Biotechnol Appl Biochem, 1993 Dec, 18 ( Pt 3), 417 - 25 Comparative studies on thermophilicity and thermostability of aspartate aminotransferases; Cubellis MV et al.; Aspartate aminotransferase from Sulfolobus solfataricus (AspATSs) is an extremely thermophilic and thermostable enzyme . In order to investigate the structural features which underlie thermophilicity and thermostability, two isoforms of AspATSs differing by a single amino acid residue were compared . The first isoform is the naturally occurring enzyme, whereas the second is a genetically engineered mutant . Thermophilicity, short-term and long-term thermostability of the isoenzymes were independently evaluated and the influence of a cysteine residue on the three properties was assessed. J Antibiot (Tokyo), 1993 Dec, 46(12), 1819 - 26 A novel quinone antibiotic from Malbranchea cinnamomea TAIM 13T54; Chiung YM et al.; A novel quinone antibiotic named malbranicin was isolated from the culture filtrate and mycelium of Malbranchea cinnamomea TAIM 13T54, a thermophilic fungus . The antibiotic was elucidated to be 6-(1-acetylethyl)-2-methoxy-2,5-cyclohexadiene-1,4-dione by spectral analysis . Malbranicin exhibited antimicrobial and cytotoxic activities against Gram-positive bacteria and mammalian cell lines, respectively. Biotechniques, 1993 Dec, 15(6), 996 - 8, 1000, 1002 Improved methods for the cultivation of strictly anaerobic, extremely thermophilic methanogens; Foster MS et al.; The strictly anaerobic, extremely thermophilic methanogens, Methanobacterium thermoautotrophicum Marburg and M . thermoautotrophicum delta H, have been cultivated in liquid culture and on solid medium in screw-top bottles, which permit continuous monitoring of the growth of the microorganisms . We have been able to routinely grow methanogens in medium containing bicarbonate, TRIS or 4-morpholinepropanesulfonic acid (MOPS) buffers and three different sulfur sources (sulfide, sulfite and thiosulfate) at temperatures up to 70 degrees C and at pressures up to 35 psi while monitoring cell density or colony formation. Genes Dev, 1993 Dec, 7(12B), 2641 - 51 Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila; Stargell LA et al.; Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus . Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle . Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hv1, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin . Polyclonal antisera were generated using whole hv1 protein and synthetic peptides from the amino and carboxyl domains of hv1 . The transcriptionally active macronuclei stained at all stages of the life cycle . Micronuclei did not stain during growth or starvation but stained with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active . Immunoblot analyses of fractionated macro- and micronuclei confirmed the micronuclear acquisition of hv1 early in conjugation . hv1 staining disappeared from developing micronuclei late in conjugation . Interestingly, the carboxy-peptide antiserum stained micronuclei only briefly, late in development . The detection of the previously sequestered carboxyl terminus of hv1 may be related to the elimination of hv1 during the dynamic restructing of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth . These studies demonstrate that the transcriptional differences between macro- and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hv1 functions in establishing a transcriptionally competent state of chromatin. Exp Cell Res, 1993 Dec, 209(2), 261 - 70 A temperature-sensitive cell cycle arrest mutation affecting H1 phosphorylation and nuclear localization of a small heat shock protein in Tetrahymena thermophila; Thatcher TH et al.; This report describes a temperature-sensitive Tetrahymena thermophila cell cycle arrest mutant that is also deficient in its heat shock response . Mutants incubated at 41 degrees C undergo rapid dephosphorylation of macronuclear histone H1, in contrast to wild-type cells which hyperphosphorylate H1 under the same conditions . Dephosphorylation is specific to H1 and is associated with a threefold decrease in the level of H1 kinase activity in macronuclei isolated from heat-shocked mutants . A small nuclear heat shock protein, sp29c, is synthesized and phosphorylated normally in the mutant cells but fails to accumulate in macronuclei . Nuclear transport of other heat shock proteins is unaffected . Mutant cells die slowly at 41 degrees C, a temperature at which wild-type cells resume normal growth after a brief lag . Wild-type cells acquire thermotolerance (competence to survive a 3-h heat shock at 43 degrees C) after a 1-h treatment at 41 degrees C, but mutant cells cannot become thermotolerant and die after the same treatment . The mutation is named chp 1 (cell cycle, heat shock, and phosphorylation defect). Dev Biol, 1993 Dec, 160(2), 333 - 54 Hypoangular: a gene potentially involved in specifying positional information in a ciliate, Tetrahymena