lung homogenates of normal mice or saline-instilled mice contained undetectable levels (less than 0.5 U/ml) of the cytokines interleukin-3 (IL-3), colony-stimulating factor-1 (CSF-1), and granulocyte/macrophage colony-stimulating factor (GM-CSF) . Mice instilled with F . rectivirgula developed a CSF cytokine response early (24 h) after the instillation that increased and plateaued 2 wk later, and stayed high thereafter . Similarly, lung homogenates of F . rectivirgula-challenged mice contained significant levels of all three CSFs from 24 h after treatment until termination of the experiment . The offending agent itself, F . rectivirgula, was found to directly induce the secretion of IL-3 and GM-CSF from isolated mouse BAL cells and mouse splenocytes, at doses ranging from 1 to 100 micrograms/ml . This was not due to contaminating endotoxin, as inclusion of polymyxin B did not modify this release . Instillation of antibodies against the CSFs in mice challenged with F . rectivirgula did not modify the increase in BAL cell number determined by the challenge (11-fold increase in BAL cell number in F . rectivirgula-instilled mice at 3 wk, whether given anti-CSFs or not) . Moreover, direct intratracheal infusion of CSFs (5,000 U of IL-3/CSF-1/GM-CSF) every week did not change the cellular response seen in challenged mice.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1992 Oct, 6(19), 2845 - 56 Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax; Tolner B et al.; The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen . The nucleotide sequences of the gltTBs and gltTBc genes were determined . In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc) . Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions . These expression signals were functional in E . coli . The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions . The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E . coli K-12 (overall 57.2% identity) . Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles . In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons. Intern Med, 1992 Oct, 31(10), 1204 - 6 Hypersensitivity pneumonitis induced by Shiitake mushroom spores; Matsui S et al.; Hypersensitivity pneumonitis due to the inhalation of Shiitake mushroom spores was demonstrated in a 38-year-old woman . Symptoms of cough, nausea and malaise, and clinical findings of cyanosis, bibasilar crackles, reduced lung volumes, hypoxemia, leukocytosis, elevated ESR, positive C-reactive protein, and bilateral diffuse reticulonodular shadows on chest roentgenogram improved after the patient was removed from exposure . Alveolitis was demonstrated by transbronchial lung biopsy, as well as an increase in lymphocytes in bronchoalveolar lavage . Serum precipitins and specific IgG antibodies to an extract of Shiitake mushroom spores, but not to other common molds or mushroom body, were detected in serum . Provocative inhalation test with the extract of mushroom spores caused the same clinical symptoms and signs as experienced in the workroom . This is the first report of typical hypersensitivity pneumonitis induced by Shiitake mushroom spores . Mushroom spores as well as thermophilic actinomycetes must be considered a causative agents for mushroom worker's lung. Appl Environ Microbiol, 1992 Oct, 58(10), 3417 - 8 Differential amplification of rRNA genes by polymerase chain reaction; Reysenbach AL et al.; The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents . We have found that this method can result in differential amplification of different rRNA genes . In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods . The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes. FEBS Lett, 1992 Sep 28, 310(2), 157 - 61 A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution; Yaremchuk AD et al.; Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant . Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA . They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A . Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers . Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA . They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution . Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer. Biochim Biophys Acta, 1992 Sep 24, 1132(2), 219 - 21 Calmodulin cDNAs from two species of Tetrahymena; Takemasa T et al.; We describe the isolation and characterization of cDNAs encoding calmodulins of Tetrahymena thermophila and Tetrahymena pyriformis . It reveals that the deduced amino acid sequences of both calmodulins are precisely the same. J Biol Chem, 1992 Sep 15, 267(26), 18361 - 4 Properties of aspartate racemase, a pyridoxal 5'-phosphate-independent amino acid racemase; Yamauchi T et al.; Aspartate racemase from Streptococcus thermophilus contains no pyridoxal 5'-phosphate or other cofactors such as FAD, NAD+, and metal ions . It was affected by neither carbonyl reagents such as hydroxylamine nor sodium borohydride but was strongly inhibited by iodoacetamide and other thiol reagents . Aspartate, cysteate, and cysteine sulfinate were the only substrates . The Km values for L- and D-aspartate were 35 and 8.7 mM, respectively . The enzyme catalyzed the exchange of alpha-hydrogen of the substrate with the solvent hydrogen . Racemization of L-aspartate in 2H2O showed an overshooting in the optical rotation of aspartate before the substrate was fully racemized . This shows that the removal of alpha-hydrogen of the substrate is at least partially rate-determining . When L- or D-aspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer . The results strongly suggest that aspartate racemase contains two hydrogen acceptors. J Mol Biol, 1992 Sep 5, 227(1), 114 - 21 Structural organization of the genes encoding the small nuclear RNAs U1 to U6 of Tetrahymena thermophila is very similar to that of plant small nuclear RNA genes; Orum H et al.; We report the sequences of the genes encoding the small nuclear RNAs (snRNAs) U1 to U6 of the ciliate Tetrahymena thermophila . The genes of the individual snRNAs exist in two to six slightly different copies per haploid genome . Sequence analyses of the gene-flanking regions indicate that there are two classes of snRNA genes . Both classes are characterized by several conserved sequence elements, some of which are unique to each class and some of which are found in both classes . Comparison of the promoter structure of the snRNA genes of T . thermophila with the promoter structures of snRNA genes of other organisms revealed several similarities to plant snRNA genes . These similarities include the overall promoter architecture as well as specific sequence elements . The structural organization of the 3' flanking region of some of the T . thermophila snRNA genes is not observed in other organisms . This finding