|
|
|
|
|
| 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-alpha-glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N-termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch- and maltodextrin-converting enzymes. Eur J Biochem, 1992 Jul 1, 207(1), 345 - 9 Characterisation of a thermostable alpha-amylase from Bacillus brevis; Stefanova M et al.; Biochemical characterization of a novel heat-stable alpha-amylase, produced by a thermophilic strain of Bacillus brevis, has been made . The pattern of the enzyme action on different substrates was studied . It was found that reducing groups were rapidly liberated from amylopectin, soluble and insoluble starch compared to amylose and glycogen . B . brevis alpha-amylase acted via endo-attack producing mainly maltopentaose during the first hour of hydrolysis . The enzyme showed high activity towards maltohexaose and maltoheptaose . The alpha-amylase from B . brevis had a neutral pI and was found to be a glycoprotein, containing 9.2% (by mass) neutral sugars . The enzyme protein possessed a unique high glycine content . Calcium or sodium ions in appropriate concentrations were required for enzyme thermostability. Plasmid, 1992 Jul, 28(1), 70 - 9 Plasmid pTB913 derivatives are segregationally stable in Bacillus subtilis at elevated temperatures; Oskam L et al.; We studied the effects of temperature on the segregational stability of derivatives of the rolling-circle-type plasmid pTB913 in Bacillus subtilis . This 4.5-kb plasmid is a deletion derivative of pTB19, which was originally isolated from a thermophilic Bacillus . pTB913 derivatives carrying large inserts or lacking the minus origin for complementary strand synthesis were segregationally unstable at 37 degrees C . In contrast, at 47 degrees C all pTB913 derivatives tested were stably maintained in B . subtilis . The increased stability at 47 degrees C was attributed, at least partly, to increased copy numbers at this temperature . Although considerable amounts of single-stranded and high-molecular-weight plasmid DNA were formed at 47 degrees C, these products did not reduce plasmid stability at this temperature . The increased stability and increased copy number of pTB913 at elevated temperatures extend the use of this plasmid as a cloning vector in B . subtilis and other bacilli. Plasmid, 1992 Jul, 28(1), 25 - 36 Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli; Solaiman DK et al.; The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E . coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18 . The pBN183 transformed E . coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA . Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA . Plasmid screening with pBN183-transformed S . thermophilus clones revealed that ca . 70% of these transformants contained deleted plasmids . Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study . It transformed E . coli to ApR and S . thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively . Screening of S . thermophilus transformants did not show the presence of deleted plasmids . Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment . Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E . coli and S . thermophilus, respectively . In contrast to the parent pBN183, only 17% of the pDBN183-transformed S . thermophilus contained deleted plasmids . Plasmid copy numbers of the three vectors in E . coli were estimated at 17-18 per chromosome . The three plasmids conferred ApR and CmR to E . coli, but only CmR to S . thermophilus . The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S . thermophilus. Mol Gen Genet, 1992 Jul, 234(1), 74 - 80 Cloning and sequencing of the melB gene encoding the melibiose permease of Salmonella typhimurium LT2; Mizushima K et al.; The nucleotide sequence of the melB gene coding for the Na+ (Li+)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced . It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52,800) . The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues . All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E . coli are conserved in the melibiose permease of S . typhimurium . In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami. Ultramicroscopy, 1992 Jul, 42-44 ( Pt B), 1194 - 9 Imaging of subunit complexes of thermophilic bacterium H(+)-ATPase with scanning tunneling microscopy; Masai J et al.; Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H(+)-ATPase of a thermophilic bacterium . The measurement was carried out in air without conductive coating on the samples deposited on a highly oriented pyrolytic graphite (HOPG) . The F1 subunit complex of the H(+)-ATPase, and an H(+)-ATPase whose F0 portion was embedded into liposomes prepared from soybean lecithin were imaged . Overall structural images of the subunit complex F1 were obtained: the structural dimensions of the STM images are in agreement with those deduced from conventional methods such as an transmission electron microscopy (TEM) and small-angle X-ray scattering (SAX) experimentation . Regarding the STM imaging of these samples, we discuss the advantages and disadvantages of the STM over those of conventional methods such as a TEM and SAX. J Protozool, 1992 Jul-Aug, 39(4), 508 - 10 The immobilization antigens of Tetrahymena thermophila are glycoproteins; Ron A et al.; The four immobilization antigens controlled by the SerH locus in Tetrahymena thermophila have been isolated and partially characterized (Doerder, F.P . & Berkowitz, M.S . 1986 . Purification and partial characterization of the H immobilization antigens of Tetrahymena thermophila . J . Protozool., 33:204-208) . We show here, using immunoprecipitation and electrophoresis after labeling with 35S-methionine, 14C-mannose, 14C-glucosamine, and N-Acetyl-D-{l-3H}glucosamine, that these proteins are glycosylated . We suggest the immobilization antigens in Tetrahymena may be anchored to the surface membrane by phosphatidylinositol glycans. Int J Syst Bacteriol, 1992 Jul, 42(3), 463 - 8 Characterization of three thermophilic strains of Methanothrix ("Methanosaeta") thermophila sp . nov . and rejection of Methanothrix ("Methanosaeta") thermoacetophila; Kamagata Y et al.; Three thermophilic Methanothrix ("Methanosaeta") strains, strains PTT (= DSM 6194T) (T = type strain), CALS-1 (= DSM 3870), and Z-517 (= DSM 4774), were characterized chemotaxonomically and compared with five mesophilic strains, Methanothrix soehngenii ("Methanosaeta concilii") GP6 (= DSM 3671), Opfikon (= DSM 2139), FE (= DSM 3013), UA, and PM . These methanogens were exclusively acetotrophic and had a characteristic sheathed structure . The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine . The thermophilic strains often had phase-refractive gas vesicles inside their cells . Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Methanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups . The difference between the thermophiles and mesophiles was also verified by comparing partial 16S rRNA sequences (ca . 30 base differences in ca . 540 bases) . On the basis of our results, we propose the name Methanothrix thermophila for the three thermophilic strains . The type strain of M . thermophila is strain PT (= DSM 6194) . We also propose that the name Methanothrix thermoacetophila ("Methanosaeta thermoacetophila"), which was given to strain Z-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate. Int J Syst Bacteriol, 1992 Jul, 42(3), 408 - 11 DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum; Touzel JP et al.; DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133) . Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting . The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M . thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp . strain THF, and Methanobacterium sp . strain FTF . The second and third groups were each represented by only one strain, Methanobacterium sp . strain CB-12 and M . thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively) . Our results indicate that the name M . thermoformicicum should be rejected as it is a synonym of M . thermoautotrophicum . The taxonomic positions of strains Marburg and CB-12 need further investigation. J Bacteriol, 1992 Jul, 174(13), 4350 - 5 Purification and characterization of DNA polymerases from Bacillus species; Sellmann E et al.; DNA polymerases from Bacillus stearothermophilus, Bacillus caldotenax, and Bacillus caldovelox were purified by chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose and obtained in high yield . The enzyme preparations are free of exo- and endonuclease activities . Additional purification steps, e.g., hydrophobic interaction chromatography and chromatography on a Mono Q column or sucrose density gradient centrifugation, are needed to obtain the enzymes in the form of homogeneous 95-kDa proteins . Each of the three organisms possesses a major DNA polymerase activity comparable to DNA polymerase I . The enzymes require Mg2+ (10 to 30 mM) for optimal activity, although 0.4 mM Mn2+ could substitute for magnesium . The optimal reaction temperatures were lowest in B . stearothermophilus (60 to 65 degrees C) and about equal in B . caldovelox and B . caldotenax (65 to 70 degrees C) . The thermal stabilities of the enzymes increased in the same order . The DNA polymerase from Thermus thermophilus was isolated for comparison by using a similar procedure . The enzyme was obtained as a homogeneous 85-kDa protein that was also free of exo- and endonucleolytic activities. FEMS Microbiol Lett, 1992 Jun 15, 72(3), 279 - 83 Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum; Eriksen SH et al.; Conidia produced by submerged cultivation of the thermophilic fungus Thermomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination . With conidia derived from submerged liquid culture at 40-45 degrees C more than 90% germination was achieved at 50 degrees C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates . The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia. FEMS Microbiol Lett, 1992 Jun 15, 72(3), 203 - 8 Plasmid maintenance in Bacillus stearothermophilus is strain-dependent; Oskam L et al.; We studied the segregational stability of plasmids based on pTB913, a 4.5-kb rolling-circle plasmid derived from the thermophilic Bacillus plasmid pTB19 . In Bacillus stearothermophilus the stability of pTB913 derivatives appeared to be strain-dependent . In strain CU21 large amounts of single-stranded pTB913 DNA were found and the plasmid was highly unstable at 57 degrees C . In strain NUB3621, however, very low amounts of single-stranded plasmid DNA were formed and pTB913-based replicons were only slightly unstable at 57 degrees C . The NUB3621/pTB913 host-vector system seems appropriate for molecular cloning . A RepA-based replicon, also derived from pTB19 but replicating by a theta mechanism, was highly unstable in B . stearothermophilus NUB3621. Science, 1992 Jun 5, 256(5062), 1416 - 9 Unusual resistance of peptidyl transferase to protein extraction procedures; Noller HF et al.; Peptidyl transferase, the ribosomal activity responsible for catalysis of peptide bond formation, is resistant to vigorous procedures that are conventionally employed to remove proteins from protein-nucleic acid complexes . When the "fragment reaction" was used as a model assay for peptide bond formation, Escherichia coli ribosomes or 50S subunits retained 20 to 40 percent activity after extensive treatment with proteinase K and SDS, but lost activity after extraction with phenol or exposure to EDTA . Ribosomes from the thermophilic eubacterium Thermus aquaticus remained more than 80 percent active after treatment with proteinase K and SDS, which was followed by vigorous extraction with phenol . This activity is attributable to peptidyl transferase, as judged by specific inhibition by the peptidyl transferase-specific antibiotics chloramphenicol and carbomycin . In contrast, activity is abolished by treatment with ribonuclease T1 . These findings support the possibility that 23S ribosomal RNA participates in the peptidyl transferase function. J Biol Chem, 1992 Jun 5, 267(16), 10999 - 1006 Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima; Rehaber V et al.; The molecular basis of thermal stability of globular proteins is a highly significant yet unsolved problem . The most promising approach to its solution is the investigation of the structure-function relationship of homologous enzymes from mesophilic and thermophilic sources . In this context, D-glyceraldehyde-3-phosphate dehydrogenase has been the most extensively studied model system . In the present study, the most thermostable homolog isolated so far is described with special emphasis on the stability of the enzyme under varying solvent conditions . D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima is an intrinsically thermostable enzyme with a thermal transition temperature around 110 degrees C . The amino acid sequence, electrophoresis, and sedimentation analysis prove the enzyme to be a homotetramer with a gross structure similar to its mesophilic counterparts . The enzyme in the absence and in the presence of its coenzyme, NAD+, exhibits no drastic structural differences except for a 3% change in sedimentation velocity reflecting slight alterations in the quaternary structure of the enzyme . At low temperature, in the absence of denaturants, neither "cold denaturation" nor subunit dissociation are detectable . Guanidinium chloride and pH-dependent deactivation precede the decrease in fluorescence emission and ellipticity, suggesting a complex denaturation mechanism . An up to 3-fold activation of the enzyme at low guanidinium concentration may be interpreted in terms of a compensation of the tight packing of the thermophilic enzyme at low temperature . Under destabilizing conditions, e.g . moderate concentrations of chaotropic agents, low temperature favors denaturation . The effect becomes important in reconstitution experiments after preceding guanidinium denaturation; the reactivation yield at low temperature drops to zero, whereas between 35 and 80 degrees C reactivation exceeds 80% . Shifting the temperature from approximately 0 degrees C to greater than or equal to 30 degrees C releases a trapped tetrameric intermediate in a fast reaction . Concentration-dependent reactivation experiments prove renaturation of the enzyme to involve consecutive folding and association steps . Reconstitution at room temperature yields the native protein, in spite of the fact that the temperature of the processes in vitro and in vivo differ by more than 60 degrees C. Mol Microbiol, 1992 Jun, 6(11), 1555 - 64 Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8; Lasa I et al.; The transcription and translation signals of the S-layer gene (slpA) from Thermus thermophilus HB8 have been used to express a thermostable kanamycin adenyl transferase gene in this organism . The chimaeric resistance gene was inserted in vitro into slpA to produce different inactive forms of the gene, which were used to transform T . thermophilus HB8 . After 48 hours of incubation at 70 degrees C, only two constructions that contained the kat gene flanked by Thermus sequences from both sides of slpA were able to produce protein layer (P100)-defective mutants . The mutants obtained with both constructions showed identical protein patterns, in which a major 50 kDa protein and two other minor proteins were tentatively identified as P100 fragments, expressed from the extreme 5' end of slpA . They also exhibited important phenotypic defects, such as slow growth in liquid broth, a tendency to aggregate as 'rotund bodies', a twisted filamentous shape, and an extreme sensitivity to lysozyme, suggesting protective and shaping roles for the S-layer in T . thermophilus HB8 . These results also demonstrate for the first time the feasibility of using selective antibiotic-resistance markers in extreme thermophiles. Biochem J, 1992 Jun 1, 284 ( Pt 2), 551 - 5 A thermostable NADH oxidase from anaerobic extreme thermophiles; Maeda K et al.; A high-abundance NADH-oxidizing enzyme (NADH: acceptor oxidoreductase, EC 1.6.99.3) has been identified and isolated from a range of anaerobic extreme thermophiles, including strains of Clostridium thermohydrosulfuricum and Thermoanaerobium brockii . By use of a pseudo-affinity salt-promoted adsorbent, a nearly pure sample was obtained in one step; remaining impurities were separated by ion-exchange . The fully active purified enzyme contains FAD (two molecules per subunit of 75-78 kDa) and iron-sulphur, and is hexameric in its most active form . The reaction with oxygen is a one- or two-electron transfer to produce superoxide radical and H2O2; other acceptors include tetrazolium salts, dichlorophenol-indophenol, menadione and ferricyanide . The role of the enzyme is not clear; it was found not to be NAD:ferredoxin oxidoreductase, which is a major NADH-utilizing enzyme in these organisms. Eur J Biochem, 1992 Jun 1, 206(2), 349 - 57 Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Colombo S et al.; A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus . Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule . An isoelectric point of 5.9 was also determined . The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+ . The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids . An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus . The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively . With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85 degrees C . Furthermore, thanks to its relatively low activation energy, i.e . 31.0 kJ/mol, it was still significantly active at room temperature . At 40 degrees C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99% . Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin . A weak similarity was only found with bovine carboxypeptidase B. Indian J Exp Biol, 1992 Jun, 30(6), 482 - 6 Induction and regulation of alpha-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14 (ATCC 48104); Sadhukhan R et al.; The alpha-amylase enzyme synthesis was higher when M . thermophila D-14 (ATCC 48104) was grown in culture medium incorporated with starch or other carbohydrates containing maltose units . Maximum enzyme production was attained with 1% starch followed by a gradual decrease with increasing concentration . Marked decrease in alpha-amylase synthesis occurred with the addition of glucose to the culture medium and this decreasing activity was proportional to the concentration of glucose . The enzyme synthesis was resumed as soon as the glucose concentration fell below a critical level . The addition of cAMP did not eliminate the repressive activity of glucose . The findings suggest that extracellular alpha-amylase synthesis in M . thermophila D-14 was inducible and subject to catabolite repression. J Med Assoc Thai, 1992 Jun, 75(6), 350 - 64 Isolation of Campylobacters from the canals of Bangkok metropolitan area; Dhamabutra N et al.; The 100 ml of canal water samples of 36 canals in Bangkok Metropolitan Area were examined in three periods starting from July-September 1988, November 1988-January 1989 and February-April 1989 . Each time the 52 water samples were checked . Of 156 water samples, 116 strains of Campylobacter species were isolated . They were 63.79 per cent (74 strains) of C . cryaerophila and 36.21 per cent (42 strains) of C . cryaerophila-like organisms . The differentiation was determined by urease activity test . C . cryaerophila was isolated from 44.23 per cent (23 strains), 51.19 per cent (27 strains) and 46.15 per cent (24 strains) and also C . cryaerophila-like organism from 28.85 per cent (15 strains), 19.23 per cent (10 strains) and 32.69 per cent (17 strains) of the 52 samples during each period respectively . Since C . cryaerophila and C . cryaerophila-like are aerotolerant Campylobacter, they grow well in aerobic conditions at 25 degrees-36 degrees C . On the contrary, thermophilic Campylobacter such as C . jejuni, C . coli and C . laridis require atmosphere containing 5 per cent O2, 10 per cent CO2, 85 per cent N2 and temperature at 36 degrees-42 degrees C, so the environment in the canals is unfavorable for their growth . The etiological role of C . cryaerophila in pathogenesis in humans is still unknown, and requires furthers study . This study shows that canals can be an important source of these two Campylobacter species that might be potential pathogens in the future. Gen Physiol Biophys, 1992 Jun, 11(3), 229 - 39 Thermally-induced delayed fluorescence of photosystem I and II chlorophyll in thermophilic cyanobacterium Synechococcus elongatus; Kaurov YuN et al.; Stationary delayed fluorescence (DF) of chlorophyll in isolated membrane preparations from thermophilic cyanobacterium Synechococcus elongatus was investigated as a function of temperature . Two peaks at different temperatures were observed . The low-temperature peak (54-60 degrees C) coincided with the main maximum of the thermally-induced delayed fluorescence of chlorophyll in intact cells and PSII-particles with active oxygen-evolving system . The high-temperature peak (78 degrees C) coincided with the minor band of delayed light emitted by intact cells . It was also observed in the delayed fluorescence emission from a PSI-enriched fraction preparation . The intensities of the DF peaks were dependent on the presence of inhibitors, donors and acceptors that cause specific effects on electron transport of the two photosystems . The low-temperature and high-temperature peaks were related to PSII and PSI, respectively . The manifestation of delayed fluorescence from PSI and PSII at different temperatures seems to be a specific property of thermophilic cyanobacteria . The reason for this may be a high thermal stability of the photosystems and the lack of the PSII antenna complex in isolated membranes . Consequently, the relative yield of delayed fluorescence from PSI markedly increases . Thermally-induced fluorescence seen in membranes of cyanobacteria showed a high sensitivity to structural and functional membrane alterations induced by pH changes, different electron transport stabilizing agents or different concentrations of MgCl2. Shi Yan Sheng Wu Xue Bao, 1992 Jun, 25(2), 165 - 72 Molecular and functional properties of EF-Tu from Calderobacterium hydrogenophilum; Qiao CL; Protein synthesis elongation factor Tu (EF-Tu) was purified from an extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum . The relative molecular mass of EF-Tu . GDP was 51,000 . The factor was heat stable and lost only 50% of its activity after heating at 80 degrees C for 5 min . Native and reduced EF-Tu or EF-Tu . GDP contained one SH-reactive group . The elongation factors from C . hydrogenophilum and E . coli were shown to be immunologically identical . From the Southern hybridization analysis seems to suggest that chromosome DNA of C . hydrogenophilum has two tuf genes. Biochimie, 1992 Jun, 74(6), 585 - 8 The 23S-5S spacer of two rRNA loci of Streptococcus salivarius subsp thermophilus includes a promoter; Guedon G et al.; The nucleotide sequence of the 3' part of a ribosomal and transfer RNA locus from Streptococcus salivarius subsp thermophilus NST1403 was determined . The sequenced DNA fragment includes the 3' end of a 23S rRNA gene, a 5S rRNA gene, a tRNA(asn) gene and a potential transcriptional terminator . The tRNA gene does not encode for the CCA 3'terminus of mature tRNA . We compared this sequence to a promoter-carrying DNA fragment sequence (P20) of Streptococcus salivarius subsp thermophilus A054 {1} . We found that the P20 sequence included the 3' end of a 23S rRNA gene, a 5S rRNA gene and the 5' part of a tRNA(val) gene . The two 23S-5S spacer sequences are identical and contain a promoter and a potential 23S rRNA processing site . Therefore, 5S rRNA and tRNA genes could be transcribed from a promoter located within the 23S-5S spacer of at least two of the six rRNA loci. J Appl Bacteriol, 1992 Jun, 72(6), 467 - 74 Differentiation between thermophilic Campylobacter species by species-specific antibodies; Griffiths PL et al.; The four species of thermophilic campylobacters, Campylobacter jejuni, C . coli, C . upsaliensis and C . lari, are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests . Those tests which do discriminate sometimes give discordant results . Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein . The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization . The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys . Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen. Mol Gen Genet, 1992 Jun, 233(3), 462 - 8 The integrated state of the rolling-circle plasmid pTB913 in the composite Bacillus plasmid pTB19; Oskam L et al.; pTB19, a 27 kb plasmid originating from a thermophilic Bacillus species, contains integrated copies of two rolling-circle type plasmids on a 10.6 kb DNA fragment . In the present study we analysed the part of pTB19 that contains the rolling-circle plasmid pTB913 and the region in between the two rolling-circle plasmids . We show that, in the integrated state, pTB913 was flanked by a 55 bp direct repeat that duplicated part of the replication initiation gene repB . Since repB was interrupted, the integrated pTB913 could not initiate rolling-circle replication . Autonomously replicating pTB913 was produced from pTB19, probably through recombination between the 55 bp direct repeats; this was a rare event . Since the second integrated rolling-circle type plasmid also contained a non-functional replication initiation gene, replication of pTB19 must be controlled by the RepA determinant . Theta-type replication, controlled by RepA is likely to account for the high stability of pTB19 . In between the two integrated rolling-circle plasmids was present an open reading frame (447 codons) which could encode a protein of unknown function. J Bacteriol, 1992 Jun, 174(11), 3487 - 93 Permeability and charge-dependent adsorption properties of the S-layer lattice from Bacillus coagulans E38-66; Sara M et al.; We investigated the permeability properties of the oblique S-layer lattice from Bacillus coagulans E38-66 after depositing cell wall fragments on a microfiltration membrane, cross-linking the S-layer protein with glutaraldehyde, and degrading the peptidoglycan with lysozyme . Comparative permeability studies on such multilayered S-layer membranes and suspended S-layer vesicles from thermophilic members of the family Bacillaceae with use of the space technique (M . Sara and U . B . Sleytr, J . Bacteriol . 169:4092-4098, 1987) revealed identical molecular exclusion limits (M . Sara and U . B . Sleytr, J . Membr . Sci . 33:27-49, 1987) . Examination of the S-layer lattice from B . coagulans E38-66 with the S-layer membrane technique revealed unhindered passage for molecules up to the size of myoglobin (M(r) 17,000) . The molecular dimensions of this protein (2.8 by 3.2 by 4.5 nm) correspond approximately to the size of the ovoid-shaped pore previously shown by high-resolution electron microscopy of negatively stained S-layer self-assembly products (D . Pum, M . Sara, and U . B . Sleytr, J . Bacteriol . 171:5296-5303, 1989) . Chemical modification of the S-layer protein and comparative labeling, adsorption, and permeability studies clearly demonstrated that (i) in the native state, free amino and carboxyl groups are present on the outer S-layer face and in the interior of the pores and (ii) electrostatic interactions between these groups prevent unspecific adsorption of the S-layer in vivo. FEBS Lett, 1992 May 25, 303(1), 64 - 8 Affinity labeling of GTP-binding proteins in cellular extracts; Low A et al.; GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized {alpha-32P}GTP as affinity label . Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction . Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography . In addition cross-linking of {alpha-32P}GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T . thermophilus and initiation factor 2 (IF2) from T . thermophilus . The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins. J Biol Chem, 1992 May 25, 267(15), 10561 - 9 The purification, characterization, and primary structure of a small redox protein from Methanobacterium thermoautotrophicum, an archaebacterium; McFarlan SC et al.; A small redox-active protein has been purified to homogeneity from cell-free extracts of the strictly anaerobic thermophilic methanogen, Methanobacterium thermoautotrophicum (strain Marburg) . The purification consisted of streptomycin sulfate and acid treatments and three chromatographic steps using Sephadex G-75, Mono Q HR 10/10, and Superose 12 HR 10/30 columns . When these procedures were carried out under strictly anaerobic conditions, approximately 3 mg of this protein could be isolated from 45 g of wet cell paste . Like the thioredoxins and glutaredoxins, it is a small acidic protein (pI = 4.2) consisting of 83 amino acids (M(r) = 9136) . In the presence of dithiothreitol or dihydrolipoate, the protein serves as a hydrogen donor for the ribonucleotide reductase from Escherichia coli, and it catalyzes the reduction of insulin . However, it does not interact with the thioredoxin reductases from E . coli or Corynebacterium nephridii and does not function as a hydrogen donor for the ribonucleotide reductase of C . nephridii . The amino acid sequences determined by automated Edman degradation of the 14C-carboxymethylated protein and of peptides derived from trypsin and chymotrypsin digestions show a redox-active site -Cys-Pro-Tyr-Cys-, typical of the glutaredoxins . Its amino acid sequence shows moderate identity with the known glutaredoxins (E . coli, yeast, rabbit bone marrow, calf thymus, and pig liver) when the proteins are aligned at the active site . The secondary structure of the glutaredoxin-like protein predicted by the Chou-Fasman procedure shows that it is similar to the known glutaredoxins . However, surprisingly, the protein does not function as a glutathione-disulfide oxidoreductase in the presence of glutathione and glutathione reductase . This glutaredoxin-like protein may be a component of a ribonucleotide-reducing system distinct from the previously described systems utilizing thioredoxin or glutaredoxin. Biochim Biophys Acta, 1992 May 21, 1106(2), 273 - 81 Molecular packing parameters of bipolar lipids; Cavagnetto F et al.; The bipolar lipid fractions extracted from the thermophilic archaeobacterium Sulfolobus solfataricus have different chemical structures and geometrical shapes . The conditions which lead to the formation of vesicles were investigated in order to study the self-assembly of these molecules . Such conditions are fulfilled when an appropriate mixture of two different molecular species (both bipolar or bipolar and monopolar) is used . According to the theory introduced by Israelachvili and co-workers, lipid self-assembly results from the balance of interaction free energy, entropy and molecular geometry . We have shown that this theory can be extended to bipolar lipids, in spite of their more complex nature, and the experimental results obtained combining 1H-NMR, light scattering and entrapped volume techniques closely match theoretical expectations . To carry out calculations, it was necessary to introduce hypotheses about the disposition of bipolar molecules in the vesicle membrane . These hypotheses have been tested indirectly by measuring the transport properties mediated by carriers or channels, whose transport mechanism can be considered to be a probe of the membrane structure. Biochim Biophys Acta, 1992 May 20, 1100(2), 125 - 36 The structure of Photosystem I from the thermophilic cyanobacterium Synechococcus sp . determined by electron microscopy of two-dimensional crystals; Bottcher B et al.; The structure of the Photosystem I (PS I) complex from the thermophilic cyanobacterium Synechococcus sp . has been investigated by electron microscopy and image analysis of two-dimensional crystals . Crystals were obtained from isolated PS I by removal of detergents with Bio-Beads . After negative staining, either single layers or two superimposed layers with a rotational different orientation were observed . The layers have a rectangular unit cell of 16.0 x 15.0 nm, which contains two PS I monomers . The monomers are arranged alternating up and down in each layer . For double-layer crystals, the images of the two layers could be separately processed by a combination of Fourier-peak-filtering and correlation averaging . Features in the two-dimensional plane can be seen with a resolution up to 1.5-1.8 nm . A model for the PS I structure was obtained by combining three-dimensional reconstructions from three tilt-series . The model shows an asymmetric PS I complex . On one side (presumably the stromal side) there is a 3 nm high ridge . This is most likely comprised of the psaC, psaD and psaE subunits . The other side (presumably the lumenal side) is rather flat, but in the center there is a 3 nm deep indentation, which possibly separates partly the two large subunits psaA and psaB. Biochem Biophys Res Commun, 1992 May 15, 184(3), 1256 - 60 Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium; Fauque G et al.; A soluble {NiFe} hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile . A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21 . This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa . This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction . The H2/HD ratio is lower than one in the D2-H+ exchange reaction . The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2- . The EPR spectrum of the native hydrogenase shows the presence of a {3Fe-4S} oxidized cluster and of a Ni(III) species. J Biol Chem, 1992 May 15, 267(14), 10184 - 92 Expression, purification, and characterization of a recombinant ribonuclease H from Thermus thermophilus HB8; Kanaya S et al.; Thermus thermophilus ribonuclease H was overexpressed and purified from Escherichia coli . The determination of the complete amino acid sequence allowed modification of that predicted from the DNA sequence, and the enzyme was shown to be composed of 166 amino acid residues with a molecular weight of 18,279 . The isoelectric point of the enzyme was 10.5, and the specific absorption coefficient A0.1%(280) was 1.69 . The enzymatic and physicochemical properties as well as the thermal and conformational stabilities of the enzyme were compared with those of E . coli RNase HI, which shows 52% amino acid sequence identity . Comparison of the far and near UV circular dichroism spectra suggests that the two enzymes are similar in the main chain folding but different in the spatial environments of tyrosine and tryptophan residues . The enzymatic activities of T . thermophilus RNase H at 37 and 70 degrees C for the hydrolysis of either an M13 DNA/RNA hybrid or a nonanucleotide duplex were approximately 5-fold lower and 3-fold higher, respectively, as compared with E . coli RNase HI at 37 degrees C . The melting temperature, Tm, of T . thermophilus RNase H was 82.1 degrees C in the presence of 1.2 M guanidine hydrochloride, which was 33.9 degrees C higher than that observed for E . coli RNase HI . The free energy changes of unfolding in the absence of denaturant, delta G{H2O}, of T . thermophilus RNase H increased by 11.79 kcal/mol at 25 degrees C and 14.07 kcal/mol at 50 degrees C, as compared with E . coli RNase HI. Arch Biochem Biophys, 1992 May 15, 295(1), 1 - 4 Crystallization and preliminary crystallographic analysis of a thermostable mutant of kanamycin nucleotidyltransferase; Kanikula AM et al.; A thermostable mutant of kanamycin nucleotidyltransferase isolated by cloning and selection for kanamycin resistance in Bacillus stearothermophilus at 70 degrees C has been crystallized in a form suitable for high-resolution diffraction analysis . This enzyme catalyzes nucleotidyl group transfer from nucleoside triphosphates such as ATP to hydroxyl groups of various aminoglycosides, thus inactivating the antibiotic . The kanamycin nucleotidyltransferase gene, originally encoded on plasmid pUB110 from the mesophile Staphylococcus aureus, was transferred to the thermophile B . stearothermophilus via shuttle plasmids and the mutant carrying the substitutions D80Y and T130K was isolated from kanamycin-resistant colonies grown at 70 degrees C . The thermostable enzyme was crystallized in two forms from solutions of polyethylene glycol 8000 (PEG8000) using batch and vapor diffusion methods . Type I crystals grown from 19% (w/v) PEG8000 and 200 mM NaCl belong to the orthorhombic space group C222(1), have unit cell dimensions of a = 128.4, b = 156.8, c = 155.8 A, and diffract to at least 2.4-A resolution . The type II form of the crystals were grown from 10% PEG8000, 200 mM KCl, and 3 mM CoCl2, and belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 78.9, and c = 220.4 A; these crystals diffract to at least 2.5-A resolution. J Biol Chem, 1992 May 5, 267(13), 9150 - 7 Lactose transport system of Streptococcus thermophilus . The role of histidine residues; Poolman B et al.; The lactose transport protein (LacS) of Streptococcus thermophilus is a chimeric protein consisting of an amino-terminal carrier domain and a carboxyl-terminal phosphoenolpyruvate:sugar phosphotransferase system (PTS) IIA protein domain . The histidine residues of LacS were changed individually into glutamine or arginine residues . Of the 11 histidine residues present in LacS, only the His-376 substitution in the carrier domain significantly affected sugar transport . The region around His-376 was found to exhibit sequence similarity to the region around His-322 of the lactose transport protein (LacY) of Escherichia coli, which has been implicated in sugar binding and in coupling of sugar and H+ transport . The H376Q mutation resulted in a reduced rate of uptake and altered affinity for lactose (beta-galactoside), melibiose (alpha-galactoside), and the lactose analog methyl-beta-D-thiogalactopyranoside . Similarly, the extent of accumulation of the galactosides by cells expressing LacS(H376Q) was highly reduced in comparison to cells bearing the wild-type protein . Nonequilibrium exchange of lactose and methyl-beta-D-thiogalactopyranoside by the H376Q mutant was approximately 2-fold reduced in comparison to the activity of the wild-type transport protein . The data indicate that His-376 is involved in sugar recognition and is important, but not essential, for the cotransport of protons and galactosides . The carboxyl-terminal domain of LacS contains 2 histidine residues (His-537 and His-552) that are conserved in seven homologous IIA protein(s) (domains) of PTSs . P-enolpyruvate-dependent phosphorylation of wild-type LacS, but not of the mutant H552Q, was demonstrated using purified Enzyme I and HPr, the general energy coupling proteins of the PTS, and inside-out membrane vesicles isolated from E . coli in which the lactose transport gene was expressed . The His-537 and His-552 mutations did not affect transport activity when the corresponding genes were expressed in E . coli. FEBS Lett, 1992 May 4, 302(1), 54 - 6 A comparative study of the relationship between thermostability and function of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus; Bobkova EV et al.; The relationship between thermostability and functional activities of phenylalanyl-tRNA synthetases (EC 6.1.1.20) from E . coli and Thermus thermophilus has been studied . In the case of the E . coli enzyme, the activity decreased after the 43 degrees C treatment, both in the {32P}PPi-ATP exchange reaction and the overall aminoacylation reaction, due to thermo-inactivation of the phenylalanyl-tRNA synthetase, whereas tRNA(Phe) preserved its native structure . In the Th . thermophilus system, the enzyme showed extreme thermostability (up to 90 degrees C), and the reduction in the tRNA aminoacylation rate after the 78 degrees C treatment was ascribed to denaturation of the tRNA(Phe) . Since the enzyme did not lose the {32P}PPi-ATP exchange activity up to 85 degrees C, the observed lower thermo-resistance of the tRNA is evidence that the native structure of ribonucleic acids should be one of the most difficult to stabilize at high temperatures. J Protozool, 1992 May-Jun, 39(3), 420 - 8 Characterization of the T, L, I, S, M and P cell surface (immobilization) antigens of Tetrahymena thermophila: molecular weights, isoforms, and cross-reactivity of antisera; Smith DL et al.; In the ciliate protist Tetrahymena thermophila the L, H, T, I, S, M and P cell surface proteins (immobilization antigens) are expressed under different conditions of temperature (L, H, T), culture media (I, S), and mutant genotype (M, P) . Immunoblot and autoradiographic studies using antisera to purified protein show that the molecular weights of these proteins range from 25,000 to 59,000 . The H, T, S, M and P antigens are recognized as single polypeptides, whereas L, I, and one allelic form of T each appear to consist of a family of polypeptides . Although antisera are specific in immobilization and immunofluorescence assays of surface protein in living cells, cross-reactivity is seen with denatured protein on immunoblots . It is hypothesized that the surface protein genes are organized into families of evolutionarily related isoloci. J Protozool, 1992 May-Jun, 39(3), 368 - 78 Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114; Dentler WL; Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins . Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved . The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase . Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase . Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities . Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface . The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase . These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme . The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved . Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles. Appl Environ Microbiol, 1992 May, 58(5), 1789 - 93 High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth; Nelson CM et al.; We describe a high-pressure reactor system suitable for simultaneous hyperbaric and hydrostatic pressurization of bacterial cultures at elevated temperatures . For the deep-sea thermophile ES4, the growth rate at 500 atm (1 atm = 101.29 kPa) and 95 degrees C under hydrostatic pressure was ca . three times the growth rate under hyperbaric pressure and ca . 40% higher than the growth rate at 35 atm. J Appl Bacteriol, 1992 May, 72(5), 400 - 9 Fungal and actinomycete spore aerosols measured at different humidities with an aerodynamic particle sizer; Madelin TM et al.; An aerodynamic particle sizer (APS) that uses laser Doppler velocimetry was used to determine aerodynamic diameters of spores of fungal and thermophilic actinomycete species common in mouldy hay, aerosolized at different humidities and temperatures . Results were compared with those obtained from inertial impaction in a cascade impactor . The APS gave slightly smaller measurements than the cascade impactor . Both methods gave aerodynamic diameters generally slightly smaller than the average spore dimensions observed on cascade impactor slides with a microscope . The latter measurements were less than axial dimensions given in the literature . Brief passage of spores through air at 95% relative humidity (RH) and 38 degrees C, compared with 40% RH and 20 degrees C, caused an immediate increase in their aerodynamic diameter and the breaking of chains of spores . Cultures maintained at 75% RH and aerosolized at 98% RH similarly produced larger spore particles than those passed through dry air . These findings have implications for mould-induced asthma and allergic alveolitis since they relate to physical behaviour of airborne spores and particle deposition sites in the lung. J Mol Evol, 1992 May, 34(5), 383 - 95 A phylogenetic analysis based on the gene encoding phosphoglycerate kinase; Vohra GB et al.; We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila . The amino acid sequence for the enzyme has also been derived from the cDNA sequence . The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids . Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes . The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences . The Tetrahymena sequence is a typical eukaryotic sequence . There is recognizable and clear homology across species that cover nearly the complete range of life forms . The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences. Eur J Biochem, 1992 May 1, 205(3), 881 - 5 Purification and characterization of a NADH oxidase from the thermophile Thermus thermophilus HB8; Park HJ et al.; A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps . The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence . It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain . The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H2O2 . The apparent Km values for NADH and NADPH are 4.14 microM and 14.0 microM (pH 7.2 at room temperature), respectively, with a sixfold greater kcat/Km values for NADH compared to NADPH . The enzyme uses O2 as an electron acceptor in the presence of either FAD, riboflavin 5'-phosphate or riboflavin as cofactor . In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochrome c, p-nitroblue tetrazolium, 2,6-dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles . No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2- as an intermediate . The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80 degrees C. Eur J Biochem, 1992 May 1, 205(3), 875 - 9 Molecular cloning and nucleotide sequence of the gene encoding a H2O2-forming NADH oxidase from the extreme thermophilic Thermus thermophilus HB8 and its expression in Escherichia coli; Park HJ et al.; The sequence of the 32 N-terminal amino acids of the NADH oxidase from the extreme thermophile, Thermus thermophilus HB8, was used to synthesize oligonucleotides to probe for the respective gene in a genomic library of T . thermophilus HB8 . The gene encoding the NADH oxidase, designated nox, was cloned, its nucleotide sequence was determined and found to be colinear with the N-terminal sequence of the enzyme . The molecular mass of 26835 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T . thermophilus HB8 (25,000 Da), as estimated by polyacrylamide gel electrophoresis under denaturing conditions . The nox gene was overexpressed in Escherichia coli and a protocol for the rapid purification of the enzyme was developed . The E . coli-borne T . thermophilus HB8 NADH oxidase has properties identical to those of the authentic T . thermophilus HB8 enzyme and possesses a high thermal stability. Arch Biochem Biophys, 1992 May 1, 294(2), 373 - 81 Mapping of antigenic sites to monoclonal antibodies on the primary structure of the F1-ATPase beta subunit from Escherichia coli: concealed amino-terminal region of the subunit in the F1; Miki J et al.; To analyze relationships between the ternary and primary structures of the beta subunit of Escherichia coli F1 ATPase, we prepared two monoclonal antibodies beta 12 and beta 31 against the beta peptide . These antibodies bind to the beta subunit but do not bind to the F1 ATPase, resulting in no inhibition of the ATPase activities . Several different portions of the beta subunit peptide were prepared by constructing expression plasmids carrying the corresponding DNA segment of the beta subunit gene amplified by the polymerase chain reaction . Western blotting analysis using these peptides revealed that the antibodies bound to a peptide of 104 amino acid residues from the amino terminal end, which is outside the previously estimated catalytic domain between residues 140 and 350 . These results indicated that the amino terminal portion of the maximal 104 residues is not exposed to the surface of the F1 ATPase . The binding spectrum of the antibodies to the subunit from various species including Vibrio alginolyticus and thermophilic bacterium PS3 indicated possible epitope sequences within the 104 residues . The ternary structure of the beta subunit, in terms of cleavage sites by endopeptidases, was analyzed using the antibodies . A 43-kDa peptide without binding ability to beta 12 and beta 31 appeared upon cleavage by lysyl endopeptidase . The results suggested that lysyl residues from around 70 to 100 from the amino terminus are exposed to the surface of the beta subunit. Curr Genet, 1992 May, 21(6), 521 - 5 Structure and expression of a plastid-encoded groEL homologous heat-shock gene in a thermophilic unicellular red alga; Maid U et al.; A gene homologous to the E . coli groEL locus was identified on the plastid genome of the unicellular red alga Cyanidium caldarium strain 14-1-1 (synonym: Galdieria sulphuraria) . The complete nucleotide sequence was determined and compared to bacterial- and nuclear-encoded counterparts of higher plants . At the amino-acid level the C . caldarium gene shows 70% homology to the corresponding gene of the cyanobacterium Synechococcus and 52% homology to nuclear-encoded counterparts of higher plants, respectively . Northern and Western blot experiments were used to investigate the dependence of the transcript- and protein-level on culture temperature and heat shock. J Dairy Sci, 1992 May, 75(5), 1192 - 6 Lithium chloride-sodium propionate agar for the enumeration of bifidobacteria in fermented dairy products; Lapierre L et al.; Lithium chloride-sodium propionate agar has been developed for the enumeration of bifidobacteria in fermented dairy products . The medium contains lithium chloride and sodium propionate to inhibit the growth of other lactic acid bacteria . Pure cultures of bifidobacteria, lactobacilli, and streptococci were tested for growth in this medium . With one exception, all bifidobacteria were able to grow in this medium and in a nonselective agar with a difference not exceeding .4 log units . However, none of the lactobacilli tested and only one strain each of Streptococcus salivarius ssp . thermophilus and Lactococcus lactis ssp . cremoris grew in lithium chloride-sodium propionate agar . In those cases, the numbers of colonies were lower in lithium chloride-sodium propionate agar by 1.26 and 2.51 log units, respectively, compared with a nonselective agar . Bifidobacteria were also selectively isolated from all fermented milks and cheeses analyzed. Nucleic Acids Res, 1992 Apr 25, 20(8), 1843 - 50 The nature of preferred hairpin structures in 16S-like rRNA variable regions; Wolters J; Variable length hairpins in 16S-like rRNA show a predominance for tetra-loops, its degree correlates with the protein content of the ribosome . The number of base-pairs adjacent to the loop (the tip size) and the nearest neighbor composition contribute to the stability of hairpin structures . The average tip size in length variable hairpins correlates with the thermophilicity of the organism, i.e . in temperate environments less stable stem structures are tolerated or even necessary . The most abundant loop families UUCG, GCAA, and CUUG occur most frequently at loop sizes 3, 2, and 7, respectively . Short tips of size less than or equal to 4 generally prefer nearest-neighbor combinations that result in CCC-GGG . Loop-specific tipmost nearest neighbors are revealed at longer tips: CUC(UUCG)GAG, GUA(GCAA)UAC with a maximum at tip sizes 5-6, and GWG(CUUG)CWC . Conserved hairpins, however, prefer variants of the UUCG and GCAA motifs with additional purines . Minor loop families and single motifs such as UUUA, UUUU, CUUGU, UUCGG, and UUU are investigated for preferable tip sizes and nearest-neighbor composition . Specific features are revealed for prominent hexa-loops. J Chromatogr, 1992 Apr 24, 597(1-2), 147 - 53 Immobilized ferredoxins for affinity chromatography of ferredoxin-dependent enzymes; Sakihama N et al.; An immobilized ferredoxin more stable than the conventional immobilized spinach ferrodoxin was prepared by reacting CNBr-Sepharose with ferredoxins isolated from barley and Synechococcus vulcanus, a thermophilic blue-green alga . The dissociation constants of immobilized ferredoxin from spinach, barley and S . vulcanus for spinach ferredoxin-NADP reductase were 0.922, 2.505 and 5.209 microM, respectively, whereas those for barley ferredoxin-NADP reductase were 1.159, 0.579 and 2.851 microM, respectively . The order of stability was S . vulcanus greater than barley greater than spinach . The immobilized ferredoxin was applied to the simultaneous detection of ferredoxin-dependent enzymes in spinach chloroplasts . Over 20 polypeptides were detected . Synechococcus ferredoxin could also be immobilized on a Toyopearl gel and repeatedly used in an automated high-performance liquid chromatographic system. FEBS Lett, 1992 Apr 20, 301(2), 145 - 9 Stoichiometric association of extrinsic cytochrome c550 and 12 kDa protein with a highly purified oxygen-evolving photosystem II core complex from Synechococcus vulcanus; Shen JR et al.; A highly purified, native photosystem II (PS II) core complex was isolated from thylakoids of Synechococcus vulcanus, a thermophilic cyanobacterium by lauryldimethylamine N-oxide (LDAO) and dodecyl beta-D-maltoside solubilization . This native PS II core complex contained, in addition to the proteins that have been well characterized in the core complex previously purified by LDAO and Triton X-100, two more extrinsic proteins with apparent molecular weights of 17 and 12 kDa . These two proteins were associated with the core complex in stoichiometric amounts and could be released by treatment with 1 M CaCl2 or 1 M alkaline Tris but not by 2 M NaCl or low-glycerol treatment, indicating that they are the real components of PS II of this cyanobacterium . N-Terminal sequencing revealed that the 17 and 12 kDa proteins correspond to the apoprotein of cytochrome c550, a low potential c-type cytochrome, and the 9 kDa extrinsic protein previously found in a partially purified PS II preparation from Phormidium laminosum, respectively . In spite of retention of these two extrinsic proteins, no homologues of higher plant 23 and 17 kDa extrinsic proteins could be detected in this cyanobacterial PS II core complex. Biochem Biophys Res Commun, 1992 Apr 15, 184(1), 31 - 5 Characterization of a cDNA clone from the haemoparasite Babesia bovis encoding a protein containing an "HMG-Box"; Dalrymple BP et al.; The complete nucleotide sequence of a Babesia bovis cDNA clone encoding a protein containing an HMG-Box has been determined . The predicted protein of 97 amino acids has a molecular weight of 11,116 . It exhibits approximately 45% overall amino acid identity with the Saccharomyces cerevisiae non-histone protein 6A (NHP6A) and approximately 57% identity in the HMG-Box . The B . bovis protein has been designated NHP1 . Like HNP6A, and unlike most other HMG1 homologues, NHP1 does not have a basic or an acidic carboxy-terminal domain . The amino acid sequence of HNP1 is much less similar to HMG1 homologues of another protozoan, Tetrahymena thermophila, than to the HMG1 homologues identified in S . cerevisiae, plants and vertebrates . This suggests that the T . thermophila proteins may not be true HMG1 homologues, or that they may be evolving at a much faster rate. Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3195 - 9 Reaction of cyanide with cytochrome ba3 from Thermus thermophilus: spectroscopic characterization of the Fe(II)a3-CN.Cu(II)B-CN complex suggests four 14N atoms are coordinated to CuB; Surerus KK et al.; Cytochrome ba3 from Thermus thermophilus reacts slowly with excess HCN at pH 7.4 to create a form of the enzyme in which CuA, cytochrome b, and CuB remain oxidized, while cytochrome a3 is reduced by one electron, presumably with the formation of cyanogen . We have examined this form of the enzyme by UV-visible, resonance Raman, EPR, and electron nuclear double resonance spectroscopies in conjunction with permutations of 13C- and 15N-labeled cyanide . The results support a model in which one CN- binds through the carbon atom to ferrous a3, supporting a low-spin (S = 0) configuration on the Fe; bridging by this cyanide to the CuB is weak or absent . Four 14N atoms, presumably donated by histidine residues of the protein, provide a strong equatorial ligand field about CuB; a second CN- is coordinated through the carbon atom to CuB in an axial position. FEBS Lett, 1992 Apr 13, 301(1), 83 - 8 Cloning and sequence analysis of the phenylalanyl-tRNA synthetase genes (pheST) from Thermus thermophilus; Keller B et al.; While crystals suitable for X-ray diffraction analyses are available of phenylalanyl-tRNA synthetase (PheRS) from the thermophilic bacterium Thermus thermophilus, neither the primary structure of its constituent alpha and beta subunits nor the nucleotide sequence of the corresponding pheS and pheT genes were known . Using specific oligonucleotides of conserved pheS regions that were adapted to the T . thermophilus codon usage, we identified, cloned and subsequently sequenced the pheST genes of this bacterium . The sequences reported here will greatly aid in the three-dimensional structure determination of T . thermophilus PheRS, a heterotetrameric (alpha 2 beta 2), class II aminoacyl-tRNA synthetase. Int J Syst Bacteriol, 1992 Apr, 42(2), 263 - 9 Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen . nov; Wisotzkey JD et al.; Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen . nov . An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species . The thermoacidophilic Bacillus species B . acidocaldarius, B . acidoterrestris, and B . cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date . This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus. J Chromatogr, 1992 Apr 10, 596(2), 203 - 9 Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography; Ide H et al.; The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E . coli in two steps while maintaining the enzymatic activity . The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S) . In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared. Biochim Biophys Acta, 1992 Apr 6, 1130(3), 339 - 42 Extended secondary structure as a basis of increased RNA stability in a thermophilic alga Cyanidium caldarium; Elela SA et al.; To identify important structural features in the intergenic sequences of ribosomal DNAs, the nucleotide sequence of the 18-25S rRNA intergenic region was determined in a thermophilic alga, Cyanidium caldarium . Although the mature 5.8S RNA is more stable to thermal denaturation, sequence comparisons reveal a longer molecule with a surprisingly low G/C nucleotide composition . Estimates of the structure further indicate that, unlike other thermophilic examples, thermostability in this organism results, at least in part, from an extended secondary structure. Biochim Biophys Acta, 1992 Apr 6, 1130(3), 289 - 96 Characteristic electron microscopical projections of the small ribosomal subunit from Thermomyces lanuginosus; Harauz G et al.; Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of small ribosomal subunits from the thermophilic fungus Thermomyces lanuginosus into their characteristic views . Three predominant modes of adsorption to the support were elucidated: right-lateral, left-lateral and asymmetric, showing reproducible detail approaching 1.8 nm resolution . The projections of the fungal complexes appeared almost identical to those of HeLa cells, rat liver and rabbit reticulocytes studied previously in this manner . This result contrasts with the greater variation in fine structural features observed between ribosomal subunits from different prokaryotic species. J Biol Chem, 1992 Apr 5, 267(10), 6531 - 40 Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus; McManus JD et al.; Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus . All three auracyanins are peripheral membrane proteins . Auracyanin A was described previously (Trost, J . T., McManus, J . D., Freeman, J . C., Ramakrishna, B . L., and Blankenship, R . E . (1988) Biochemistry 27, 7858-7863) and is not glycosylated . The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa . The amino acid sequences of the B forms are presented . All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different . Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins . Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein . All three auracyanins exhibit a midpoint redox potential of +240 mV . Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer . Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center. Biochimie, 1992 Apr, 74(4), 327 - 36 Ribosomal proteins from Thermus thermophilus for structural investigations; Garber MB et al.; In parallel with crystallographic studies of ribosomes from Thermus thermophilus, a long-term program on the crystallization and structural investigations of ribosomal proteins from the same microorganism has been started at the Institute of Protein Research (Pushchino, Russia) . At present, more than half of the individual ribosomal proteins from T thermophilus have been purified without denaturating agents on a preparative scale and some of them have been obtained in the crystalline form . X-ray structural analysis of two ribosomal proteins, L1 and S6, is being carried out jointly with the Institute of Molecular Biology (Moscow, Russia) and laboratory of professor A Liljas (Lund University, Sweden) . L1 is the large protein of the large ribosomal subunit . It can bind not only to a specific site on the 23S rRNA, but also to the mRNA that codes for L1 and L11, thereby acting as a translational repressor for the synthesis of these proteins . The crystals of L1 are orthorhombic and diffract to about 2 A resolution . Native data and data for several heavy atom derivatives have been collected . S6 is a small acidic protein from the small ribosomal subunit . The crystals of S6 are orthorhombic and diffract to 2 A resolution . Native data and derivatives' data have been collected. FEMS Microbiol Lett, 1992 Apr 1, 71(1), 73 - 7 A non-covalent NH2-terminal pro-region aids the production of active aqualysin I (a thermophilic protease) without the COOH-terminal pro-sequence in Escherichia coli; Lee YC et al.; The precursor of aqualysin I, an extracellular protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a C-terminal pro-sequence . In an Escherichia coli expression system, mature and active aqualysin I is formed by treatment at 65 degrees C and the N-pro-sequence is required for its production . Complete deletion of the C-pro-sequence did not affect the production of active aqualysin I, indicating that the C-pro-sequence is not essential . A non-covalent N-pro-region was separately synthesized from the protease domain with or without the C-pro-sequence . In this system, mature and active aqualysin I was detected only when the C-pro-sequence was deleted. J Vet Diagn Invest, 1992 Apr, 4(2), 181 - 5 Fungi associated with bovine abortion in the northern plains states (USA); Knudtson WU et al.; Mycotic infection was diagnosed in 6.8% of 6,858 cases of bovine abortion and stillbirth examined during a 9-year period . Aspergilli were associated with approximately 5% of all abortion cases and 71% of 446 cases that were cultured for fungi and diagnosed as mycotic abortion . Aspergillus fumigatus was the most frequent isolate (62%), followed by A . terreus (6.7%), Emericella (Aspergillus) nidulans (3.0%), A . flavus (2.9%), and E . rugulosus (less than 1.0%) . Zygomycetes (Absidia, Mortierella, Rhizomucor, Rhizopus) accounted for 21% of the cases . Pseudallescheria boydii and yeasts (Candida, Torulopsis) were each identified in 2% of the cases . Fungi that uncommonly cause infection accounted for 2% of the cases and included Curvularia geniculata, Exophilia jeanselmei, Hendersonula toruloidea, Lecythosphora hoffmannii, Talaromyces flavus var . flavus (Penicillium vermiculatus), T . (Penicillium) thermophilus, and Wangiella dermatitidis . About 10% of the mycotic cases were mixed fungal infections involving A . fumigatus (87%), A . flavus (12.5%), or E . nidulans (12.5%) coexisting with Absidia corymbifera (72%), Rhizomucor pusillus (4.3%), or Rhizopus arrhizus (4.3%) . In each mixed infection, both septate and nonseptate hyphae were observed in placental tissues . Twelve percent of the mycotic abortion cases were diagnosed by histologic examination alone because isolation attempts were negative or only formalin-preserved tissues were available. Appl Environ Microbiol, 1992 Apr, 58(4), 1128 - 33 Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca; Burchhardt G et al.; A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism . The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals . This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555) . Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production . Cells containing xylanase were harvested at the end of fermentation and added to a xylan solution at 60 degrees C, thereby releasing xylanase for saccharification . After cooling, the hydrolysate was fermented to ethanol with the same organism (30 degrees C), thereby replenishing the supply of xylanase for a subsequent saccharification . Recombinant E . coli metabolized only xylose, while recombinant K . oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose . Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis . By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process . The yield appeared to be limited by the digestibility of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity . In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers. Biochem J, 1992 Apr 1, 283 ( Pt 1), 69 - 73 Purification and characterization of a new endoglucanase from Clostridium thermocellum; Romaniec MP et al.; An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants . No carbohydrate is associated with the endoglucanase . A molecular mass of 76,000 Da was determined by SDS/PAGE . The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05 . The enzyme has a temperature optimum of 70 degrees C and retains approx . 50% of its activity after 48 h at 60 degrees C . Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity . The endoglucanase shows little ability to hydrolyse highly ordered cellulose . Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity . The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent. J Bacteriol, 1992 Apr, 174(8), 2449 - 53 Glucose catabolism by Spirochaeta thermophila RI 19.B1; Janssen PH et al.; Spirochaeta thermophila RI 19.B1 (DSM 6192) fermented glucose to lactate, acetate, CO2, and H2 with concomitant formation of cell material . The cell dry mass yield was 20.0 g/mol of glucose . From the fermentation balance data and knowledge of the fermentation pathway, a YATP of 9.22 g of dry mass per mol of ATP was calculated for pH-uncontrolled batch-culture growth on glucose in a mineral medium . Measurement of enzyme activities in glucose-grown cells revealed that glucose was taken up by a permease and then subjected to ATP-dependent phosphorylation by a hexokinase . Glucose-6-phosphate was further metabolized to pyruvate through the Embden-Meyerhof-Parnas pathway . The phosphoryl donor for phosphofructokinase activity was PPi rather than ATP . This was also found for the type strain of S . thermophila, Z-1203 (DSM 6578) . PPi was probably formed by pyrophosphoroclastic cleavage of ATP, with recovery of the resultant AMP by the activity of adenylate kinase . All other measured kinase activities utilized ATP as the phosphoryl donor . Pyruvate was further metabolized to acetyl coenzyme A with concomitant production of H2 and CO2 by pyruvate synthase . Lactate was also produced from pyruvate by a fructose-1,6-diphosphate-insensitive lactate dehydrogenase . Evidence was obtained for the transfer of reducing equivalents from the glycolytic pathway to hydrogenase to produce H2 . No formate dehydrogenase or significant ethanol-producing enzyme activities were detected. Eur J Biochem, 1992 Apr 1, 205(1), 369 - 73 Inclusion bodies of the thermophilic endoglucanase D from Clostridium thermocellum are made of native enzyme that resists 8 M urea; Chaffotte AF et al.; Endoglucanase D from Clostridium thermocellum was purified from inclusion bodies formed upon its overproduction in Escherichia coli, using 5 M urea as a solubilizing solution . We examined the effects of denaturing agents upon the stability of the pure soluble enzyme as a function of the temperature . At room temperature, guanidinium chloride induces an irreversible denaturation . By comparison, we observed no structural or functional effects at room temperature using high concentrations of urea as denaturing agent . The irreversible denaturation process observed with guanidinium chloride also occurs with urea but only at elevated temperature (greater than or equal to 60 degrees C); in 6 M urea, the activation energy of the denaturation reaction is decreased by a factor of only 1.8 . We interpret the high resistance of this protein to urea as reflecting a reduced flexibility of its structure at normal temperatures which should be correlated to the thermophilic origin of this protein. Eur J Biochem, 1992 Apr 1, 205(1), 249 - 56 Assignment of Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase I to an exo-alpha-1,4-glucosidase, and its striking similarity to bacillary oligo-1,6-glucosidases in N-terminal sequence and in structural parameters calculated from the amino acid composition; Suzuki Y et al.; alpha-Glucosidase I of Bacillus thermoamyloliquefaciens KP1071 (FERM P8477, facultative thermophile) was purified to homogeneity . The relative molecular mass was estimated to be 62,000 Da . From its catalytic properties, the enzyme has been assigned to an exo-alpha-1,4-glucosidase . The enzyme shares its antigenic groups in part with Bacillus stearothermophilus ATCC12016 (obligate thermophile) exo-alpha-1,4-glucosidase . These exo-alpha-1,4-glucosidases strikingly resemble oligo-1,6-glucosidases from B . thermoamyloliquefaciens KP1071 and from Bacillus cereus ATCC7064 in the molecular properties tested, including relative molecular mass, N-terminal sequence of 15 residues, amino acid composition and structural parameters calculated from amino acid composition . We have suggested that bacillary exo-alpha-1,4-glucosidases take the same folded conformation, i.e . an (alpha/beta)8-barrel super-secondary structure in its N-terminal domain, as bacillary oligo-1,6-glucosidases. Indian J Chest Dis Allied Sci, 1992 Apr-Jun, 34(2), 49 - 56 Farmer's lung disease in north-western India--a preliminary report; Gaur SN et al.; A study of farmer's lung (FL) disease was carried out in 197 subjects engaged in farming and having respiratory complaints of varying duration . It revealed that 13.2% of the subjects had precipitating antibodies against thermophilic actinomycetes, with Faenia rectivirgula (Micropolyspora faeni) alone accounting for 85% of the positive reactions . Precipitating antibodies against Thermoactinomyces vulgaris and T . thalpophilus were observed only in 1.5% and 0.5% of the subjects, respectively . Two subjects concomitantly demonstrated F . rectivirgula and T . vulgaris-specific serum precipitins . Sixty (30%) of the subjects related their respiratory symptoms to exposure to wheat straw/thresher's dust or other vegetable substrata in the working environment . Based upon a suggestive clinical history, roentgenography, pulmonary function studies and demonstration of serum precipitins against F . rectivirgula, FL was diagnosed in 4 subjects whose salient features are presented and discussed . To the best of our knowledge, this is the first authentic report on FL from India . A comprehensive epidemiological survey is indicated to determine the prevalence of FL in different geo-climatic regions of the country. Biochimie, 1992 Apr, 74(4), 353 - 6 Determination of tRNA(Phe) recognition nucleotides for phenylalanyl-tRNA synthetase from Thermus thermophilus; Moor N et al.; The tRNA(Phe) recognition nucleotides for phenylalanyl-tRNA synthetase from an extreme thermophile Thermus thermophilus were investigated . Using yeast tRNA(Phe) T7 transcripts with various point mutations it was shown that four recognition points (G34, A35, A36 from anticodon and A73 from acceptor stem) are important for aminoacylation at 37 degrees C . In the case of the 73rd discriminator base A----U, but not A----C, substitution suppresses aminoacylation . Position 20, which proved to be essential for all phenylalanyl-tRNA synthetases so far studied, is not involved in the recognition of tRNA(Phe) by the T thermophilus enzyme. Mol Cell Biol, 1992 Apr, 12(4), 1443 - 50 Drugs affecting microtubule dynamics increase alpha-tubulin mRNA accumulation via transcription in Tetrahymena thermophila; Stargell LA et al.; In cultured mammalian cells, an increase in the amount of tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in tubulin synthesis . This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of tubulin message with no change in transcriptional activity of tubulin genes . Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of tubulin . Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in alpha-tubulin synthesis and alpha-tubulin message in starved Tetrahymena thermophila . This increase is paralleled by an increase in transcription of alpha-tubulin sequences measured by run-on transcription, while the half-life of tubulin message measured by decay in the presence of actinomycin D does not change appreciably . Treatment of starved cells with taxol also produces an increase in alpha-tubulin synthesis via an increase in message abundance due to an increase in transcription of the alpha-tubulin gene . These results indicate that tubulin synthesis in T . thermophila is regulated very differently than in cultured mammalian cells. Biochim Biophys Acta, 1992 Mar 25, 1124(3), 249 - 52 Glycolipids from Thermotoga maritima, a hyperthermophilic microorganism belonging to Bacteria domain; Manca MC et al.; Two novel glycolipids with a very rare alpha(1-->4) diglucosyl structure have been isolated from the thermophilic bacterium Thermotoga maritima . The structures of these compounds, on the basis of chemical procedures and spectroscopic studies (FAB-MS and NMR), were shown to be: 1(3),2-dipalmitoyl-3(1)-{glucopyranosyl-(6-decanoyl)-alpha-D-(1-->4)- glucopyranosyl-alpha-D}-glycerol (Glycolipid 1) and 1(3),2-dipalmitoyl-3(1)-{glucopyranosyl-alpha-D-(1-->4)-glucopyranosyl- alpha-D}-glycerol (Glycolipid 2). Biochemistry, 1992 Mar 24, 31(11), 2977 - 82 NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu; Lowry DF et al.; The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods . Five proton resonances were found below 10.5 ppm . One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins . The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen . The amide protons from the homologous lysines in N-ras p21 {Redfield, A.G., & Papastavros, M.Z . (1990) Biochemistry 29, 3509-3514} and the catalytic domain of Escherichia coli elongation factor Tu {Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A . (1991) Biochemistry 30, 10872-10877} also resonate downfield in similar positions . We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins . We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C. Biochemistry, 1992 Mar 24, 31(11), 2970 - 7 Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR; Limmer S et al.; Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm) . Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide . However, three downfield resonances appear even in the nucleotide-free EF-Tu . The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein . In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region . Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra . NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out . The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP . Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded . The progress of the GTP hydrolysis could be monitored using this method . The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site. J Mol Biol, 1992 Mar 20, 224(2), 519 - 22 Crystallization of the seryl-tRNA synthetase-tRNA(Ser) complex from Thermus thermophilus; Yaremchuk AD et al.; The complex between seryl-tRNA synthetase and its cognate tRNA from the extreme thermophile Thermus thermophilus has been crystallized from ammonium sulphate solutions . Two different tetragonal crystal forms have been characterized, both diffracting to about 6 A using synchrotron radiation . One form grows as large bipyramids and has cell dimensions a = b = 127 A, c = 467 A, and the second form occurs as long, thin square prisms with cell dimensions a = b = 101 A, c = 471 A . Analysis of washed and dissolved crystals demonstrates the presence of both protein and tRNA. Nature, 1992 Mar 12, 356(6365), 148 - 9 Novel major archaebacterial group from marine plankton; Fuhrman JA et al.; Marine bacteria often dominate the plankton biomass and are responsible for much of the cycling of organic matter, but bacterial diversity is poorly understood because conventional identification methods (requiring culturing) miss about 99% of the organisms . Recent advances permit characterization of microbial communities by analysis of 16S ribosomal RNA gene sequences directly from biomass without the need to culture the organisms; such studies from surface ocean samples have found only eubacteria, not archaebacteria (or Archaea), which are profoundly different . Here we report 16S rRNA sequences obtained from Pacific Ocean bacterioplankton samples collected from depths of 100 m and 500 m . Among these we found sequences only distantly related to those of any organisms previously characterized by 16S rRNA sequences, with similarities to the nearest such relatives (extreme thermophiles) approximately the same as those between animals and plants . We suggest that these sequences are from a previously undescribed archaebacterial group that may have diverged from the ancestors of characterized organisms very early in evolution. J Biol Chem, 1992 Mar 5, 267(7), 4551 - 6 Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP; Hisabori T et al.; The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated . When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit . Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1 . Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM . When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed . The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP . Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP . However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase. J Biol Chem, 1992 Mar 5, 267(7), 4592 - 9 Isoleucyl-tRNA synthetase from the ciliated protozoan Tetrahymena thermophila . DNA sequence, gene regulation, and leucine zipper motifs; Csank C et al.; We have determined the nucleotide sequence of a protozoan aminoacyl-tRNA synthetase . The isoleucyl-tRNA synthetase (ileRS) gene {ilsA; formerly cupC, Martindale, D . W., Martindale, H . M., and Bruns, P . J . (1986) Nucleic Acids Res . 14, 1341-1354} from the ciliate Tetrahymena thermophila was sequenced and found to have eight introns, four transcription start sites, and a putative polypeptide of 1081 amino acids . A polypeptide 20 amino acids longer could be made if a transcribed in-frame ATG close to the start sites and with suboptimal sequence context is used . This gene was identified through hybridization and amino acid sequence similarity to the previously cloned and sequenced ileRS (cytoplasmic) gene from Saccharomyces cerevisiae {Englisch, U., Englisch, S., Markmeyer, P., Schischkoff, J., Sternbach, H., Kratzin, H., and Cramer, F . (1987) Biol . Chem . Hoppe-Seyler 368, 971-979; Martindale, D . W., Gu, Z . M., and Csank, C . (1989) Curr . Genet . 15, 99-106} with which it shares 47% of its amino acids . We also compared it to ileRS genes from E . coli and an archaebacterium . Two leucine zippers motifs were identified in the carboxyl-terminal domain of the polypeptide; one of these motifs is in the same area as the zinc finger motif found in the E . coli enzyme . The transcription pattern of the ilsA gene was monitored under various culture conditions and parallels changes in protein synthesis. J Protozool, 1992 Mar-Apr, 39(2), 343 - 5 Porphyrin rings and phospholipids: stimulators of cloning efficiency in certain species of Tetrahymena; Schousboe P et al.; Species of Tetrahymena, including T . vorax, T . thermophila, T . pyriformis, and T . pigmentosa, were tested for cloning efficiency in proteose peptone and in synthetic nutrient media to which were added hemin, protoporphyrin IX, chlorophyllin, or asolectin, an impure mixture of phospholipids . All species could be cloned with high efficiency in the crude media . In unsupplemented synthetic medium the cloning efficiencies were 0-10%, around 50%, around 50%, and 90-100% for T . thermophila, T . vorax, T . pyriformis, and T . pigmentosa, respectively . The first three were all stimulated to 90-100% by addition of the porphyrin or phospholipid compounds mentioned above . Uroporphyrin III and coproporphyrin I and III had no effect . We suggest that cells unable to form clones suffer from a lack of cellular energy . This situation may be alleviated by our additions, certain porphyrin rings may be built into cytochromes and phospholipids may be used as fuel . Thus, the synthetic media used so far for these ciliates have not been optimal. J Protozool, 1992 Mar-Apr, 39(2), 323 - 8 A genetic screen for vegetative gene expression in the micronucleus of Tetrahymena thermophila; Kaney AR et al.; The presence of a micronucleus with at least a small portion of the micronuclear genome appears to be indispensable for vegetative viability in the ciliate Tetrahymena thermophila . A genetic screen was devised to detect evidence of expression of essential genes in the vegetative micronucleus by identification of thermosensitive-lethal mutations expressed in the absence of nuclear reorganization . Although control experiments demonstrated the efficacy of the method for induction and recovery of thermosensitive lethal mutations in micronuclear genes, no expressed mutations were recovered in the absence of nuclear reorganization . This finding complements the existing lack of convincing biochemical evidence for gene expression in the vegetative micronucleus and suggests that the essential function may involve genomic DNA sequences for which thermosensitive mutant alleles are not recoverable, or perhaps a non-genomic component of the organelle. Appl Environ Microbiol, 1992 Mar, 58(3), 862 - 8 Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium; Schmidt JE et al.; The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied . The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method . Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+ . The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+ . The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation . These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules. J Appl Bacteriol, 1992 Mar, 72(3), 227 - 32 Purification and partial characterization of an intracellular aminopeptidase from Streptococcus salivarius subsp . thermophilus strain ACA-DC 114; Tsakalidou E et al.; An intracellular aminopeptidase from Streptococcus salivarius subsp . thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100 . The enzyme had a molecular weight of 89,000 . It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35 degrees C with Km = 1.80 mmol/l; above 55 degrees C the enzyme activity declined rapidly . The aminopeptidase was capable of degrading substrates by hydrolysis of the N-terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity . The enzyme was strongly inactivated by EDTA . Serine and sulphydryl group reagents had no effect on enzyme activity. Eur J Biochem, 1992 Mar 1, 204(2), 751 - 8 The archaebacterial hypusine-containing protein . Structural features suggest common ancestry with eukaryotic translation initiation factor 5A; Bartig D et al.; The amino acid hypusine is formed by post-translational modification of a lysine residue in eukaryotes and archaebacteria but up to now only the eukaryotic translation initiation factor eIF-5A has been known to contain this unique component . We isolated and purified a hypusine-containing protein from the thermophilic archaebacterium Sulfolobus acidocaldarius . The mainly cytosolic protein comprised about 0.03% of the post-ribosomal supernatant protein . No other hypusine-containing protein could be detected in S . acidocaldarius . The molar ratio of hypusine/hypusine-containing protein was 1:1 . SDS/PAGE showed a molecular mass of 16.8 kDa; a pI of 7.8 for the native protein resulted from IEF . The N-terminus was blocked . Four cyanogen bromide fragments were partially sequenced and used to derive two 17-base oligonucleotide probes . A 3-kb HindIII fragment of genomic DNA hybridizing with both probes was cloned . By sequencing of exonuclease III deletion clones an open reading frame of 405 nucleotides was found coding for a protein of 135 amino acids with a molecular mass of 15 kDa . It contained all cyanogen bromide sequences analysed . Sequence alignment revealed that seven of eight residues around Lys40 in the Sulfolobus hypusine-containing protein were identical to the nonapeptides centered by hypusine in the three eIF-5A proteins sequenced so far . The Edman procedure gave no phenylthiohydantoin derivative for this position . For a central region of 44 residues a sequence similarity of 54% between the archaebacterial and eukaryotic proteins was calculated; for the total sequence about 33% similarity resulted . In addition, there were a number of conservative changes . The unique lysine modification surrounded by a conserved sequence strongly suggests a common ancestry of archaebacterial hypusine-containing protein and eIF-5A . Together with similarities in molecular mass and intracellular localization, it may point to an analogous biochemical function. Eur J Biochem, 1992 Mar 1, 204(2), 575 - 81 Structural analysis of three prokaryotic 5S rRNA species and selected 5S rRNA--ribosomal-protein complexes by means of Pb(II)-induced hydrolysis; Ciesiolka J et al.; Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli . Based on the distribution of Pb(II)-induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed . They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region . The 'prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure . Hydrolysis of E . coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109 . It supports the concept that the major L18-induced conformational change involves the junction of helices A, B and D. Eur J Biochem, 1992 Mar 1, 204(2), 483 - 90 Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium . 2 . Denaturation and structural stability; Ragone R et al.; The thermal stability of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium, has been characterized thermodynamically by a Van't Hoff analysis . Conformational transitions induced by guanidine hydrochloride, as well as by temperature, have been linked together in a scheme involving six equilibria, which arise from both dissociation and unfolding . The mechanism by which the protein achieves thermal stabilization is quite unusual . It is driven by a conformational equilibrium between two forms of different stability . The stability of each form towards denaturation is characterized by a specific temperature dependence . The low-temperature form, indicated as 'form A', is stable over 12-89 degrees C . Its stability maximum is 36.8 kJ/mol at 50 degrees C . 'Form B', which is populated at higher temperature, spans the interval 28-146 degrees C . Its stability maximum is 71.6 kJ/mol at 87 degrees C . A possible explanation for the mechanism underlying this behaviour is discussed assuming that two major terms contribute to stability, i.e . hydrophobic interactions arising from burying of the accessible surface residues as well as conformational entropy . The thermal stabilization of the enzyme seems to depend on effects related to both an overall increase of flexibility and a concomitant decrease of the area buried upon folding . In this regard proteins from extreme thermophilic organisms appear to be a useful model to shed new light on the general problem of protein stability. Eur J Biochem, 1992 Mar 1, 204(2), 473 - 82 Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium . 1 . Conformational behavior of the oligomeric enzyme in solution; Facchiano F et al.; The effect of temperature on the molecular structure of propylamine transferase from Sulfolobus solfataricus has been investigated . Sulfolobus solfataricus is an extreme thermophilic archaebacterium with an optimum living condition at 90 degrees C . The enzyme is an oligomeric (trimer) protein of molecular mass 112 kDa . The frictional ratio for the native protein suggests an irregularly shaped compact globular structure . The protein matrix is well organized as suggested by far ultraviolet circular dichroism at 25 degrees C (18% alpha helix, 43% beta structure, 19% beta bends and 20% unordered: root mean square = 7) . Structural effects of temperature were investigated over 25-85 degrees C . The protein retains its quaternary structure in this temperature range . A highly reversible subtle conformational transition was detected by numerous structure-dependent techniques over 40-50 degrees C, with a midpoint centered at 45 degrees C . Functional data also support this view . In fact, two enzyme forms, characterized by different catalytic properties, are present in solution . The Arrhenius plot suggests the occurrence of two different activation-energy-dependent processes, one at a temperature higher and one at a temperature lower than 45 degrees C . The transition has been considered as a molecular switch between two protein populations at equilibrium with different functional and structural properties, temperature modulated . A physiological role for the molecular switch has also been postulated . The protein also shows some subtle and reversible spectroscopic changes around 75 degrees C . The molecular basis of the thermophilic nature of this enzyme seems to reside in its capability to dynamically couple catalytic and structural events to the thermal properties of the ambient medium. Eur J Biochem, 1992 Mar 1, 204(2), 465 - 72 Glutamyl-tRNA synthetase from Thermus thermophilus HB8 . Molecular cloning of the gltX gene and crystallization of the overproduced protein; Nureki O et al.; The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase . Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901) . Codon usage in the T . thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94% . In contrast, the amino acid sequence of T . thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved . The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter . The recombinant T . thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography . Single crystals of T . thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique . The crystals diffract X-rays beyond 0.35 nm . The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm. J Protozool, 1992 Mar-Apr, 39(2), 261 - 6 A temperature-sensitive mutation affecting synthesis of outer arm dyneins in Tetrahymena thermophila; Attwell GJ et al.; We have characterized a novel, temperature-sensitive mutation affecting motility in Tetrahymena thermophila . Mutants grew and divided normally at the restrictive temperature (38 degrees C), but became nonmotile . Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length . Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad 1 (outer arm deficient) . Motile mutants shifted to 38 degrees C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28 degrees C) the outer arm dyneins remain functional at 38 degrees C . Starved, deciliated mutants regenerated a full complement of functional cilia at 38 degrees C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad 1 mutation . Starved, nonmotile mutants regained motility when shifted back to 28 degrees C, but not when incubated with cycloheximide . We interpret these results to rule out the hypothesis that the oad 1 mutation affects the site on the microtubules to which the outer arm dyneins bind . Axonemes isolated from mutants grown for one generation at 38 degrees C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38 degrees C had a mean of 3.2 outer arm dyneins . Taken together, these results indicate that the oad 1 mutation affects the synthesis of outer arm dyneins in Tetrahymena. Vopr Virusol, 1992 Mar-Apr, 37(2), 94 - 5 {The use of DNA polymerase from Thermus thermophilus}; Kuz'min AN et al.; National DNA polymerase from Thermus thermophilus was used in polymerase chain reaction (PCR) . The synthetic oligonucleotides (primers) to the basic structural HIV genes GAG, ENV, the genome DNA of donor peripheral blood lymphocytes were used, and the controls included the plasmid DNA with cloned HIV genome and the genome DNA of peripheral blood lymphocytes from HIV-infected persons confirmed by ELISA and Western blot analysis . The PCR technique and evaluation of the obtained results are described . The expediency of using PCR for different contingents is discussed. Appl Microbiol Biotechnol, 1992 Mar, 36(6), 727 - 32 Efficient production of thermostable Thermus thermophilus xylose isomerase in Escherichia coli and Bacillus brevis; Dekker K et al.; The xylose (glucose) isomerase from the thermophile Thermus thermophilus seems to have potential for the development of new isomerization processes using high temperatures and slightly acidic pH . The isomerase has an optimum temperature at 95 degrees C, and is also very stable at high temperatures . The optimum pH is around 7.0, close to where by-product formation is minimal . Since Thermus produces only a little of this useful isomerase, the production of the cloned gene in Escherichia coli and Bacillus brevis were compared . Especially B . brevis was able to produce the isomerase efficiently, more than 1 g/l, in spite of the high G + C content (67%) of the Thermus gene, and the presence of codons not frequently used in E . coli or B . brevis. Biokhimiia, 1992 Mar, 57(3), 430 - 7 {Comparative characteristics of cyclodextrin glycosyltransferases from various groups of microorganisms}; Abelian VA et al.; Cyclodextrin glycosyltransferases (CGT-ase, 1.4-alpha-glucanotransferase, cyclizing, EC 2.4.1.19) produced by some thermophilic, alkalophilic and mesophilic bacterial strains, were isolated and characterized . It was shown that thermophilic and mesophilic CGT-ases represent a mixture of alpha-, beta- and gamma-cyclodextrins (CD), alpha-cyclodextrin being the predominant component . Alkalophilic enzymes produce only beta-CD and are able to produce CD not only from starch but also from maltose, melibiose, maltotriose, amylose and amylopectin . The optimal conditions for the catalytic activity of the enzymes were determined . It was found that calcium, magnesium and zinc ions have a beneficial effect on the specific activity of these enzymes . The amino acid composition of the enzymes was studied. Gene, 1992 Mar 1, 112(1), 3 - 12 Cloning and sequencing of genes encoding the TthHB8I restriction and modification enzymes: comparison with the isoschizomeric TaqI enzymes; Barany F et al.; Genes encoding the TthHB8I restriction and modification (R-M) system from Thermus thermophilus HB8 (recognition sequence T decreases CGA) were cloned in Escherichia coli . The genes have the same transcriptional orientation, with the last 13 codons of the methyltransferase (MTase) overlapping the first 13 codons of the endonuclease (ENase) . Nucleotide sequence analysis of the TthHB8I ENase revealed a single chain of 263 amino acid (aa) residues that share a 77% identity with the corrected isoschizomeric TaqI ENase . Likewise, the Tth MTase (428 aa) shares a 79% identity with the corrected sequence of the TaqI MTase . This high degree of aa conservation suggests a common origin between the Taq and Tth R-M systems . However, codon usage and G+C content for the R-M genes differed markedly from that of other cloned Thermus genes . This suggests that these R-M genes were only recently introduced into the genus Thermus. J Bacteriol, 1992 Feb, 174(3), 873 - 82 Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression; Moracci M et al.; The lacS gene from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus encodes an enzyme with beta-galactosidase activity that, like other enzymes from this organism, is exceptionally thermophilic (optimal activity above 90 degrees C), thermostable, and resistant to common protein denaturants and proteases . Expression of the gene in mesophilic hosts is needed to uncover the molecular nature of these features . We have obtained expression of beta-galactosidase in Saccharomyces cerevisiae under the control of the galactose-inducible upstream activating sequence of the yeast genes GAL1 and GAL10 . The expressed enzyme is identical in molecular mass, thermostability, and thermophilicity to the native enzyme, showing that these features are intrinsic to the primary structure of the enzyme . We also present a new promoter for the expression of thermostable proteins in S . cerevisiae . This promoter contains a sequence isolated from the nematode Caenorhabditis elegans that works as a strong, heat-inducible upstream activating sequence in S . cerevisiae . Transcription of the lacS gene under the control of this sequence is rapidly and efficiently induced by heat shock . The availability of a plate assay for monitoring beta-galactosidase activity in S . cerevisiae may allow screening for mutants affecting the efficiency and activity of the enzyme. Am J Vet Res, 1992 Feb, 53(2), 194 - 7 Adhesion of bacteria to the cecal mucosal surface of conventional and germ-free chickens infected with Eimeria tenella; Baba E et al.; When Salmonella typhimurium and Clostridium perfringens were tested in conventional chickens, larger numbers of S typhimurium and C perfringens adhered to Eimeria tenella-infected ceca than to uninfected ceca . In germ-free chickens, S typhimurium and C perfringens adhered to the E tenella-infected cecal mucosa more than to the uninfected cecal mucosa, but fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the E tenella-infected ceca than to the uninfected ceca . Many bacteria adhered to the lesions caused by E tenella as observed by scanning electron microscopy . On the basis of our findings, we suggest that infection with E tenella upsets the balance of competitive adherence of bacteria, allowing more colonization of S typhimurium and C perfringens. J Dairy Res, 1992 Feb, 59(1), 71 - 9 Evidence and characterization of temperate bacteriophage in Streptococcus salivarius subsp . thermophilus St18; Carminati D et al.; Lysogenic strains of Streptococcus salivarius subsp . thermophilus were studied using induction with mitomycin C (MC) . The induction and presence of temperate phage were investigated carrying out tests on sensitive strains, electron microscopy and phage DNA analysis . Forty-five Str . salivarius subsp . thermophilus strains were subjected to induction with MC and growth of the various cultures was evaluated . Only one strain of those tested showed lysis after adding MC, thus indicating a possible lysogenic state, 0.5 micrograms MC/ml being the optimal dose . Two phi 18 phage-sensitive strains out of 45 were isolated in which this phage behaved as virulent, causing lysis of the culture in broth, but no lysis plaques on agar medium were detected . The St18 strain was cured by u.v . irradiation but no mutants sensitive to the phi 18 phage were found among the clones non-inducible by MC . The presence of phages having a hexagonal isometric head and a long non-contractile tail in the lysate obtained after inducing the St18 strain was confirmed by examination under the electron microscope . Study of the phage DNA showed a genome size of 40.9 +/- 0.5 kb without cohesive end fragments . In addition, the restriction map of the phage genome was constructed . This study has demonstrated lysogeny in Str . salivarius subsp . thermophilus and also that several phage infections of Str . salivarius subsp . thermophilus starters may have an 'endogenous' origin. J Dairy Res, 1992 Feb, 59(1), 65 - 9 Detection of propionic acid bacteria in cheese; Drinan FD et al.; Mesophilic lactic starters and thermophilic lactobacilli but not Streptococcus salivarius subsp . thermophilus grew on the sodium lactate agar (SLA) used for estimating the numbers of propionic acid bacteria (PAB) in cheese . The addition of cloxacillin (4 micrograms/ml) to SLA inhibited the starter bacteria but had no effect on the PAB . It was possible to count low numbers of PAB in the presence of high numbers of starter bacteria . A correlation coefficient of 0.9 was obtained between the level of propionic acid and the counts of PAB in cheese (n = 40) . A disadvantage of the medium is that other bacteria found in cheese (mesophilic lactobacilli, enterococci, Clostridium tyrobutyricum) also grow on it; however, these bacteria are easily distinguishable from PAB on the basis of size, colour and absence of catalase. Wei Sheng Wu Xue Bao, 1992 Feb, 32(1), 23 - 9 {Purification and characterization of alpha-glucosidase from an extreme thermophile, Thermus thermophilus HB 8}; Yang S et al.; A highly thermostable alpha-glucosidase (E C.3.2.1.20) from an extreme thermophile, Thermus thermophilus HB 8, was purified to homogeneous by ammonium sulfate fractionation, DEAE-cellulose chromatography and preparative slab gel electrophoresis . The enzyme was purified 17 fold with 21% recovery of activity . The enzyme had a molecular weight of 67000 by SDS-PAGE . The isoelectric point was pH4.5 by IEF on PAG . The enzyme hydrolized p-nitrophenyl-alpha-glucoside (PN-PG), sucrose and maltose, but not cellobiose, melibiose and soluble starch . The km value for PNPG was 0.4mmol/L, the Vmax was 0.29 mumol.min-1.mg-1 . The enzyme exhibited high thermostability . After incubation at 90 degrees C for 10 h in 50 mmol/L acetate buffer pH 5.8, the enzyme retained 90% of its original activity . The half-live (t1/2) at 95 degrees C was 108 min . The enzyme was activated by Mg2+, Mn2+, Ca2+, Ba2+ and strongly inhibited by Hg2+, Cu2+ . Modification of the enzyme by EDC or DEPC led to complete loss of activity, which suggests that carboxyl group(s) and histidine residue(s) are essential for activity of alpha-glucosidase. J Gen Microbiol, 1992 Feb, 138 ( Pt 2), 383 - 93 The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli; Sanangelantoni AM et al.; The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb HindIII DNA fragment . Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50,088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus) . The T . maritima glnA gene was expressed in E . coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051 . The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation . The molecular mass of the Thermotoga GS (590,000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type . Comparison of the amino acid sequence of T . maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability. Vet Microbiol, 1992 Feb, 30(2-3), 257 - 66 Profiles of toxin production by thermophilic Campylobacter of animal origin; McFarland BA et al.; Seventy-five strains of Campylobacter jejuni and C . coli, which were isolated from a variety of animal species, primarily poultry, were examined for production of toxin . Polymyxin extracts were tested in in vitro assays using CHO-KI, FCL (foetal calf lung), Vero, HeLa and CEF (chicken embryo fibroblast) cells . The toxic effects observed were cell rounding and death . Extracts from almost all C . jejuni and C . coli strains were toxic to both CHO-KI and FCL cells and 69.0% of C . jejuni isolates and 75% of C . coli isolates were also toxic to CEF cells . 50.7% of C . jejuni extracts were toxic to Vero cells and 46.5% toxic to HeLa cells . None of the C . coli isolates were toxic to either of these cell lines . None of the strains tested produced cytotonic enterotoxin . No differences in toxigenicity patterns were evident between Campylobacter isolated from different sources. Biochem Biophys Res Commun, 1992 Jan 15, 182(1), 425 - 31 Ribulose-1,5-bisphosphate carboxylase of thermophilic hydrogen-oxidizing microorganism Bacillus schlegelii; Mikulik K et al.; Ribulose-1,5-bisphosphate carboxylase was isolated from thermophilic hydrogen-oxidizing Bacillus schlegelii . Molecular mass of the native enzyme is 560,000 and optimal reaction temperature is 70 degrees C . Km value for ribulose 1,5-bisphosphate is 0.27 mM . The carboxylase activity of the enzyme is dependent on Mg2+ with the optimum at 10 mM . The enzyme is an oligomer of L8S8 type with Mr of large subunits and small subunits of 56,000 and 14,000, respectively . Negatively stained enzyme has regular polygonal shape in top view, 12 nm in diameter, with central electron dense patch. J Biol Chem, 1992 Jan 15, 267(2), 1375 - 81 Functional reconstitution of membrane proteins in monolayer liposomes from bipolar lipids of Sulfolobus acidocaldarius; Elferink MG et al.; Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of unusual bipolar lipids . They consist of macrocyclic tetraethers with two polar heads linked by two hydrophobic C40 phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membrane . Fractionation of a total lipid-extract from S . acidocaldarius yielded a lipid fraction which forms closed and stable unilamellar liposomes in aqueous media . Beef heart cytochrome c-oxidase could be functionally reconstituted in these liposomes . In the presence of reduced cytochrome c, a protonmotive force (delta p) across the liposomal membrane was generated of up to -92 mV . Upon fusion of these proteoliposomes with membrane vesicles of Lactococcus lactis, the delta p generated by cytochrome c-oxidase activity was capable to drive uphill transport of leucine . Electron microscopic analysis indicated that the tetraether lipids form a single monolayer liposome . The results demonstrate that tetraether lipids of archaebacteria can form a suitable matrix for the function of exogenous membrane proteins originating from a regular lipid bilayer. J Bacteriol, 1992 Jan, 174(1), 269 - 78 Evolutionary relationships among sulfur- and iron-oxidizing eubacteria; Lane DJ et al.; Some 37 reverse transcriptase, partial 16S rRNA sequences from sulfur- and/or iron-oxidizing eubacteria, including sequences from species of the genera Thiobacillus, Thiothrix, Thiomicrospira, Acidophilium, "Leptospirillum," Thiovulum, and Chlorobium, have been determined . In addition, 16S sequences from a number of unnamed sulfur- and/or iron-oxidizing bacteria from hydrothermal vent sites, from invertebrate-bacterial endosymbioses, and from various mineral recovery operations also have been determined . The majority of sequences place their bacterial donors in one or another of the subdivisions of the Proteobacteria . However, three unnamed facultatively thermophilic iron-oxidizing isolates, Alv, BC, and TH3, are affiliated with the gram-positive division . One H2S-oxidizer, from the genus Thiovulum, is affiliated with Campylobacter, Wolinella, and other genera in what appears to be a new subdivision of the Proteobacteria . Three "Leptospirillum"-helical vibrioid isolates, BU-1, LfLa, and Z-2, exhibit no clear phylum level affiliation at all, other than their strong relationship to each other . A picture is emerging of an evolutionary widespread capacity for sulfur and/or iron oxidation among the eubacteria. Biochim Biophys Acta, 1992 Jan 6, 1129(2), 207 - 14 Electron image analysis of ribosomal subunits from Thermus aquaticus; Harauz G et al.; Electron micrographs of ribosomal subunits from the thermophilic bacterium Thermus aquaticus were analysed using multivariate statistical analysis and characteristic views constructed to reproducible spatial resolutions ranging from 1.9 to 3.6 nm . These views were comparable to morphological classes of Escherichia coli ribosomal subunits, albeit with differences in fine features also found in archaebacterial ribosomes. Crit Rev Biotechnol, 1992, 12(1-2), 45 - 63 Biochemistry and genetics of actinomycete cellulases; Wilson DB; The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species . Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca . Fractionation of M . bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized . Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases . The structural genes of five M . bispora cellulases have been cloned and one was sequenced . Fractionation of T . fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized . One of the T . fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T . fusca endocellulases and with Trichoderma reesei CBHI but not with T . reesei CBHII . Each T . fusca cellulase contains distinct catalytic and cellulose binding domains . The structural genes of four of the T . fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T . curvata" . The T . fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans . Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T . fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families . There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated . This work has shown that the different T . fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source. Genetics, 1992 Jan, 130(1), 97 - 104 Multiple effects of mutation on expression of alternative cell surface protein genes in Tetrahymena thermophila; Smith DL et al.; Genes at the SerH locus of the ciliated protist Tetrahymena thermophila specify the major (H) surface protein on cells grown at 20-36 degrees . Alternative proteins L, T, S and I are expressed under different conditions of temperature and culture media . Mutants unable to express SerH genes were examined for expression of these proteins, also called immobilization or i-antigens, at both H and non-H conditions . In all instances, one or more i-antigens were expressed in the absence of H, and, in most instances, expression of i-antigens under non-H conditions was also affected . Examples of the latter include both the continued expression of H-replacement antigens and the inability to express certain other i-antigens . Such multiple effects were observed in mutants with trans-acting (rseA, rseB, rseC, RseD) and cis-acting (H1-1 and H1-2) mutations, but not in mutants in which SerH is affected developmentally (B2092, B2101, B2103, B2107) . These interactions suggest that the wild-type genes identified by mutation exert both positive and negative effects in the regulation of i-antigen gene expression. Res Microbiol, 1992 Jan, 143(1), 37 - 46 Detection of intraspecific DNA polymorphism in Streptococcus salivarius subsp . thermophilus by a homologous rDNA probe; Pebay M et al.; Three ribosomal probes from Streptococcus salivarius subsp . thermophilus were cloned . Sequence data demonstrate that their juxtaposition corresponds to an entire operon . They were used in order to study ribosomal operon number and organization . rRNA genes were shown to be clustered in the order 5'-16S-23S-5S-3' and the number of rrn loci to vary within the subspecies . The smallest of the 3 probes was used for strain characterization . Substantial variability in hybridization patterns was observed among strains, resulting not only from, restriction fragment length polymorphism (RFLP) but also from the variability of ribosomal operon number. Appl Biochem Biotechnol, 1992 Spring, 34-35, 247 - 59 Purification and characterization of a xylanase from the thermophilic ascomycete Thelavia terrestris 255B; Gilbert M et al.; Thielavia terrestris 255B, a thermophilic ascomycete, produced two major forms of xylanase with pIs of 4.6 (xylanase I) and 6.1 (xylanase II) . The latter enzyme could be purified to greater than 99% homogeneity using anion-exchange chromatography and gel filtration . Xylanase II had a mol wt of 25.7 kDa (SDS-PAGE) and a pH and a temperature optimum of 3.6-4.0 and 60-65 degrees C, respectively . The ratio of the enzyme's activity against xylan and carboxymethylcellulose was 500-1000 to 1, indicating a possible application of this enzyme in biobleaching processes . The amino acid sequence of this protein is being determined, and initial data suggest that the enzyme belongs to a group of low-mol wt xylanases that have been isolated from both bacteria and fungi. Adv Biochem Eng Biotechnol, 1992, 45, 57 - 98 The enzymes from extreme thermophiles: bacterial sources, thermostabilities and industrial relevance; Coolbear T et al.; This review on enzymes from extreme thermophiles (optimum growth temperature greater than 65 degrees C) concentrates on their characteristics, especially thermostabilities, and their commercial applicability . The enzymes are considered in general terms first, with comments on denaturation, stabilization and industrial processes . Discussion of the enzymes subsequently proceeds in order of their E.C . classification: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases . The ramifications of cloned enzymes from extreme thermophiles are also discussed. Pneumologie, 1992 Jan, 46(1), 32 - 5 {Exogenous allergic alveolitis caused by mouldy hazel nut leaves}; Erkan F et al.; A 62-year old farmer woman from the northeastern, very rainy part of Turkey has been collecting large amounts of green and brown involucral hazel-nut leaves for subsequent use as fuel . For the last 20 years she had been complaining of cough, respiratory distress and intermittent fever . In the course of years of continual antigen exposure she developed the clinical and x-ray signs of fibrosis of the lung . Bronchoalveolar lavage produced the typical cell pattern of chronic exogenous allergic alveolitis with predominant CD8 cells . Serum analysis yielded high titres of IgG antibodies against mould fungi partly obtained from hazel-nut husk cultures, as well as thermophilic actinomycetes. J Forensic Sci, 1992 Jan, 37(1), 6 - 20 A polymerase chain reaction (PCR) method for sex and species determination with novel controls for deoxyribonucleic acid (DNA) template length; Gaensslen RE et al.; Human X and Y chromosome alpha-satellite sequences lying within higher order repeats were amplified by the polymerase chain reaction (PCR) in genomic deoxyribonucleic acid (DNA) isolated from blood, bone, and several other tissues and specimens of potential forensic science interest . X and Y sequences could be coamplified under some of the PCR conditions employed . Monomorphic sequences in the 3'-apolipoprotein B gene (designated "H") and in an alpha-satellite higher order repeat on Chromosome 17 (p17H8, D17Z1) were likewise amplified in the specimens . X and Y sequence amplification can provide information about the sex of origin . Amplification of the X, H, and D17Z1 sequences was found to be primate-specific among the common animals tested and can thus provide species of origin information about a specimen . The authors suggest that amplification of X and D17Z1 or H sequences might provide "relaxed" and "stringent" controls for appropriate PCR amplification tests on forensic science specimens . Testing was carried out using PCR protocols that employed Thermophilus aquaticus (Taq) and Thermus flavis (Replinase) thermostable DNA polymerases. Appl Environ Microbiol, 1992 Jan, 58(1), 93 - 8 Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8; Miyazaki K et al.; The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced . This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site . Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively . The G + C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3% . On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A comparison of the amino acid sequence of the T . thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E . coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T . thermophilus enzyme . T . thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme. Appl Environ Microbiol, 1992 Jan, 58(1), 371 - 5 Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520; Tsujibo H et al.; Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520 . The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR . The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively . The pIs were 4.2 (STX-I) and 8.0 (STX-II) . The optimum pH levels for the activity of STX-I and STX-II were pH 7.0 . The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C . The enzymes were completely inhibited by N-bromosuccinimide . The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases . STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology). Biol Chem Hoppe Seyler, 1992 Jan, 373(1), 5 - 11 Characterization of DNA-directed RNA polymerases in isolated macronuclei of the ciliated protozoan Tetrahymena thermophila . Effects of purified ornithine decarboxylase and amine compounds; Eichler W et al.; The possible regulatory interactions of purified ornithine decarboxylase with DNA-directed RNA polymerases in isolated macronuclei from the ciliated protozoan Tetrahymena thermophila were studied . It has been found that highly purified ODC (specific activity 10.2 mumols CO2 x h-1 x mg-1), even at activities of 37,500 nmol CO2 x h-1 per ml failed to alter RNA polymerase activity in the in vitro transcription assay in the presence or absence of the substrate L-ornithine at 20mM . The naturally occurring di- and polyamines putrescine, spermidine, and spermine stimulated in-vitro-transcription in isolated macronuclei more at optimal Mg2+/Mn(2+)-concentrations than at suboptimal concentrations, suggesting that polyamines act via a mechanism which is distinct from that of the inorganic cations . Of the monovalent amine compounds tested, (NH4)+ at high concentrations between 40 and 50mM slightly stimulated activity whereas the onset of stimulation by the organic amine compounds, piperidine and cyclohexylamine, was inversely related to the hydrophobicity of each particular compound . In the series of divalent amines, the correct distance between the N-atoms appeared to be very important since ethylenediamine and piperazine did not stimulate significantly but did inhibit at concentrations above 5 mM . 1,3-Diaminopropane stimulated slightly but inhibited above 10 mM, whereas the 1,4-diamino compounds putrescine and 1,4-diaminocyclohexane (DAC) were equally potent stimulators with the more hydrophobic one, DAC, reaching the maximum at lower concentrations than putrescine . For the trivalent amines, the influence of correct spacing seems not to be as important: N-(2-aminoethyl)piperazine stimulated very similar to spermidine.(ABSTRACT TRUNCATED AT 250 WORDS) Zentralbl Mikrobiol, 1992, 147(1-2), 41 - 4 Comparison of two media for the isolation of thermophilic Campylobacters from waste waters of different quality; Jacob J et al.; Counts of thermophilic campylobacters from 31 different waste water samples were parallel estimated with two different cultivation media . The resulting increased isolation sale obtained with an modified Charcoal-Cefoperazone-Deoxycholat-Medium (MCCD-Medium), was statistically significant . An 15.8 fold increased isolation rate, compared with the standard medium, could be estimated with the help of the geometric mean. Mol Biol (Mosk), 1992 Jan-Feb, 26(1), 173 - 8 {Nucleotide sequence of the pNB2 plasmid from thermophilic bacteria Clostridium thermosaccharolyticum}; Belogurova NG et al.; The complete nucleotide sequence of pNB2, a 1.9-kilobases cryptic plasmid from thermophilic Clostridium thermosaccharolyticum has been determined . The plasmid consists of 1882 base pairs and has a G+C composition of 27.2% . The sequence contains three open reading frames capable of coding for polypeptides two of which were identified in maxicell Escherichia coli extracts . Our future studies are directed toward a construction of pNB2-derivatives as vectors for Clostridia. Dev Genet, 1992, 13(3), 216 - 22 The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila; Kaczanowska J et al.; During cytokinesis, furrowing creates new boundaries for daughter cells . Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis . When maintained under weak restrictive conditions (35 degrees C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains) . In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain . The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle . These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes . They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity. Dev Genet, 1992, 13(2), 174 - 9 Locus-dependent profiles of the rescue of nonexcitable behavioral mutants during conjugation in Tetrahymena thermophila; Takahashi M; The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes . When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about 1 h after pair formation . The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2 h, they were not distinguishable from the wild-type homotypic pairs . On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs . When tnrA was mated with tnrB, more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect . Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca2+ channels. Dev Genet, 1992, 13(2), 167 - 73 Genetic characterization of the secretory mutant MS-1 of Tetrahymena thermophila: vacuolarization and block in secretion of lysosomal hydrolases are caused by a single gene mutation; Hunseler P et al.; The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS-1 of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed . Although blocked constitutively in secretion, MS-1 contains active lysosomal hydrolases in amounts equivalent to the wild type . The 3:1 segregation in F-2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec . The sec allele of MS-1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength . Deletion mapping by crossing MS-1 with nullisomic strains, all secreting lysosomal hydrolases at wild-type rates, was performed . The sec phenotype was expressed in monosomic-4 progeny only, indicating that the sec allele is located on chromosome 4 of T . thermophila. Dev Genet, 1992, 13(2), 160 - 6 Genetic characterization of Tetrahymena thermophila mutants unable to secrete capsules; Gutierrez JC et al.; Under appropriate conditions, Alcian Blue-induced exocytosis of Tetrahymena mucocysts leads to formation of a capsule that surrounds the cell . This phenomenon is an example of regulated secretion, a mechanism of fundamental significance in eukaryotic cells . In order to dissect genetically the mechanism of mucocyst biogenesis and regulated exocytosis, mutants unable to form capsules (Caps-) were isolated . In this paper we report a genetic characterization of Caps- mutants in this collection . The mutations in mutants SB255 and SB281 behave as single recessive Mendelian mutations . The mutation in SB251 is restricted to the macronucleus, and could not be further characterized by the genetic methods we used . Complementation tests suggest the existence of at least 2 genes, named exoA and exoB; additional mutant loci are likely to be included in the mutant collection . Deletion mapping using nullisomic strains showed that exoA and exoB are located on the left arm of chromosome 4 . The exo-3 mutation, which behaves as recessive and complements with exoA1 in SB255 and exoB2 in SB281, maps to chromosome 3 . These Caps- mutants may be useful for the elucidation of the developmental pathway of mucocyst biogenesis and the control of regulated secretion in eukaryotic cells. Dev Genet, 1992, 13(2), 151 - 9 Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody; Turkewitz AP et al.; Dense-core granules represent an adaptation of specialized secretory cells to facilitate stimulus-regulated release of stored proteins . Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates . In Tetrahymena thermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis . We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors . We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells . The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes . In addition, the assay represents a powerful technique for diagnosis of new mutants. Dev Genet, 1992, 13(2), 133 - 42 Inheritance of the group I rDNA intron in Tetrahymena pigmentosa; Nielsen H et al.; We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution . We have now approached the question of intron mobility experimentally by crossing intron+ and intron- strains looking for a strong polarity in the inheritance of the intron (intron homing) . Based on the genetic analysis we find that the intron in T . pigmentosa is inherited as a neutral character and that intron+ and intron- alleles segregate in a Mendelian fashion with no sign of intron homing . In an analysis of vegetatively growing cells containing intron+ and intron- rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing . During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA . One possible explanation is that T . pigmentosa has two rdn loci in contrast to the single locus found in T . thermophila . Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis. Dev Genet, 1992, 13(2), 126 - 32 Rate of phenotypic assortment in Tetrahymena thermophila; Doerder FP et al.; During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both . Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus . The primary tools to study assortment are Rf, the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes . Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number . Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development . Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured . Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis . Here we examine the experimental determination of Rf . First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf . Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele . Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with approximately 45 assorting units, as has been asserted. Dev Genet, 1992, 13(2), 103 - 10 DNA elimination and its relation to quantities in the macronucleus of Tetrahymena; Bodenbender J et al.; The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size . They belong to about 270 species each one occurring at an average number of 45 copies . Macronuclei divide unequally and nothing is known of segregation control . This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level . We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains . This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA . The following results were found: Elimination of DNA occurs at almost every division . The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content . In T . thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state . It varies with species, amounting to as much as 8% in T . pigmentosa . During conditions which increase the macronuclear DNA content, very little DNA is eliminated . On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease . DNA to be eliminated at division is synthesized at the same time as bulk DNA . We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.(ABSTRACT TRUNCATED AT 250 WORDS) Antisense Res Dev, 1992 Fall, 2(3), 251 - 6 Cellular uptake and degradation of phosphorothioate oligonucleotides by marine and freshwater ciliates; Thomas LL et al.; To date, no antisense studies have been reported with either ciliated protozoa or marine organisms . This study examines the feasibility of using antisense oligonucleotides to alter gene expression in marine and freshwater ciliates . Radiolabeled, phosphorothioate-modified oligonucleotides were used to investigate whether ciliates take up and degrade oligonucleotides present in the culture medium . With all three ciliates examined, Euplotes crassus, Tetrahymena thermophila, and Oxytricha nova, the oligonucleotide in the culture medium was degraded very rapidly (> 90% in 8 h) . The degradation probably occurred when the cells filtered the culture medium through the oral apparatus . Our results indicate that experiments involving the uptake of oligonucleotides from the culture medium are likely to be successful with ciliated protozoa . In studies designed to examine the uptake of fluorescent oligonucleotide by Euplotes cells, we found that dead or dying cells have a high affinity for fluorescein-labeled oligonucleotide . These results illustrate the importance of careful studies when only certain cell populations are found to have a high affinity for oligonucleotide . Although the seawater culture medium used to grow Euplotes causes some oligonucleotide to precipitate, this problem is not serious at concentrations > or = 1 microM oligonucleotide . Thus, it should be possible to use antisense oligonucleotides to manipulate gene expression in other marine organisms. Cytobios, 1992, 71(284), 37 - 50 Protein phosphorylation and the regulation of basal body microtubule organizing centres in Tetrahymena; Huelsman DA et al.; Previous work suggests that changes in the phosphorylation state of some centrosomal proteins regulate centrosomal activity . The hypothesis that changes in the phosphorylation state of one or more basal body microtubule organizing centre (MTOC) components regulate its ability to nucleate cilia assembly in Tetrahymena thermophila was tested . The MPM-2 antibody, which recognizes phosphorylated epitopes in MTOCs in a variety of organisms, was used to probe immunoblots of cytoskeletal frameworks prepared from starved Tetrahymena, from starved deciliated Tetrahymena, and from a starved deciliated mutant Tetrahymena which failed to initiate ciliogenesis following deciliation . The MPM-2 antibody recognized an identical array of proteins in all blots . These results suggest that, unlike centrosomes, basal body MTOC activity is not regulated by changes in the phosphorylation state of component proteins. Biochem Soc Symp, 1992, 58, 149 - 69 Enzymes from thermophilic archaebacteria: current and future applications in biotechnology; Cowan DA; The one guaranteed property of enzymes isolated from extremely thermophilic micro-organisms is their thermostability . Most significantly, almost any such enzyme will be more thermostable than the functionally similar enzyme from a lower temperature source . Thermostability is not an isolated property: resistance to heat denaturation imparts stability to a number of other denaturing influences (detergents, organic solvents, etc) . These characteristics of hyperthermophilic enzymes are the most likely basis for the development of new biotechnological applications . A limited number of hyperthermophilic enzymes have found application in specialist biotechnological applications; others have visible potential in growing areas of biotechnology . Existing and potential applications are discussed using DNA manipulation enzymes, dehydrogenases, and esterases as examples. Annu Rev Microbiol, 1992, 46, 165 - 91 Molecular biology of methanogens; Reeve JN; Methanogens are a very diverse group of the Archaea (Archaebacteria) . Their genomic DNAs range from 26 to 68 mol% G+C; they exhibit all known prokaryotic morphologies and inhabit anaerobic environments as varied as the human gut and deep-sea volcanic vents . They are, nevertheless, unified by their ability to gain energy by reducing CO, CO2, formate, methanol, methylamines, or acetate to methane . Methanogen genes are reviewed and analyzed in terms of their organization, structure, and expression and are compared with their bacterial (eubacterial) and eukaryal (eukaryotic) counterparts . Many methanogens are thermophiles, and some are hyperthermophiles . The influence of these extreme environments on their macromolecular structures is also addressed . Methanogens are oxygen-sensitive, fastidious anaerobes, and therefore their experimental manipulation in research laboratories has been very limited . The majority of the information currently available describing their molecular biology has been gained by gene cloning . With improvements in anaerobic handling procedures, this is beginning to change, and several experimentally tractable regulated systems of gene expression in methanogens are discussed . Anaerobic biodegradation terminating in methane biogenesis is an established, economically very important biotechnology used world-wide both to reduce waste and to generate fuel-grade biogas . The substantial progress made over the past decade, reviewed here, in understanding the molecular biology of methanogens should now provide a data base for considering genetic approaches to improving this process. Arch Microbiol, 1992, 158(4), 282 - 6 Transformation of synthetic pyrethroid insecticides by a thermophilic Bacillus sp; Maloney SE et al.; Employing a mineral salts medium containing Tween 80 as the primary carbon source, a strain of Bacillus stearothermophilus was isolated which was able to hydrolyse selected second and third-generation pyrethroids to non-insecticidal products . Of a range of pyrethroid insecticides the trans-isomer of permethrin was the most readily transformed by this microbial isolate, whilst flumethrin was the least . 3-Phenoxybenzoic acid and the respective halovinyl or haloacid moieties were detected as the major hydrolytic products of the pyrethroids . It is believed that 3-phenoxybenzoic acid was formed from 3-phenoxybenzyl alcohol which was not however detected as an intermediate in these systems . 3-Phenoxybenzoic acid was further transformed to 4-hydroxy-3-phenoxybenzoic acid . A potential metabolic pathway has been described. Dev Genet, 1992, 13(1), 87 - 93 Stochastic developmental variation in the ratio of allelic rDNAs among newly differentiated, heterozygous macronuclei of Tetrahymena thermophila; Orias E et al.; Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei . The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell . The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle . Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification . Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR . We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC . We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs . Either rDNA type can be in the majority . Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself . The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4-8 at the most, in a MAC anlage . In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA . Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification.(ABSTRACT TRUNCATED AT 250 WORDS) Dev Genet, 1992, 13(1), 58 - 65 Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: early expression of the zygotic genotype; Kaczanowski A; A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest . Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei . In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1-2 hr later . In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them . After addition of 10 mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants . The calcium induced separation of the mra conjugants occurred later than the separation of control pairs . During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macronuclear DNA . Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. Dev Genet, 1992, 13(1), 34 - 40 Mapping the mating type locus of Tetrahymena thermophila: meiotic linkage of mat to the ribosomal RNA gene; Bleyman LK et al.; Tetrahymena thermophila has a multiple mating type system . While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types . The set of allowed mating types is specified by the mat locus . The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus) . In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping . We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L . This represents another (rare) case of meiotic linkage in Tetrahymena. Dev Genet, 1992, 13(1), 26 - 33 Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: starved versus nonstarved; Cheng LJ et al.; To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation . In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent ("initiated") cells were compared with those from non-starved, mating-incompetent ("non-initiated") cells, by gel electrophoresis and lectin blotting . In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains . In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated . In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings) . In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides . Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glycoconjugate as having a potential role in control of pair formation during mating. Dev Genet, 1992, 13(2), 143 - 50 In vitro transcription in isolated nucleoli of Tetrahymena pyriformis; Matsuura T et al.; An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template . Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl . Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila . The transcription product was examined for the presence of transcripts from T . pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T . pyriformis rDNA clone . A sequence difference between T . pyriformis and T . thermophila in the 5' region of their 35S pre-rRNAs permitted exclusive detection of T . pyriformis transcripts . The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone . This system may offer unique advantages for future studies of transcriptional control during development and differentiation. Appl Microbiol Biotechnol, 1992 Jan, 36(4), 503 - 6 In vitro mutagenesis of a xylanase from the extreme thermophile Caldocellum saccharolyticum; Luthi E et al.; Six mutant xylanases were obtained by in vitro mutagenesis of a xylanase gene from the extremely thermophilic bacterium Caldocellum saccharolyticum . The temperature stability of all enzymes was affected by mutation to various degrees and one of the xylanases had an altered temperature optimum . The mutations had no effect on the pH optimum . The C . saccharolyticum xylanase showed strong homology to several thermophilic and mesophilic xylanases, and comparison of primary sequences allowed the localization of probable active sites and residues involved in thermostability. Nucleic Acids Symp Ser, 1992, (27), 171 - 2 Enzymatic synthesis of PAPS with an ATP-regeneration system; Ibuki H et al.; Sulfate activating enzymes, ATP sulfurylase and APS kinase, were newly isolated from a thermophile, Bacillus stearothermophilus . Adenosine 3'-phosphate 5'-phosphosulfate (PAPS) was synthesized by these enzymes . The reaction proceeded more efficiently when an ATP-regeneration system, using acetate kinase, was coupled to the reaction system. Microbios, 1992, 71(286), 7 - 14 Analysis of chromosome-sized DNA from the bacterial genome of thermophilic Campylobacter laridis by pulsed-field gel electrophoresis and physical mapping; Matsumoto K et al.; Three restriction enzymes ApaI, SalI and SmaI, among nine enzymes tested, were found to produce distributions of DNA fragments which were useful for analysis of chromosome-sized DNA from thermophilic Campylobacter laridis by pulsed-field gel electrophoresis . From experiments with C . laridis JCM2530T and four isolates of C . laridis, the size of the genome of C . laridis was calculated to range from 1,590 to 1,700 kb, with a mean of 1,640 kb . An SmaI restriction map was derived by the partial digestion of the DNA from C . laridis JCM2530T. Dev Genet, 1992, 13(1), 75 - 9 A micronucleus-limited sequence family in Tetrahymena thermophila: organization and sequence conservation; Tsao NN et al.; During macronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs . Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families . We have previously identified a moderately repetitive micronucleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA . The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element . Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences . We have nucleotide sequence data for three cloned members of the X-H family . This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less . The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited. Can J Microbiol, 1992 Jan, 38(1), 65 - 8 A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria); Jarrell KF et al.; High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS . DNA can subsequently be purified by the usual phenol-chloroform extractions . The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation . The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell. Appl Environ Microbiol, 1992 Jan, 58(1), 421 - 5 Development of plasmid cloning vectors for Thermus thermophilus HB8: expression of a heterologous, plasmid-borne kanamycin nucleotidyltransferase gene; Mather MW et al.; While several Thermus genes have been cloned and T . thermophilus has been shown to be transformable, molecular genetic studies of these thermophiles have been hampered by the absence of selectable cloning vectors . We have constructed a selectable plasmid by random insertion of a heterologous gene encoding a thermostable kanamycin nucleotidyltransferase activity into a cryptic, multicopy plasmid from T . thermophilus HB8 . This plasmid should serve as a suitable starting point for the development of a gene expression system for T . thermophilus. DNA Seq, 1992, 3(4), 221 - 32 The glgB gene from the thermophile Bacillus caldolyticus encodes a thermolabile branching enzyme; Kiel JA et al.; We have cloned the structural gene for the Bacillus caldolyticus glycogen branching enzyme (glgB) in Escherichia coli . The glgB gene consisted of a 1998 bp open reading frame (ORF) encoding a 78,087 Da protein, which was highly similar to the Bacillus stearothermophilus branching enzyme . The 5' end of a second gene that encoded a protein with extensive similarity to E . coli ADP-glucose pyrophosphorylase (ADPGP) partly overlapped the 3' end of the glgB gene . A putative promoter recognized by Bacillus subtilis RNA polymerase containing the sigma factor H (E-sigma H) preceded the genes . These data suggest that in contrast to the situation observed in B . stearothermophilus, the genes involved in glycogen synthesis in B . caldolyticus are clustered on the chromosome, and are presumably coordinately expressed during the early stages of sporulation . An incomplete third gene started upstream of B . caldolyticus glgB . This gene was highly similar to a gene found directly upstream of B . stearothermophilus glgB, which encodes a putative membrane protein with unknown function . The B . caldolyticus glgB gene was expressed in E . coli and B . subtilis . Surprisingly, the branching enzyme appeared to be thermolabile, the temperature of optimal activity being only 39 degrees C. Nucleic Acids Symp Ser, 1992, (27), 141 - 2 Purification and characterization of tRNA(adenosine-1-)-methyltransferase from Thermus thermophilus HB27; Yamazaki N et al.; A58, the conserved adenosine residue in the T psi C loop of tRNAs, is methylated to m1A 58 in an extreme thermophile, Thermus thermophilus HB27 . The enzyme catalyzing this methyltransfer reaction was purified from the thermophle . The substrate specificity of the enzyme was investigated by using tRNA fragments . The enzyme can transfer the methyl group to the 3'-half fragment of E . coli initiator tRNA, indicating that the main recognition site of the enzyme exists in the 3' half of tRNA including the T-loop and the T-stem. J Biol Chem, 1991 Dec 25, 266(36), 24287 - 94 Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006; Watanabe K et al.; The gene encoding for an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) was sequenced . The amino acid sequence deduced from the nucleotide sequence of the gene (1686 base pairs) corresponded to a protein of 562 amino acid residues with a Mr of 66,502 . Its predicted amino acid composition, Mr, and N-terminal sequence of 12 residues were consistent with those determined for B . thermoglucosidasius oligo-1,6-glucosidase . The deduced sequence of the enzyme was 72% homologous to that of a thermolabile oligo-1,6-glucosidase (558 residues) from Bacillus cereus ATCC7064 (mesophile) . B . cereus oligo-1,6-glucosidase contained 19 prolines . Eighteen of these were conserved at the equivalent positions of B . thermoglucosidasius oligo-1,6-glucosidase . This enzyme contained 14 extra prolines besides the conservative prolines . The majority of extra prolines was replaced by polar or charged residues (Glu, Thr, or Lys) in B . cereus oligo-1,6-glucosidase . The extra prolines were responsible for the difference in thermostability between these two enzymes . We suggested that 11 of the extra prolines in B . thermoglucosidasius oligo-1,6-glucosidase occur in beta-turns or in coils within the loops binding adjacent secondary structures. Gene, 1991 Dec 20, 109(1), 39 - 45 Sequence and expression of the gene encoding 3-phosphoglycerate kinase from Bacillus stearothermophilus; Davies GJ et al.; The structural gene (pgk) encoding 3-phosphoglycerate (PGK) from Bacillus stearothermophilus NCA1503, has been cloned in Escherichia coli and its complete nucleotide sequence determined . The gene consists of an open reading frame corresponding to a protein of 394 amino acids (aa) (calculated Mr 42,703) and, in common with other prokaryotic pgk genes, is preceded by the structural gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . Constructs containing the B . stearothermophilus pgk gene and its flanking sequences in the high-copy plasmid, pUC9, co-express both PGK and GAPDH at high levels in transformed E . coli cells, typically producing PGK at levels of up to 30% of the soluble cell protein . The deduced aa sequence of B . stearothermophilus PGK is compared with those of the mesophilic (yeast) and extreme thermophilic (Thermus thermophilus) enzymes since the crystal structure of these PGKs are known or are in the process of being determined . Changes in the sequences of the three enzymes, as they appear to relate to the enhancement of thermal stability, are discussed. Gene, 1991 Dec 20, 109(1), 1 - 11 Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene; Barany F et al.; Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction {Barany, Proc . Natl . Acad . Sci . USA 88 (1991) 189-193} . The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E . coli host . Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E . coli ligase . Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E . coli cellular proteins . Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11105 - 9 Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core; Heuer TS et al.; Fe(II)-EDTA, a solvent-based cleavage reagent that distinguishes between the inside and outside surfaces of a folded RNA molecule, has revealed some of the higher-order folding of the group IB intron from Tetrahymena thermophila pre-rRNA . This reagent has now been used to analyze the bacteriophage T4 sunY and td introns, both of which are members of the group IA subclass . Significant portions of the phylogenetically conserved secondary structure are protected from Fe(II)-EDTA cleavage . However, the P4 secondary structure element, which is substantially protected in the Tetrahymena intron, is available for cleavage in the two T4 introns . We conclude that a family of catalytic RNAs (ribozymes) that possess similar secondary structures and have similar activities fold into similar but nonidentical tertiary structures that nevertheless serve to internalize portions of the catalytic center . Furthermore, comparison of cleavage patterns of the sunY and td intron RNAs indicates that conserved nucleotides outside as well as within the catalytic core participate in the tertiary structure. Nature, 1991 Dec 12, 354(6353), 490 - 3 A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1; Trent JD et al.; There is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol . We postulated that the major heat-shock protein of the thermophilic archaebacterium, Sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol . Acquired thermotolerance in S . shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic factor 55 (TF55) . TF55 is a homo-oligomeric complex of two stacked 9-membered rings, closely resembling the 7-membered-ring complexes of the chaperonins, groEL, hsp60 and Rubisco-binding protein . The TF55 complex binds unfolded polypeptides in vitro and has ATPase activity-features consistent with its being a molecular chaperone . The primary structure of TF55, however, is not significantly related to the chaperonins . On the other hand, it is highly homologous (36-40% identity) to a ubiquitous eukaryotic protein, t-complex polypeptide-1 (TCP1) . In Saccharomyces cerevisiae, TCP1 is an essential protein that may play a part in mitotic spindle formation . We suggest that TF55 in archaebacteria and TCP1 in the eukaryotic cytosol are members of a new class of molecular chaperones. Eur J Biochem, 1991 Dec 5, 202(2), 625 - 34 Temperature dependence of charge recombination from the P+QA- and P+QB- states in photosynthetic reaction centers isolated from the thermophilic bacterium Chloroflexus aurantiacus; Venturoli G et al.; The temperature dependence of charge recombination from the P+QA- and from the P+QB- states produced by a flash was studied in reaction centers isolated from the photosynthetic thermophilic bacterium Chloroflexus aurantiacus . P designates the primary electron donor; QA and QB the primary and secondary quinone electron acceptors respectively . In QB-depleted reaction centers the rate constant (kAP) for P+QA- recombination was temperature independent between 0-50 degrees C (17.6 +/- 0.7 s-1 at pH 8 and pH 10) . The same value was obtained in intact membranes in the presence of o-phenanthroline . Upon lowering the temperature from 250 K to 160 K, kAP increased by a factor of two and remained constant down to 80 K . The overall temperature dependence of kAP was consistent with an activationless process . Ubiquinone (UQ-3) and different types of menaquinone were used for QB reconstitution . In UQ-3 reconstituted reaction centers charge recombination was monoexponential (rate constant k = 0.18 +/- 0.03 s-1) and temperature independent between 5-40 degrees C . In contrast, in menaquinone-3- and menaquinone-4-reconstituted reaction centers P+ rereduction following a flash was markedly biphasic and temperature dependent . In menaquinone-6-reconstituted reaction centers a minor contribution from a third kinetic phase corresponding to P+QA- charge recombination was detected . Analysis of these kinetics and of the effects of the inhibitor o-phenanthroline at high temperature suggest that in detergent suspensions of menaquinone-reconstituted reaction centers a redox reaction removing electrons from the quinone acceptor complex competes with charge recombination . Instability of the semiquinone anions is more pronounced when QB is a short-chain menaquinone . From the temperature dependence of P+ decay the activation parameters for the P+QB- recombination and for the competing side oxidation of the reduced menaquinone acceptor have been derived . For both reactions the activation enthalpies and entropies change markedly with menaquinone chain length but counterbalance each other, resulting in activation free energies at ambient temperature independent of the menaquinone tail . When reaction centers are incorporated into phospholipid vesicles containing menaquinone-8 a temperature-dependent, monophasic, o-phenanthroline-sensitive recombination from the P+QB- state is observed, which is consistent with the formation of stable semiquinone anions . This result seems to indicate a proper QB functioning in the two-subunit reaction center isolated from Chlorflexus aurantiacus when the complex is inserted into a lipid bilayer. J Mol Biol, 1991 Dec 5, 222(3), 725 - 38 Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution; Imada K et al.; The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution . The final R-factor is 0.185 for 20,307 reflections . The crystal asymmetric unit has one subunit consisting of 345 amino acid residues . The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs . The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases . A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry . Close subunit contacts are also present at two alpha-helices in the second domain . These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer . Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding . These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme. Biochim Biophys Acta, 1991 Dec 3, 1098(1), 13 - 20 Molecular cloning of genes encoding major two subunits of a eubacterial V-type ATPase from Thermus thermophilus; Tsutsumi S et al.; The atpAB genes which encode the alpha and beta subunits of membrane ATPase from a thermophilic eubacterium, Thermus thermophilus HB8, were cloned . The deduced amino-acid sequences of the alpha subunit (583 amino acids) and the beta subunit (478 amino acids) are only moderately similar to the alpha beta subunits of the F0F1-ATPases, while they are highly similar to the major two subunits of the V-type ATPases, a family of ATPases which have been so far found in eukaryotic endomembrane vacuolar vesicles and archaebacterial plasma membranes . Thus, T . thermophilus ATPase belongs to the V-type ATPase family, even though this bacterium is a eubacterium . The hypothesis that the differentiation of an ancestral ATPase into V-type and F0F1-ATPase occurred after the evolution of a primordial cell into archaebacteria and eubacteria should be modified accordingly. Am J Clin Nutr, 1991 Dec, 54(6), 1041 - 6 Strains and species of lactic acid bacteria in fermented milks (yogurts): effect on in vivo lactose digestion; Martini MC et al.; Lactose in yogurt with live bacteria is better tolerated than lactose in other dairy foods, partly because of the activity of microbial beta-galactosidase (beta-gal), which digests lactose in vivo . To evaluate the ability of different strains and species of lactic acid bacteria to digest lactose in vivo, yogurts (containing mixtures of strains of Streptococcus salivarius subsp thermophilus and Lactobacillus delbrueckii subsp bulgaricus) and fermented milks (containing individual species of S thermophilus, L bulgaricus, L acidophilus, or Bifidobacterium bifidus) that varied in microbial beta-gal activity were produced . Selected products were fed to healthy people who cannot digest lactose, and breath hydrogen production was monitored . All yogurts dramatically and similarly improved lactose digestion, regardless of their total or specific beta-gal activity . The response to fermented milks varied from marginal improvement with B bifidus milk to nearly complete lactose digestion with L bulgaricus milk . The results suggest that total beta-gal was not the limiting factor in promoting lactose digestion, perhaps because of a limited rate of intracellular substrate transport. J Bacteriol, 1991 Dec, 173(23), 7725 - 7 Selectable mutant phenotypes of the extremely thermophilic archaebacterium Sulfolobus acidocaldarius; Grogan DW; As a first step toward developing the genetic potential of extremely thermophilic archaebacteria, mutant strains of Sulfolobus acidocaldarius were selected by plating cells directly on solid medium containing one of several growth inhibitors . Three spontaneous resistance phenotypes were observed (5-fluorouracil resistance, novobiocin resistance, and L-ethionine resistance), each at a different average frequency . Characterization of representative strains showed each of the three mutant phenotypes to provide a potentially useful genetic marker. J Bacteriol, 1991 Dec, 173(23), 7698 - 700 Positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius by use of 5-fluoroorotic acid; Kondo S et al.; Uracil auxotrophs of Sulfolobus acidocaldarius were positively selected by using 5-fluoroorotic acid . The wild-type strain was unable to grow in medium containing 5-fluoroorotic acid, whereas the mutants grew normally . Positive selection could be done for the auxotrophs . Mutants deficient in orotidine-5'-monophosphate pyrophosphorylase activity were isolated. Wei Sheng Wu Xue Bao, 1991 Dec, 31(6), 438 - 42 {Studies on the thermostable L-lactate dehydrogenase from thermophilic bacteria}; Yang S et al.; About 200 strains of extreme thermophilic bacteria were isolated from hot springs in Guandong province . A strain, HG25, was found to produce thermostable intracellular L-lactate dehydrogenase (EC . 