thermophila; Frankel J et al.; In Tetrahymena, two unique cell-surface structures, the oral apparatus and the cytoproct, are formed at opposite ends of one ciliary row, the reference meridian, which is propagated longitudinally during clonal growth . A third set of unique structures, the contractile vacuole pore(s) (CVP), is located at a nearly constant proportion of the cell circumference to the cell's right of the reference meridian . Three allelic recessive temperature-sensitive mutations, collectively named hypoangular (hpo), alter both the geometry of propagation of the reference meridian and the location of the CVPs . In mutant cells, the reference meridian typically undergoes a steady rightward shift in successive cell generations ("cortical slippage"); concomitantly, CVP sets come to lie closer to the reference meridian . Although CVP location is still proportional to the cell circumference, the constant of proportionality (the "CVP angle") is reduced . Another effect is an alteration in the widths of morphogenetic domains within the cortex . As the temperature is raised (made more restrictive), these effects are accentuated and the CVP angle becomes reduced further . At the extreme, the CVP angle collapses to zero and less, i.e., there is a topological switch such that CVPs come to lie to the left of the reference meridian, and the direction of cortical slippage reverses from rightward to leftward . These observations are hard to reconcile with existing formal models of pattern specification in this system and suggest that the hpo locus might specify a key component of the intracellular positional system. Mol Cell Biol, 1993 Dec, 13(12), 7689 - 97 AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster; McCullough AJ et al.; cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are . The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei . In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron . Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron . To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei . We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco . Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences . In contrast to tobacco, in which the activities of two competing 5' splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D . melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides . The enhanced version of the cryptic intronic site is efficiently selected in D . melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted . Selection of this internal site in tobacco requires more drastic disruption of these motifs . We conclude that 5' splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco. Microbiologia, 1993 Dec, 9(2), 77 - 89 Thermophilic enzymes and their biotechnological potential; Lasa I et al.; The ability of many microorganisms to grow at high temperatures has held a particular fascination for microbiologists and biochemists since a long time . As any of their cellular components, their proteins are inherently more stable to heat than those of conventional organisms . This thermal stability is not due to any specific characteristic, but results a consequence of various changes which contribute to the whole stability of the protein in an additive manner . These enzymes are not only more thermostable, but also more resistant to chemical agents than their mesophilic homologous, what makes them extremely interesting for industrial processes . Despite this, most of the enzymes used at present in industrial processes have been isolated from mesophiles due to the limited knowledge and difficulties to grow thermophiles in high scale . The objective of this review is to consider briefly the importance of the thermostability in order to apply enzymes in the industry, and to overview the most recent advances in the identification of new thermophilic organisms and enzymes . Furthermore, the recent development of genetic model systems for moderate and extreme thermophiles are referred. J Bioenerg Biomembr, 1993 Dec, 25(6), 679 - 84 The TF1-ATPase and ATPase activities of assembled alpha 3 beta 3 gamma, alpha 3 beta 3 gamma delta, and alpha 3 beta 3 gamma epsilon complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the alpha 3 beta 3, and alpha 3 beta 3 delta complexes; Paik SR et al.; The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 microM where 70% stimulation is observed at 36 degrees C . Half maximal stimulation is observed at about 3 microM dye . At rhodamine 6G concentrations greater than 10 microM, ATPase activity declines with 50% inhibition observed at about 75 microM dye . The ATPase activities of the alpha 3 beta 3 gamma and alpha 3 beta 3 gamma delta complexes assembled from isolated subunits of TF1 expressed in E . coli deleted of the unc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1 . In contrast, the ATPase activities of the alpha 3 beta 3 and alpha 3 beta 3 delta complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 