is discussed in relation to a possible role in snRNA 3'-end formation. FEMS Microbiol Lett, 1992 Sep 1, 75(1), 55 - 9 Reductive dechlorination of trichloroethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila; Jablonski PE et al.; Trichloroethylene (TCE) was reductively dechlorinated to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila; the apparent Km and Vmax values were 1.7 +/- 0.3 mM TCE and 26.2 +/- 1.7 mol TCE dechlorinated/min/mmol factor III . Factor III also catalysed the dechlorination of TCE when in the presence of titanium(III) citrate; the apparent Km and Vmax values were 1.2 +/- 0.3 mM TCE and 34.9 +/- 3.6 mol TCE dechlorinated/min/mmol factor III . The enzyme complex was resolved into the two-subunit nickel/iron-sulfur (Ni/Fe-S) component and the two-subunit factor III-containing corrinoid/iron-sulfur (Co/Fe-S) component . The Ni/Fe-S component was unable to dechlorinate TCE in the presence of CO; however, reconstitution with the Co/Fe-S component yielded the same dechlorinated products as with the CO dehydrogenase enzyme complex. J Protozool, 1992 Sep-Oct, 39(5), 628 - 35 Exceptions to mutual exclusion among cell surface i-antigens of Tetrahymena thermophila during salt stress and stationary phase; Smith DL et al.; In ciliates, only one of the alternative forms of the immunodominant membrane glycoprotein usually coats the external surface of the cell . Such mutual exclusion is regulated at the pretranslational level by mechanisms that result in the expression of a single protein gene . In the holotrich Tetrahymena thermophila five alternative cell surface immobilization proteins (i-antigens) are expressed under different conditions of temperature (L, H, T) and culture media (I, S) . Using polyclonal and monoclonal antibodies to these proteins and a cDNA probe derived from the SerH3 gene, we have reinvestigated expression of i-antigens in media supplemented with 0.2 M NaCl . We find that in addition to S, the H and L antigens are also present on the cell surface . While all three i-antigens may be simultaneously present on the cell surface, the combinations S/L and S/H are more frequent . Compared to cells expressing H and L singly, the level of H3 mRNA is diminished, and a subset of the L family of polypeptides is variably expressed . The expression of S begins within 30 min after transfer to NaCl-supplemented medium, while the expression of L begins three days to several weeks after transfer . When cells are transferred out of NaCl-supplemented medium, S is turned off within 24 h, and L is expressed for at least 1 wk prior to the return of full H expression . Although these differences in kinetics suggest differences in control mechanism(s), the absence of I and T on the surface of NaCl-grown cells suggests that there is also a common regulatory link among H, S and L.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1992 Sep, 174(18), 5848 - 53 Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus; Shao W et al.; A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus . The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) . The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside . T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5) . The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates . An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75% . At 86 degrees C the half-life was around 15 min . With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined . The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences. Eur J Biochem, 1992 Sep 1, 208(2), 411 - 7 Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution; Reshetnikova L et al.; The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution . Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm . Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed . However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units . This implies 73% solvent content in the unit cell . Three heavy-atom derivatives {K2PtCl4, KAu(CN)2 and Hg(CH3COO)2} and the solvent-flattening procedure were used for electron-density-map calculations . This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit . The phenylalanyl-tRNA synthetase from T . thermophilus molecule has a 'quasi-linear' subunit organization . As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer. J Bacteriol, 1992 Sep, 174(17), 5719 - 26 Characterization of the archaeal, plasmid-encoded type II restriction-modification system MthTI from Methanobacterium thermoformicicum THF: homology to the bacterial NgoPII system from Neisseria gonorrhoeae; Nolling J et al.; A restriction-modification system, designated MthTI, was localized on plasmid pFV1 from the thermophilic archaeon Methanobacterium thermoformicicum THF . The MthTI system is a new member of the family of GGCC-recognizing restriction-modification systems . Functional expression of the archaeal MthTI genes was obtained in Escherichia coli . The mthTIR and mthTIM genes are 843 and 990 bp in size and code for proteins of 281 (32,102 Da) and 330 (37,360 Da) amino acids, respectively . The deduced amino acid sequence of M.MthTI showed high similarity with that of the isospecific methyltransferases M.NgoPII and M.HaeIII . In addition, extensive sequence similarity on the amino acid level was observed for the endonucleases R.MthTI and R.NgoPII . Moreover, the endonuclease and methyltransferase genes of the thermophilic MthTI system and those of the Neisseria gonorrhoeae NgoPII system show identical organizations and high (54.5%) nucleotide identity . This finding suggests horizontal transfer of restriction-modification systems between members of the domains Bacteria and Archaea. Biochemistry, 1992 Sep 1, 31(34), 7796 - 801 Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'; Braxton S et al.; A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis . Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis . These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures . These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops . To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase . Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49 . When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM) . This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA . However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna . Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165) . However, all of these mutants were less stable than wild-type subtilisin . Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease. Appl Environ Microbiol, 1992 Sep, 58(9), 2806 - 14 Cloning and nucleotide sequence of the gene coding for aspartokinase II from a thermophilic methylotrophic Bacillus sp; Schendel FJ et al.; The structural gene coding for the lysine-sensitive aspartokinase II of the methylotrophic thermotolerant Bacillus sp . strain MGA3 was cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking all three aspartokinase isozymes . The nucleotide sequence of the entire 2.2-kb PstI fragment was determined, and a single open reading frame coding for the aspartokinase II enzyme was found . Aspartokinase II was shown to be an alpha 2 beta 2 tetramer (M(r) 122,000) with the beta subunit (M(r) 18,000) encoded within the alpha subunit (M(r) 45,000) in the samea reading frame . The enzyme was purified, and the N-terminal sequences of the alpha and beta subunits were identical with those predicted from the gene sequences . The predicted amino acid sequence was 76% identical with the sequence of the Bacillus subtilis aspartokinase II . The transcription initiation site was located approximately 350 bp upstream of the translation start site, and putative promoter regions at -10 (TATGCT) and -35 (ATGACA) were identified . A 300-nucleotide intervening sequence between the transcription initiation and translational start sites suggests a possible attenuation mechanism for the regulation of transcription of this enzyme in the presence of lysine. Protein Eng, 1992 Sep, 5(6), 535 - 41 Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86); Turner SL et al.; The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein . Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C) . The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence . The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken . Both enzymes had similar sp . act . in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM . The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C. Mol Gen Genet, 1992 Sep, 234(3), 489 - 93 Development of a transformation system for the thermophilic fungus Talaromyces sp . CL240 based on the use of phleomycin resistance as a dominant selectable marker; Jain S et al.; A transformation system for the thermophilic cellulolytic fungus Talaromyces sp . CL240 has been developed, using the phleomycin resistance gene from Streptoalloteichus hindustanus (Sh ble) as a dominant selectable marker . The plasmids (pAN8-1 and pUT720) carrying the Sh ble gene under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter, allowed selection of phleomycin-resistant transformants . A new promoter sequence cloned from chromosomal DNA of Trichoderma reesei (pUT737) was also able to drive efficient expression of the Sh ble gene in Talaromyces sp . CL240, resulting in the selection of transformants that were highly resistant to phleomycin. Enzyme Microb Technol, 1992 Sep, 14(9), 696 - 701 Heterologous expression of thermopsin, a heat-stable acid proteinase; Lin X et al.; We have previously reported the isolation, characterization, and gene sequence of a new thermostable acid protease, thermopsin, from Sulfolobus acidocaldarius, a thermophilic archaebacterium . Thermopsin is similar to aspartic protease pepsin in specificity and pH dependence . However, it optimally catalyzes in the temperature range of 85 to 90 degrees C and it is not structurally related to pepsin . The current report describes the synthesis of recombinant thermopsin in E . coli and in insect cells . Several recombinant thermopsin fusion proteins were expressed as "inclusion bodies" in the cytosol of E . coli . Active thermopsin preparations were obtained by refolding from urea solutions . Recombinant thermopsin was also expressed in insect cells using a baculovirus expression system . The thermostability of recombinant thermopsin is similar to that of the native enzyme. Nucleic Acids Res, 1992 Aug 25, 20(16), 4173 - 8 Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli; Kreutzer R et al.; A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined . The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively . Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit . Secondary structure predictions indicate that an arm composed of two anti-parallel alpha-helices similar to that reported for the E.coli seryl-tRNA synthetase may be present in its N-terminal portion . In the beta-subunit clusters of hydrophilic amino acids and a leucine zipper motif were identified, and several pronounced alpha-helical regions were predicted . The particular arginine and lysine residues in the N-terminal portion of the beta-subunit, which were found to participate in tRNA binding in the yeast and E.coli PheRSs, have their counterparts in the T.thermophilus protein . The 5'-portion of an open reading frame downstream of pheT was found and codes for a yet unidentified, extremely hydrophobic peptide . The pheST genes are presumably cotranscribed and translationally coupled . A novel type of a putative transcriptional terminator in Thermus species was identified immediately downstream of pheT and other Thermus genes . The genes pheS and pheST were expressed in E.coli. Biochim Biophys Acta, 1992 Aug 21, 1122(3), 283 - 92 A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Burlini N et al.; A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus . Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule . An isoelectric point of 5.6 was also determined . The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin . Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin . This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent . Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator . The enzyme also displayed a broad pH optimum in the range 6.5-8.0 . Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C) . At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds . None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases. Biochim Biophys Acta, 1992 Aug 17, 1132(1), 58 - 66 Electron microscopical projections of the large ribosomal subunit from Thermomyces lanuginosus; Harauz G; Multivariate statistical analysis and hierarchical ascendant classification have been used to construct averages of two fundamental electron microscopical views of large ribosomal subunits from the thermophilic fungus Thermomyces lanuginosus . The first was roughly pentagonal in shape and corresponded to the canonical crown view seen in images of large subunits from prokaryotic species . The second and more prevalent projection was elliptical in shape, and by matching protuberances could be interpreted as the complex rotated from the crown orientation . Because of its ubiquity and consistency, this elongated view could potentially serve as the standard for structural comparisons of the large ribosomal subunit from eukaryotic organisms, and as the basis for a three-dimensional reconstruction. FEMS Microbiol Lett, 1992 Aug 15, 74(2-3), 175 - 80 Sequencing and characterization of pST1, a cryptic plasmid from Streptococcus thermophilus; Janzen T et al.; Eighty-six strains of S . thermophilus were examined for their plasmid content . Thirteen strains were found to contain one or two plasmids ranging in size from 2.1 to 7.4 kb . DNA-DNA hybridization analysis revealed the presence of five distinct groups of DNA homology . The complete nucleotide sequence of plasmid pST1 (Accession number X65856), which belongs to the major homology group, was determined . It has a molecular size of 2093 bp, a GC content of 35% and contains one major open reading frame of 945 bp (ORF A) . The predicted protein, designated Rep A, showed sequence homology with replication proteins from a group of plasmids which are known to replicate via single-stranded DNA intermediates (ssDNA plasmids). Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7645 - 9 The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA; Schleper C et al.; The lemon-shaped "virus-like" particle SSV1 produced by the thermophilic archaeon Sulfolobus shibatae has not previously been observed to infect any host . Using a plaque assay suitable for the extreme growth conditions of this archaeon, we have shown infection of Sulfolobus solfataricus by SSV1 . Upon infection, the viral genome was always found integrated into a tRNA gene of the host chromosome, a situation similar to that in S . shibatae, proving that site-specific integration is involved in establishing the lysogenic state . As in S . shibatae, UV-irradiation of lysogenized S . solfataricus led to virus production apparently not accompanied by cell lysis . We have also demonstrated the efficient uptake of exogenous DNA and its expression in Sulfolobus by transfecting S . solfataricus with SSV1 DNA by electroporation . Transfection efficiencies of up to 10(6) transfectants per microgram of DNA were obtained. J Biol Chem, 1992 Aug 15, 267(23), 16484 - 90 Molecular dissection of the beta subunit of F1-ATPase into peptide fragments; Tozawa K et al.; Partial digestion of the native beta subunit of F1-ATPase from the thermophilic Bacillus strain PS3 by three different proteases produced a limited number of peptide fragments . In most cases, the peptides remained associated, and the gross structure of the beta subunit was not destroyed . Furthermore, most peptides were able to reassociate into the form of the beta subunit after denaturating urea treatment . Therefore, the cleaved sites are most likely located in water-exposed loop regions in the tertiary structure of the protein . Almost all peptides were analyzed, and 17 cleaved sites were determined . From the analysis of the distribution of cleaved sites and deletions or insertions in the multiple amino acid sequence alignment of proteins homologous to the beta subunit, locations of five loops and four candidate loops in the beta subunit are suggested . There are two large loops in the central region of the beta subunit sequence, and dicyclohexylcarbodiimide-reactive Glu190 is located in one of them . Tyr341, involved in putative catalytic ATP binding, is also found in one of the loops . Then, taking cleaved sites as a reference, two kinds of expression plasmids, each of which carried genes of two complementary peptide fragments, 1-193 and 198-473 or 1-284 and 285-473, were constructed and expressed in Escherichia coli . For each plasmid, two peptides were coexpressed, associated into a stable beta subunit form in E . coli cells, and purified without dissociation . When these beta subunits were denatured by urea and applied to polyacrylamide gel without denaturant, a protein band with the same mobility as that of the beta subunit appeared, indicating that reassociation of peptide fragments into the form of the beta subunit occurred upon removal of urea . These beta subunits retained the ability to reconstitute the alpha 3 beta 3 gamma complexes even though the efficiency of reconstitution and the recovered ATPase activities were decreased . These complexes were stable at high or low temperature, and ATPase activities were sensitive to inhibition by N3-. J Bacteriol, 1992 Aug, 174(16), 5400 - 5 Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli; Podkovyrov SM et al.; Clostridium thermohydrosulfuricum 39E, a gram-positive thermophilic anaerobic bacterium, produced a cyclodextrin (CD)-degrading enzyme, cyclodextrinase (CDase) (EC 3.2.1.54) . The enzyme was purified to homogeneity from Escherichia coli cells carrying a recombinant multicopy plasmid that contained the gene encoding for thermophilic CDase . The purified enzyme was a monomer with an M(r) of 66,000 +/- 2,000 . It showed the highest activity at pH 5.9 and 65 degrees C . The enzyme hydrolyzed alpha-, beta-, and gamma-CD and linear maltooligosaccharides to yield maltose and glucose . The Km values for alpha-, beta-, and gamma-CD were 2.5, 2.1, and 1.3 mM, respectively . The rates of hydrolysis for polysaccharides (starch, amylose, amylopectin, and pullulan) were less than 5% of the rate of hydrolysis for alpha-CD . The entire nucleotide sequence of the CDase gene was determined . The deduced amino acid sequence of CDase, consisting of 574 amino acids, showed some similarities with those of various amylolytic enzymes. Gene, 1992 Aug 1, 117(1), 39 - 44 The DNA-binding protein HU from mesophilic and thermophilic bacilli: gene cloning, overproduction and purification; Padas PM et al.; The major histone-like bacterial protein (HU)-encoding genes (hup) from five different Bacilli have been cloned, sequenced and overexpressed in Escherichia coli . The five Bacilli selected are closely related, but have different optimum growth temperatures: greater than 70 degrees C for Bacillus caldolyticus and B . caldotenax; 60-65 degrees C for B . stearothermophilus (Bst); 37 degrees C for B . subtilis and 30 degrees C for B . globigii . The deduced amino acid (aa) sequences from the three thermophiles are identical . Those from the two mesophiles are also identical and differ from those of the thermophiles at eleven aa positions . The mesophilic proteins have an extra two aa at the C terminus . Cells harbouring plasmids containing the hup genes can produce HU . An efficient purification scheme using cation-exchange chromatography and fast protein liquid chromatography is presented . This gives approx . 30-40 mg of more than 95% pure Bst HU per litre of E . coli culture. Exp Cell Res, 1992 Aug, 201(2), 477 - 84 Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis; Christopher GK et al.; Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone . Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites . Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 micrograms/ml for 1 h (receptor induced, RI) . After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis . Observations indicate that T . pyriformis does bind insulin whether or not the cells have prior exposure to insulin . Staining intensity increased at the two highest RI concentrations over 0 microgram/ml (P less than 0.01) but the staining intensity at 0 microgram/ml was not different from that at 3 micrograms/ml . The results confirm that T . pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner . Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular . Both techniques produced very similar labeling patterns when optical sections through the cells were viewed . Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin . Further studies with the exo- mutant of T . thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding. J Bacteriol, 1992 Aug, 174(15), 5171 - 5 A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction; Duwat P et al.; An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction . The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp . thermophilus were amplified, cloned, and sequenced . The G + C contents of the DNA from these species range from 28 to 52% . The sequences of the bacterial recA genes show strong relatedness . This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning. Appl Environ Microbiol, 1992 Aug, 58(8), 2654 - 9 Growth-promoting effect of thermophilic fungi on the mycelium of the edible mushroom Agaricus bisporus; Wiegant WM et al.; The growth-promoting effect of the thermophilic fungus Scytalidium thermophilum in mushroom compost on the mycelium of the edible mushroom Agaricus bisporus was investigated . Results obtained by others were confirmed by showing that S . thermophilum leads to an increased hyphal extension rate of the mushroom mycelium . However, it was demonstrated that hyphal extension rates were not clearly related to mushroom biomass increase rates . A number of experiments pointed strongly towards CO2 as the determinant of hyphal extension rates . In compost, CO2 is produced mainly by thermophilic fungi . Several experiments did not reveal any other specific compound produced by S . thermophilum that increases the hyphal extension rate of the mushroom mycelium. J Leukoc Biol, 1992 Aug, 52(2), 197 - 201 Interleukin-6 in mouse hypersensitivity pneumonitis: changes in lung free cells following depletion of endogenous IL-6 or direct administration of IL-6; Denis M; In this study, we examined the role of interleukin-6 (IL-6) in the development of chronic lung inflammatory conditions, using a mouse model of hypersensitivity pneumonitis established by intranasal instillation of the thermophilic actinomycete Faeni rectivirgula . Challenged mice developed an early neutrophilic response at 24 h, followed by a macrophage/lymphocyte recruitment . The impact of IL-6 on the development of the inflammatory response was assessed by giving infusions of a monoclonal antibody against IL-6 so as to deplete endogenous levels of this cytokine or by giving exogenous IL-6 to challenged mice . Mice challenged intranasally with the actinomycete and given the anti-IL-6 antibody developed a strong, sustained neutrophilic response, with a significantly higher lung free cell number than control mice . Assessment of fibrosis by measuring lung hydroxyproline levels showed that challenged mice given anti-IL-6 developed more significant fibrosis than control mice . Conversely, infusions with IL-6 diminished F . rectivirgula-induced cell recruitments and the fibrotic response in the lungs . Moreover, alveolar macrophages from mice given 2 weeks of F . rectivirgula treatment released high levels of tumor necrosis factor alpha (TNF-alpha) bioactivity upon in vitro lipopolysaccharide challenge, compared to mice instilled with saline only . This TNF-alpha activity produced by macrophages was decreased by in vivo IL-6 treatment and enhanced by in vivo neutralization with anti-IL-6 . These observations suggest that IL-6 may play a role in regulating the cellular recruitment in the lungs during an inflammatory response, with dramatic consequences for the cellular profile in the bronchoalveolar lavage and the subsequent fibrosis. Eur J Biochem, 1992 Aug 1, 207(3), 839 - 46 Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli; Voss RH et al.; The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined . The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%) . The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues . Three exchanges are located in the additional loop of ten amino acids (182-191) . The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli . Since EF-Tu from E . coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization . Comparison of the EF-Tu domain I from E . coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome . Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability . The T . aquaticus EF-Tu was overproduced in E . coli using the tac expression system . Identity of the recombinant T . aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays. Am J Respir Cell Mol Biol, 1992 Aug, 7(2), 156 - 60 Transforming growth factor-beta is generated in the course of hypersensitivity pneumonitis: contribution to collagen synthesis; Denis M et al.; Mice of the C57BL/6 strain were instilled with optimal doses (150 micrograms/day for 3 days/wk) of the thermophilic actinomycete Faeni rectivirgula (also known as Saccharopolyspora rectivirgula or Micropolyspora faeni) to induce a hypersensitivity pneumonitis inflammation that mimics the human disease affecting certain occupational groups . This mouse model was characterized by a very significant alveolitis (3-fold increase in bronchoalveolar lavage {BAL} cell number at 48 h and a 10-fold increase at 3 wk) . Also, total lung transforming growth factor (TGF-beta) was shown to be elevated in treated mice as early as 1 wk after the first instillation and increased gradually to 2.5 micrograms/lung at 3 wk (approximately 0.3 microgram/lung in saline-instilled controls) . Intranasal instillation with F . rectivirgula was also associated with very significant increases in lung fibroblast collagen synthesis, starting at 2 wk . BAL macrophages from mice instilled with F . rectivirgula were found to release significantly more TGF-beta upon in vitro stimulation with zymosan beads than did BAL macrophages from saline-instilled mice . Finally, we show that supernatants from activated BAL macrophages of mice given F . rectivirgula increased quite significantly collagen synthesis in normal mouse lung fibroblasts . This increase could be abrogated by treating conditioned medium with a rabbit antibody against TGF-beta . Collectively, these data suggest that TGF-beta is generated in the course of experimental mouse hypersensitivity pneumonitis and contributes significantly to collagen synthesis. Chem Pharm Bull (Tokyo), 1992 Aug, 40(8), 2210 - 1 Studies on thermophile products . IV . Structural elucidation of cytotoxic substance, BS-1, derived