1.1.1.27) . It has the characteristic of Thermus sp . The cells were gram-negative, non-sporulating, nonmotile, aerobic rods containing yellow pigment . The optimum temperature for growth was between 65 degrees C to 75 degrees C, the maximum 85 degrees C, and minimum 40 degrees C . The generation time at the optimum was about 80 min . Starch was not hydrolyzed . Acid was not produced from glucose . The G+C content in DNA was 62-65 mol% (Tm) . As the properties of strain HG25 is similar to those of Thermus aquaticus and T . thermophilus HB 8 belonging to the genus Thermus . The thermostable L-lactate dehydrogenase was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography . For pyruvate reduction, the optimum temperature of the enzyme was 60 degrees C and pH 8.0 . After incubation in 0.1 mol/L phosphate buffer pH 7.4 at 70 degrees C for 10 min, the enzyme retained about 85% of its original activity . The half-live time (t1/2) at 85 degrees C was 10 min. Protein Seq Data Anal, 1991 Dec, 4(6), 327 - 31 Amino acid sequence of the 8-kDa protein in photosystem I reaction center complex from a thermophilic cyanobacterium, Synechococcus elongatus; Jone CS et al.; The 8-kDa protein in Photosystem I (PS I) reaction center complex was isolated from a thermophilic cyanobacterium, Synechococcus elongatus, by SDS-polyacrylamide gel electrophoresis using TRIS-Tricine buffer system . The complete amino acid sequence of the protein was determined . The 8-kDa protein consisted of 73 amino acid residues giving a calculated molecular weight of 7,472 . No significant sequence homology were observed with the known other small subunits in PS I reaction center complex, except for the 6.5-kDa protein in PS I from another thermophilic cyanobacterium, S . vulcanus . The 8-kDa protein was characteristically rich in hydrophobic amino acid residues, especially the content of leucine . These suggest that the 8-kDa subunit is an intrinsic structure component in PS I core complex for stabilization of the reaction center. Yeast, 1991 Dec, 7(9), 963 - 70 Cloning and disruption of the LEU2 gene of Kluyveromyces marxianus CBS 6556; Bergkamp RJ et al.; The LEU2 gene, coding for beta-isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced . An open reading frame, coding for a protein with a molecular weight of 38 kDa was found . Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities . The cloned gene could complement a leuB mutation of Escherichia coli and a leu2 mutation of Saccharomyces cerevisiae . Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII . Analysis of the 5'-untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine-specific regulation of transcription . We show that the cloned gene can be used for the construction of a non-reverting K . marxianus leu2 mutant. Appl Environ Microbiol, 1991 Dec, 57(12), 3677 - 8 Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp . thermophilus; Benbadis L et al.; SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp . thermophilus strain BSN 45 . SslI is an isoschizomer of BstNI . SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C . Activity against phage DNA in vitro was demonstrated. Appl Environ Microbiol, 1991 Dec, 57(12), 3593 - 9 Purification and characterization of an endopeptidase from Lactococcus lactis subsp . cremoris Wg2; Tan PS et al.; An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp . cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column . Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da . The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides . The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity . It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C . The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+ . The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+ . Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp . and Streptococcus salivarius subsp . thermophilus. J Clin Microbiol, 1991 Dec, 29(12), 2746 - 51 Detection of variable DNA repeats in diverse eukaryotic microorganisms by a single set of polymerase chain reaction primers; Riley DE et al.; We cloned and sequenced a variable DNA repeat from Trichomonas vaginalis, a flagellated protozoan parasite . Targeting of this repeat in the polymerase chain reaction resulted in complex and intense product patterns for a wide variety of eukaryotic microorganisms, including the pathogenic protozoan parasites T . vaginalis, Giardia lamblia, Leishmania donovani, three species of Trypanosoma, and four species of Acanthamoeba; the nonpathogenic protozoans, Paramecium tetraurelia and Tetrahymena thermophilia; and a yeast, Saccharomyces cerevisiae . Each microorganism exhibited a distinctive pattern of repeats . For example, a characteristic pattern was exhibited by six clinical T . vaginalis isolates . Eight G . lamblia isolates exhibited either one of two characteristic pattern types . There was no reaction with human DNA or DNA from the prokaryotes Ureaplasma urealyticum and Mycoplasma hominis . This approach may facilitate detection of a wide variety of eukaryotic microorganisms by use of a single primer set and holds promise for the development of typing schemes for both T . vaginalis and G . lamblia. Dev Biol, 1991 Dec, 148(2), 403 - 19 Conjugal blocks in Tetrahymena pattern mutants and their cytoplasmic rescue . I . Broadened cortical domains (bcd); Cole ES; The cortical pattern mutant broadened cortical domains (bcd) in Tetrahymena thermophila is unable to complete the nuclear events associated with conjugation . bcd x bcd pairs become arrested at the "nuclear exchange" configuration . Genetic analysis reveals that the bcd conjugal block is 100% penetrant, under macronuclear control, and rescueable (a) by outcrossing to a wild-type partner, (b) by administration of a hyperosmotic shock 5 hr after cells are mixed for mating, or (c) by cytoplasmic transfusion from a wild-type donor . Cytological analysis reveals that the conjugal block is primarily the result of failure in pronuclear fusion (karyogamy) . bcd pairs also exhibit reduced nuclear exchange efficiency and a failure of macronuclear anlagen formation . The hypothesis is proposed that the bcd+ gene codes for a microtubule-based organelle "motor" similar to kinesin. An Acad Bras Cienc, 1991 Dec, 63(4), 409 - 14 Amylolytic activity of Humicola sp; de Oliveira AR et al.; The thermophilic and cellulolytic fungus Humicola sp . secretes amylase in the liquid culture medium . This activity is induced by starch, maltose and cellobiose . Glucose impairs accumulation of amylolytic activity in the culture medium . The enzyme hydrolyzes starch, maltose and pullulan to glucose as the end-product. Biochimie, 1991 Dec, 73(12), 1465 - 72 Translation and ribosome assembly in extremely thermophilic archaebacteria; Londei P et al.; Several features of translation and ribosome structure in extremely thermophilic, sulfur-dependent archaebacteria are described, including: i) a peculiar mechanism of transfer RNA-mediated 70S ribosome formation from free subunits; ii) poly(U)translation by hybrid ribosomes composed by one archaebacterial and one eucaryotic subunit; iii) ribosome assembly and homologous and heterologous RNA/protein recognition. J Biochem (Tokyo), 1991 Dec, 110(6), 905 - 8 Image analysis by electron microscopy of two-dimensional crystals developed on a mercury surface of chaperonin from Thermus thermophilus; Ishii N et al.; Two-dimensional crystals of functional chaperonin molecules, which are protein complexes of cpn60 and cpn10, isolated from Thermus thermophilus were prepared on a mercury surface under oxygen atmosphere and were observed by electron microscope after transferring them to carbon coated specimen grids . The crystals showed the hexagonal lattice with unit cell dimensions of a = b = 12.4 nm and gamma = 120 degrees . The averaged image at a 3 nm resolution of the chaperonin has a doughnut-like shape which has seven peripheral masses and a central cavity . Preincubation of the chaperonin with MgATP changed the mobility in non-denaturating PAGE but did not cause distinguishable change of shape . The location of cpn10 in the chaperonin molecule is discussed. Wei Sheng Wu Xue Bao, 1991 Dec, 31(6), 426 - 32 {Cloning and orientation of a promoter of thermophilic Thiobacillus sp.}; Yao A et al.; Thiobacillus sp . is an obligate autotrophic thermophilic bacterium which was isolated from an acidic hot spring in Yunnan Province . Its optimum growth temperature is 45-50 degrees C and its optimum pH is 2.0-3.0 . Using DNA recombinant technique, we inserted the HindIII fragments of the Thiobacillus sp . chromosomal DNA into the HindIII site of promoter-probe plasmid pSDSI (AprTcs, 5.65 kb) . Transformants resistant to tetracycline were obtained on Tc plates (12 micrograms/ml) . Of these, twenty transformants were able to grow on 120 micrograms/ml Tc plates, and two of them, designated pSDH7 and pSDH11, were able to grow on plates containing Tc at concentration up to 360 micrograms/ml . With HindIII, pSDH11 produced a 0.95kb fragment which had the function of promoter and a PstI site besides the 5.65 kb fragment of pSDSI . Southern blot hybridization showed that the 0.95 kb insert was from the Thiobacillus sp . chromosomal DNA . After restriction mapping, a 2.85 kb fragment of pSDH11 (which contained 0.7 kb of the inserted fragment) was removed with the aid of Pst1, and the remained fragment was used to construct a 3.75 kb plasmid (named pSDH114) which was resistant to a higher level of tetracycline (360 micrograms/ml) than for pBR322 (120 micrograms/ml) . The remained 0.25 kb foreign fragment in pSDH114 still retained full function of the promoter contained in the original 0.95 kb . Thus we could orient the cloned promotor function fragment (0.25 kb) from Thiobacillus sp . in pSDH114. J Biochem (Tokyo), 1991 Dec, 110(6), 854 - 5 Crystallization and preliminary X-ray diffraction study of cytochrome c552 from Hydrogenobacter thermophilus; Fukuyama K et al.; Cytochrome c552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, exhibits remarkable thermostability . The oxidized cytochrome c552 has been crystallized in an ethanol/water mixture by means of the vapor diffusion method . The crystals belong to the orthorhombic system, space group P2(1)2(1)2, with unit cell dimensions of a = 93.4 A, b = 52.9 A, and c = 32.4 A . Most probably the asymmetric unit contains two molecules of cytochrome c552 . The crystals diffract X-rays to better than 2.5 A resolution and are stable to X-ray irradiation. J Biochem (Tokyo), 1991 Dec, 110(6), 1016 - 21 Effects of aeration during growth of Bacillus stearothermophilus on proton pumping activity and change of terminal oxidases; Sone N et al.; The effects of aeration during bacterial growth on the proton translocating activity of the respiratory chain of B . stearothermophilus ATCC 8005, which is stable enough for measurement of the H+/O ratio by an oxygen pulse method, were examined . For endogenous and ascorbate-N,N,N',N'-tetramethyl p-phenylene diamine (TMPD) respiration, H+/O ratios of around 6 and 2 were obtained using resting cells grown under highly aerated conditions . The values were about 4 and 0 when cells were grown under limited-air conditions . Spectrophotometric and enzyme kinetical analyses revealed that both cytochrome caa3 and pigment-432 (cytochrome cao) were acting as terminal oxidases, while cytochrome b-558 (corresponding to the "cytochrome o-type oxidase" of the thermophilic bacterium PS3 in the previous paper {Sone, N., Kutoh, E., & Sato, K . (1990) J . Biochem . 107, 597-602}) was mainly serving in the cells grown under limited-air conditions . Measurement of the pH change upon ferrocytochrome c pulse with proteoliposomes reconstituted from the membrane extract of vigorously aerated cells and that of limited-air cells suggested that both cytochrome caa3, and pigment-432 (cytochrome cao) pump protons, while cytochrome b-558 does not. Agric Biol Chem, 1991 Dec, 55(12), 3059 - 66 Purification and properties of thermostable tryptophanase from an obligately symbiotic thermophile, Symbiobacterium thermophilum; Suzuki S et al.; A thermostable tryptophanase was extracted from a thermophilic bacterium, Symbiobacterium thermophilum strain T, which is obligately symbiotic with the thermophilic Bacillus strain S . The enzyme was purified 21-fold to homogeneity with 19% recovery by a series of chromatographies using anion-exchange, hydroxylapatite, hydrophobic interaction, and MonoQ anion-exchange columns . The molecular weight of the purified enzyme was estimated to be approximately 210,000 by gel filtration, while the molecular weight of its subunit was 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the native enzyme is composed of four homologous subunits . The isoelectric point of the enzyme was 4.9 . The tryptophanase was stable to heating at 65 degrees C for 20 min and the optimum temperature for the enzyme activity for 20 min reaction was 70 degrees C . The optimum pH was 7.0 . The NH2-terminal amino acid sequence of this tryptophanase shows similarity to that of Escherichia coli K-12, despite a great difference in the thermostability of these two enzymes . The purified enzyme catalyzed the degradation (alpha, beta-elimination) of L-tryptophan into indole, pyruvate, and ammonia in the presence of pyridoxal-5'-phosphate . The Km value for L-tryptophan was 1.47 mM . 5-Hydroxy-L-tryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, and L-serine were also used as substrates and converted to pyruvate . The reverse reaction of alpha, beta-elimination of this tryptophanase produced L-tryptophan from indole and pyruvate in the presence of a high concentration of ammonium acetate. Agric Biol Chem, 1991 Dec, 55(12), 3027 - 32 Involvement of NH2-terminal pro-sequence in the production of active aqualysin I (a thermophilic serine protease) in Escherichia coli; Lee YC et al.; Aqualysin I is a heat-stable subtilisin-type protease produced by Thermus aquaticus YT-1 . The precursor of aqualysin I consists of four domains: an NH2-terminal signal peptide, an NH2-terminal pro-sequence, a protease domain, and a COOH-terminal pro-sequence . In Escherichia coli cells harboring recombinant plasmid carrying the aqualysin I gene, proteolytic activity is obtained on treatment at 65 degrees C and mature enzyme is detected . In the case of mutant genes containing partial deletions in the NH2-terminal pro-sequence, no proteolytic activity was detected and the precursor protein was found to be unstable in E . coli . These results indicate that the NH2-terminal pro-sequence is required to produce the active enzyme by stabilizing the precursor structure . Amino acid substitutions in the conserved sequence of the NH2-terminal pro-sequence found among subtilisin-type proteases made the processing faster compared with the wild type. Biochem Biophys Res Commun, 1991 Nov 27, 181(1), 437 - 42 The cytochrome C oxidase genes in blue-green algae and characteristics of the deduced protein sequence for subunit II of the thermophilic cyanobacterium Synechococcus vulcanus; Tano H et al.; Blue-green algae (cyanobacteria) contain both primitive photosynthetic and respiratory systems in their membranes . The controversial genes coding for an alpha alpha 3-type cytochrome oxidase in cyanobacteria were examined . The DNA probe coding for the most conserved part of subunit I hybridized with DNA fragments from four cyanobacterial species . We have cloned the genes coding for subunits I and II from the genomic library of the thermophilic cyanobacterium Synechococcus vulcanus and determined the nucleotide sequence of the subunit II gene . The deduced protein sequence (327 amino acid residues) indicates that there are two hydrophobic segments near the N-terminus and a hydrophilic intermembrane domain containing ligands for CuA (the ESR-active Copper) similar to other subunit IIs . The S . vulcanus subunit II does not contain the cytochrome c moiety that is present in bacilli and thermophiles. Biochem Biophys Res Commun, 1991 Nov 27, 181(1), 342 - 7 Spectroscopic studies on APS reductase isolated from the hyperthermophilic sulfate-reducing archaebacterium Archaeglobus fulgidus; Lampreia J et al.; Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP . A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304 . Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme . The A . fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct {4Fe-4S} clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas . Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential. J Biol Chem, 1991 Nov 25, 266(33), 22411 - 8 A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes; Taguchi H et al.; A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus . It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view . Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate . ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis . The T . thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria . At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner . No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution . An excess amount of the chaperonin is inhibitory for refolding . At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented . At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding. J Mol Biol, 1991 Nov 20, 222(2), 219 - 32 Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions; Orum H et al.; We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila . With the exception of U4 snRNA, the sizes of the T . thermophila snRNAs are closely similar to their metazoan homologues . The T . thermophila snRNAs all have unique 5' ends, which start with an adenine residue . In contrast, with the exception of U6, their 3' ends show some size heterogeneity . The primary sequences of the T . thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other organisms . Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T . thermophila data . Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data . Two of these occur between U2 and U6, whereas the third occurs between U1 and U2 . The proposed interactions locate the intron 5' splice-site close to the intron branch-site nucleotide as well as to the most highly conserved domain of U6 . We envisage that these interactions may facilitate the first step of pre-mRNA splicing. Biochim Biophys Acta, 1991 Nov 15, 1080(3), 198 - 204 The active site of Sulfolobus solfataricus aspartate aminotransferase; Birolo L et al.; Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241 . This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro{3H}hydride and sequencing the specifically labeled peptic peptides . The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes . An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues . Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases . These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates . Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids) . It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates . Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate . The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes. Nucleic Acids Res, 1991 Nov 11, 19(21), 5957 - 64 Analysis of the gene encoding the RNA subunit of ribonuclease P from T . thermophilus HB8; Hartmann RK et al.; The gene for the RNA subunit of ribonuclease P from the extreme thermophilic eubacterium T . thermophilus HB8 was cloned using oligonucleotide probes complementary to conserved regions of RNase P RNA subunits from proteobacteria . The monocistronic gene and its flanking regions were sequenced . The gene is enclosed by a promoter and a rho-independent terminator . Nuclease S1 protection analyses showed that the primary transcript is identical with the mature RNA, i.e . no processing events are involved . The stem and loop structure of the terminator remains part of the mature molecule . In vitro transcription of the cloned gene with purified RNA polymerase from T . thermophilus yields the same RNA product as in vivo, indicating that no other components except RNA polymerase are involved in the synthesis of the RNA . RNase P RNA from T . thermophilus cleaved a pre-tRNA(Tyr) from E . coli with highest efficiency between 55 degrees C and 65 degrees C . The T . thermophilus RNA, which has a G-C content of 86% in helical regions, displays several structural idiosyncrasies, although its secondary structure is similar to that of proteobacteria . Numerous invariable nucleotides in the structural core of eubacterial RNase P RNAs are also conserved in the RNA from the extreme thermophilic eubacterium. Biochemistry, 1991 Nov 5, 30(44), 10632 - 40 Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups; Bevilacqua PC et al.; Dissociation constants at 15 degrees C were measured by equilibrium dialysis for the binding of rCrUrCrU, dCrUrCrU, rCdUrCrU, rCrUdCrU, and rCrUrCdU to the L-21 ScaI form of the self-splicing group I LSU intron from Tetrahymena thermophila . Substitution of deoxyribose for ribose in each of the middle two positions makes the free energy change for binding 1-2 kcal/mol less favorable, compared to about 0.3 kcal/mol less favorable for each of the terminal positions . Dissociation constants for binding of the same oligomers to rGGAGAA were measured by optical melting methods . Substitution of a single deoxyribose for ribose makes the free energy change for binding less favorable by 0.4-0.9 kcal/mol for this simple duplex formation . Comparison of the effects for binding to ribozyme and to rGGAGAA indicate that ribozyme-specific tertiary interactions dependent on the middle two 2' OH groups of rCrUrCrU add about 2 kcal/mol of favorable free energy for binding to L-21 ScaI . Comparisons are made with results from gel retardation studies {Pyle, A . M., McSwiggen, J . A., & Cech, T . R . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 8187-8191; Pyle, A . M., & Cech, T . R . (1991) Nature (London) 350, 628-631}.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