from Bacillus stearothermophilus; Kohama Y et al.; A new cytotoxic substance designated as BS-1 was isolated from the autolysate and culture filtrate of Bacillus stearothermophilus UK563 . On the basis of spectral data, the structure of BS-1 was determined as bis(2-hydroxyethyl) trisulfide and confirmed by direct comparison with the synthetic compound . BS-1 exhibited potent cytotoxicity against leukemia P388-D1, leukemia P388, mastocytoma P815, lymphoma EL4 and lymphoma MOLT4. J Biochem (Tokyo), 1992 Aug, 112(2), 173 - 4 Crystallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HB8 chimeric 3-isopropylmalate dehydrogenase and thermostable mutants of it; Sakurai M et al.; A new type of chimeric 3-isopropylmalate dehydrogenase (2T2M6T) was produced by expressing the fused gene of Bacillus subtilis and Thermus thermophilus . The enzyme shows heat stability intermediate between those of the parents . The crystal of the enzyme belongs to the space group of P3(2)21, with cell dimensions of a = b = 78.9 A and c = 158.9 A . Two thermostable mutants of the chimeric enzyme were prepared by site-directed mutagenesis and then crystallized. Exp Cell Res, 1992 Aug, 201(2), 522 - 5 Differential increase in activity of acid phosphatase induced by phosphate starvation in Tetrahymena; Rasmussen L et al.; We have studied the effects of phosphate starvation on the levels and distributions of activities of acid phosphatase and beta-hexosaminidase in cultures of Tetrahymena thermophila . The cells were grown in synthetic nutrient medium and refed every day with fresh medium . After 4 days of growth in the complete medium, the cultures were divided into two portions . One received complete medium and the other phosphate-free, but otherwise complete, medium . Population densities and activities of acid phosphatase and beta-hexosaminidase in cells plus medium and in cell-free samples were determined in aliquots removed every day before medium replacement . In cultures having complete medium the enzyme levels remained fairly constant; in the phosphate-starved cultures both total and extracellular activities of acid phosphatase increased sixfold . beta-Hexosaminidase levels remained essentially unaltered in both cases . These results indicate that phosphate starvation can induce differential increase in acid phosphatase activity in cultures of Tetrahymena . Somewhat less than 50% of the total activities of both enzymes are found in the cell-free extracellular fluid at any time. FEMS Microbiol Lett, 1992 Aug 1, 74(1), 87 - 93 Ribosomal RNA gene restriction fragment diversity amongst Lior biotypes and Penner serotypes of Campylobacter jejuni and Campylobacter coli; Fayos A et al.; Diversity based on ribosomal RNA gene-restriction endonuclease digest patterns was detected amongst 42 strains of Campylobacter jejuni and 18 strains of C . coli including representatives of 53 different Penner serotypes . HaeIII ribopatterns were coded for numerical analysis which showed that all except two were different including those of several strains of the same serotype (P2 and P20) . At the 30% similarity level, four groupings were formed in the analysis of which three corresponded to C . jejuni, C . coli and C . lari phenotypes respectively . Eight strains (13%) were atypical as their phenotypic and ribopattern associations did not correspond . Ribopattern fragments of 3.0, 5.0 and 9.3 kb were characteristic of the majority of C . jejuni, whereas 1.5, 2.2-, 2.3- and 4.7-kb fragments were commonly present in C . coli . These fragments provided novel species-specific markers . We conclude that HaeIII ribotyping was as discriminatory as Penner serotyping of C . jejuni and C . coli and may even provide a basis for distinguishing between strains of the same serotype and for identifying new groups within the thermophilic campylobacters. J Bacteriol, 1992 Aug, 174(16), 5244 - 50 Cloning, nucleotide sequence, and transcriptional analyses of the gene encoding a ferredoxin from Methanosarcina thermophila; Clements AP et al.; A mixed 17-mer oligonucleotide deduced from the N terminus of a ferredoxin isolated from Methanosarcina thermophila was used to probe a lambda gt11 library prepared from M . thermophila genomic DNA; positive clones contained either a 5.7- or 2.1-kbp EcoRI insert . An open reading frame (fdxA) located within the 5.7-kbp insert had a deduced amino acid sequence that was identical to the first 26 N-terminal residues reported for the ferredoxin isolated from M . thermophila, with the exception of the initiator methionine . fdxA had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 2{4Fe-4S} ferredoxins . An open reading frame (ORF1) located within the 2.1-kbp EcoRI fragment also had the potential to encode a 2{4Fe-4S} bacterial-type ferredoxin (5,850 Da) . fdxA and ORF1 were present as single copies in the genome, and each was transcribed on a monocistronic mRNA . While the fdxA- and ORF1-specific mRNAs were detected in cells grown on methanol and trimethylamine, only the fdxA-specific transcript was present in acetate-grown cells . The apparent transcriptional start sites of fdxA and ORF1, as determined by primer extension analyses, lay 21 to 28 bases downstream of sequences with high identity to the consensus methanogen promoter. Appl Environ Microbiol, 1992 Aug, 58(8), 2633 - 42 Cloning, nucleotide sequences, and overexpression in Escherichia coli of tandem copies of a tryptophanase gene in an obligately symbiotic thermophile, Symbiobacterium thermophilum; Hirahara T et al.; Symbiobacterium thermophilum, a thermophilic bacterium, is a thermostable tryptophanase producer that can grow only in coculture with a specific Bacillus strain . Two thermostable tryptophanase genes, tna-1 and tna-2, that are located close to each other were cloned into Escherichia coli from S . thermophilum by the DNA-probing method . The nucleotide and deduced amino acid sequences indicate that Tna1 and Tna2 share 92% identical amino acids in a total of 453 amino acids . By means of DNA manipulation with E . coli host-vector systems, Tna1 and Tna2 were produced in very large amounts in enzymatically active forms . Comparison of the NH2-terminal amino acid sequences and the enzymatic properties of the tryptophanases purified from the original S . thermophilum strain and these two tryptophanases from recombinant E . coli cells suggest that in S . thermophilum, only Tna2 is produced and tna-1 is silent . Notwithstanding the great similarity in amino acid sequence between Tna1 and Tna2, the two enzymes differ markedly in activation energy for catalysis and thermostability. PCR Methods Appl, 1992 Aug, 2(1), 51 - 9 Optimization of long-distance PCR using a transposon-based model system; Ohler LD et al.; The ability to amplify routinely long PCR products (5-25 kb) with high specificity and fidelity, regardless of target template sequence or structure, would provide significant benefits to genome mapping and sequencing endeavors . Although occasional reports have described the generation of long PCR products, such results have been difficult to replicate and have frequently utilized probe hybridization to identify the specific product from nonspecific amplified DNA . Production of specific PCR products has generally been limited to target templates of less than 3 kb . To extend the effective range of standard PCR amplification, it may be necessary to utilize alternative reaction conditions and/or components, such as novel thermostable DNA polymerases or accessory proteins . We describe the use of a model system to evaluate systematically methodological changes that might enable efficient long-range PCR . Specifically, the transposon Tn5supF has been used to introduce randomly identical, known primer binding sites within separate isolates of phage clones carrying identical inserts . Transposon-based PCR allows us to study amplification of DNA fragments that vary in size and sequence using only a single set of primers . In the present studies, we describe conditions that enable PCR amplification of specific DNA templates ranging in size up to 9 kb . Some of the key features of our methodology include the use of recombinant Thermus thermophilus (rTth) DNA polymerase, the addition of gelatin to the reaction mixture, the use of wax-mediated "hot starts" and, lastly, the use of auto-segment extension thermocycling . These results also provide insights into additional approaches that might further enhance our ability to perform long-distance PCR.(ABSTRACT TRUNCATED AT 250 WORDS) J Dairy Res, 1992 Aug, 59(3), 349 - 57 Different bacteriophage resistance mechanisms in Streptococcus salivarius subsp . thermophilus; Larbi D et al.; Streptococcus salivarius subsp . thermophilus strain NST5 exhibited a temperature-dependent defence mechanism against the virulent bacteriophages phi B1.2 and phi A1.1 . It was active at 42 degrees C but not at 30 degrees C as demonstrated by a significant increase of both plaque size and efficiency of plaquing . This defence mechanism did not affect host-dependent phage replication and did not interfere with phage adsorption to NST5 . These results suggest that it interfered with phage development . The phages phi T33, phi T58, phi D1, phi T21 and phi T9, belonging to the same phage type as phi B1.2, were examined for their ability to infect NST3 and NST5 . Restriction modification systems of different specificity were detected in NST3 and NST5; host-dependent phage replication was detected at 30 and 42 degrees C; an abortive defence mechanism was detected in NST5 which was active at 42 degrees C, but not 30 degrees C, and was independent of restriction modification action or interference with phage adsorption . Our investigations of phage-host interactions showed that the two Str . salivarius subsp . thermophilus strains studied avoided attack by related bacteriophages by evolving at least three different resistance systems. Biochemistry, 1992 Jul 28, 31(29), 6856 - 64 Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes; Elhag GA et al.; A tobacco (Nicotiana tabacum cv . Petite Havana) leaf cDNA library was constructed in the expression vector lambda gt11 . Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an M(r) 19,641 precursor to an M(r) 14,420 mature protein which is homologous to Escherichia coli ribosomal protein L27 . One cDNA (L27-1; 882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly(A)29 tail . A beta-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins . Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins . The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27 protein . The mature L27 protein has 66%, 61%, 56%, and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E . coli, Bacillus stearo-thermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions . The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence . Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corresponding portion of yeast MRP7.(ABSTRACT TRUNCATED AT 250 WORDS) Biochim Biophys Acta, 1992 Jul 21, 1117(1), 71 - 7 Structure of the glycan chain from the surface layer glycoprotein of Clostridium thermohydrosulfuricum L77-66; Altman E et al.; The thermophilic eubacterium Clostridium thermohydrosulfuricum L77-66 is covered by a crystalline surface layer composed of identical glycoprotein subunits which are arranged in a hexagonal lattice with centre-to-centre spacings of approx . 14.3 nm . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cell wall preparations showed the presence of several broadened, carbohydrate-containing bands in a molecular mass range of 90 to 200 kDa . A total carbohydrate content of approx . 14% was determined in the purified surface layer glycoprotein . Chemical deglycosylation of this material by trifluoromethanesulfonic acid resulted in the disappearance of the complex banding pattern . Only a single band with a molecular mass of 82 kDa remained visible upon Coomassie staining . After proteolytic digestion of the surface layer glycoprotein a single glycopeptide fraction with an apparent molecular mass of approx . 25 kDa was obtained by gel filtration . Composition analysis, methylation, periodate oxidation and a combination of homonuclear and 1H-detected heteronuclear shift-correlated nuclear magnetic resonance experiments established the following structure for the glycan chain of the surface layer glycoprotein. Eur J Biochem, 1992 Jul 15, 207(2), 559 - 65 A tungsten-containing active formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei; Schmitz RA et al.; Methanobacterium wolfei is a thermophilic methanogenic archaeon which requires tungsten or molybdenum for growth . We have found that the organism contains two formylmethanofuran dehydrogenases, one of which is a tungsten enzyme . Indirect evidence indicates that the other formylmethanofuran dehydrogenase is a molybdenum enzyme . The tungsten enzyme was purified and characterized . The native enzyme had an apparent molecular mass of 130 kDa . SDS/PAGE revealed a composition of three subunits of apparent molecular mass 35, 51 and 64 kDa, the N-terminal amino acid sequences of two of which were determined . 0.3-0.4 mol tungsten/mol enzyme was found but no molybdenum . The pterin cofactor was identified as molybdopterin guanine dinucleotide . The purified enzyme exhibited a specific activity of 8.3 mumol.min-1.mg protein-1 and an apparent Km for formylmethanofuran and methylviologen of 13 microM and 0.4 mM, respectively . The optimum temperature for activity was 65 degrees C . At 40-60 degrees C, the rate increased with a Q10 of 1.9; the activation energy of the reaction was 45 kJ/mol . The enzyme was found to require potassium ions for thermostability . The oxygen-sensitive enzyme was not inactivated by cyanide. Eur J Biochem, 1992 Jul 15, 207(2), 413 - 8 Molecular cloning of a glucoamylase gene from a thermophilic Clostridium and kinetics of the cloned enzyme; Ohnishi H et al.; Clostridium sp . G0005 produces a cell-bound glucoamylase (CGA) . The gene encoding CGA has been sequenced . The deduced amino acid sequence begins with a putative 21-residue signal sequence for secretion of bacterial lipoproteins, which suggests that a putative CGA precursor is modified and secreted like other bacterial lipoproteins in Clostridium sp . G0005, and that the modified residue is important in the cell-bound form of mature CGA . Comparison of the amino acid sequence of the CGA precursor with known eukaryotic enzymes showed several regions of high similarity in spite of low similarity throughout the overall primary structure . CGA is the first bacterial glucoamylase to be cloned . The CGA gene was expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5' non-coding region and the N-terminal coding region of the gene were replaced with the lac promoter . Kinetic studies of the cloned enzyme purified from E . coli were performed with a set of linear malto-oligosaccharides as substrates, and the subsite affinity was calculated from the kinetic parameters . CGA had typical kinetic properties for a glucoamylase, but this bacterial enzyme had higher isomaltose-hydrolyzing activity than other eukaryotic glucoamylases. FEMS Microbiol Lett, 1992 Jul 15, 73(3), 235 - 9 Purification and properties of pyruvate kinase from Thermoplasma acidophilum; Potter S et al.; Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens) . To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker . The enzyme has been purified and has a native M(r) of 250,000 . It consists of four, apparently identical subunits each of M(r) 60,000 . No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability . Its amino acid composition has been determined and some partial sequencing has been done. J Biol Chem, 1992 Jul 15, 267(20), 14068 - 72 The Rieske FeS center from the gram-positive bacterium PS3 and its interaction with the menaquinone pool studied by EPR; Liebl U et al.; The Rieske 2Fe2S center from Bacillus PS3, a Gram-positive thermophilic eubacterium, has been studied by EPR spectroscopy . Its redox midpoint potential at pH 7.0 was determined to be +165 +/- 10 mV and was found to decrease with an apparent slope of -80 mV/pH unit above pH 7.9 . The Qo-site inhibitor stigmatellin induced spectral changes analogous to those reported for Rieske centers from mitochondria and chloroplasts . The redox midpoint potential of the PS3 Rieske cluster was not affected by stigmatellin . The orientation of the g tensor was similar to other Rieske centers (gz and gy are oriented parallel, gx is oriented perpendicular to the membrane plane) . The shape of the EPR spectrum of the Rieske cluster from PS3 changed as a function of the redox state of the menaquinone (MK) pool . This permitted the redox midpoint potential of the MK pool to be determined in the membrane . Values of -60 +/- 20 mV at pH 7.0 and of -130 +/- 20 mV at pH 8.0 were obtained . The results are compared with already published data from other Rieske centers . It is proposed that all Rieske centers that function in electron transport chains using MK as pool quinone show common features that distinguish them from Rieske centers operating in ubiquinone- or plastoquinone-based electron transfer chains. Biochim Biophys Acta, 1992 Jul 9, 1127(1), 22 - 7 Tetrahydropyran ring-containing fatty acid-combined taurine (tetrathermoyltaurine) in the taurolipid fraction of Tetrahymena thermophila; Kaya K et al.; A tetrahydropyran ring-containing fatty acid-combined taurine (tetrathermoyltaurine) was found in the taurolipid fraction of Tetrahymena thermophila . Tetrathermoyltaurine accounted for about 0.6% of the total taurolipid of the cells . The compound was subjected to methanolic hydrochloric acid hydrolysis, and the structures of the hydrolysis products were identified by nuclear magnetic resonance and mass spectrometries, as taurine and 2,3-dihydroxy-9,13-oxy-7-trans-octadecenoic acid (tetrathermic acid) . The chemical structure of tetrathermoyltaurine was identified as 2-(2,3-dihydroxy-9,13-oxy-7-trans-octadecenoylamino) ethanesulfonic acid . This structure suggests that tetrathermoyltaurine may be derived from taurolipid B as the major taurolipid of the cells . When cells of HL 60, as a human lymphoma, were cultured with tetrathermoyltaurine, 88% of the cell growth was inhibited at the concentration of 100 micrograms/ml. Science, 1992 Jul 3, 257(5066), 74 - 6 Evidence that eukaryotes and eocyte prokaryotes are immediate relatives; Rivera MC et al.; The phylogenetic origin of eukaryotes has been unclear because eukaryotic nuclear genes have diverged substantially from prokaryotic ones . The genes coding for elongation factor EF-1 alpha were compared among various organisms . The EF-1 alpha sequences of eukaryotes contained an 11-amino acid segment that was also found in eocytes (extremely thermophilic, sulfur-metabolizing bacteria) but that was absent in all other bacteria . The related (paralogous) genes encoding elongation factor EF-2 and initiation factor IF-2 also lacked the 11-amino acid insert . These data imply that the eocytes are the closest surviving relatives (sister taxon) of the eukaryotes. Eur J Biochem, 1992 Jul 1, 207(1), 81 - 8 Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli; Liebl W et al.; Maltodextrin glycosyltransferase (4-alpha-glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T . maritima MSB8 chromosomal gene . T . maritima 4-alpha-glucanotransferase, an approximately 53-kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date . It retained more than 90% of its maximum activity at temperatures from 55 degrees C up to 80 degrees C . The proposed action modus is the transfer of 1,4-alpha-glucanosyl chains, thus resulting in the disproportionation of 1,4-alpha-glucans . It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates . The addition of low-molecular-mass malto-oligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size . Use of either of the malto-oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues . On the other hand, maltose and maltotriose were not disproportionated by 4-alpha-glucanotransferase, although both were good acceptors for glucanosyl transfer . Glucose did not function as an acceptor in transfer reactions . Glucose also never appeared as a reaction product . The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues . Comparison of the N-terminal amino acid sequence of 4-alph