microM dye at 36 degrees C . The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO . In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 microM dye at 30 degrees C . One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO. J Biochem (Tokyo), 1993 Dec, 114(6), 926 - 9 RecA protein from an extremely thermophilic bacterium, Thermus thermophilus HB8; Kato R et al.; The recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T.th.) HB8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe . The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein . Characteristic amino acid changes between the two RecA proteins were found . In the amino acid composition of the T.th . RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu . These changes are supposed to stabilize the native protein conformation against heat denaturation . The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved . The T.th . recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E . coli recA deletion mutant . Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E . coli RecA protein. J Protein Chem, 1993 Dec, 12(6), 725 - 34 The complete primary structure of ribosomal protein L1 from Thermus thermophilus; Amons R et al.; The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit of Thermus thermophilus ribosomes has been elucidated by direct protein sequencing of selected peptides prepared by enzymatic and chemical cleavage of the intact purified protein . The polypeptide chain contains 228 amino acids and has a calculated molecular mass of 24,694 D . A comparison with the primary structures of the corresponding proteins from Escherichia coli and Bacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively . With respect to both proteins, L1 from T . thermophilus contains particularly less Ala, Lys, Gln, and Val, whereas its content of Glu, Gly, His, Ile, and Arg is higher . In addition, two fragments obtained by limited proteolysis of the intact, unmodified protein were characterized. Zentralbl Veterinarmed B, 1993 Dec, 40(9-10), 727 - 9 Microbiological quality of French yogurts commercialized in Spain; Lopez C et al.; The microbiological quality has been evaluated in 6 batches of yogurt (plain, flavoured and fruit-added) produced commercially in France and purchased in Spain . The essential microflora: Streptococcus salivarius subsp . thermophilus and Lactobacillus delbrueckii subsp . bulgaricus, and contaminants (coliform bacteria, yeasts and molds) were checked . The pH was also measured . The totality of the samples tested fulfilled French and Spanish regulation with respect to the presence of viable yogurt organisms . Likewise, in 100% of the yogurts, counts of contaminants were under 10 cfu/g . The pH ranged between 3.92 and 4.19. Appl Microbiol Biotechnol, 1993 Dec, 40(4), 508 - 14 A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus; Gilbert M et al.; Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans . Partial amino acid sequencing showed that these two enzymes belonged to different families of beta-1,4-glycanases . Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylo-oligomers . An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action . The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone . The two xylanases were able to remove about 12% of the xylan remaining in an aspen kraft pulp . This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps. J Clin Microbiol, 1993 Dec, 31(12), 3340 - 3 Discrimination among thermophilic Campylobacter species by polymerase chain reaction amplification of 23S rRNA gene fragments; Eyers M et al.; By comparing nucleic acid sequences determined for one of the most variable areas of 23S rRNA genes of 23 Campylobacter strains, we were able to identify regions specific for thermophilic Campylobacter strains . Oligonucleotide primers corresponding to these unique regions were synthesized and used in the polymerase chain reaction . One primer pair selectively detected all thermophilic Campylobacter species, while four other primer pairs allowed discrimination among the thermophilic species Campylobacter coli, Campylobacter jejuni subsp . jejuni, Campylobacter lari, and Campylobacter upsaliensis . All primer sets were tested successfully on a large number of clinical isolates. Gene, 1993 Nov 30, 134(1), 137 - 8 Sequence of the triosephosphate isomerase-encoding gene isolated from the thermophile Bacillus stearothermophilus; Rentier-Delrue F et al.; By analysis of genomic clones, we have determined the complete nucleotide sequence of the gene encoding triosephosphate isomerase (TIM; EC 5.3.1.1) in the thermophilic bacterium, Bacillus stearothermophilus . The gene encodes a 253-amino-acid TIM which is 39% identical to that of the mesophile, Escherichia coli. Biochim Biophys Acta, 1993 Nov 16, 1216(2), 213 - 20 ATP-independent DNA topoisomerase from Fervidobacterium islandicum; Bouthier de la Tour C et al.; Thermotogales are thermophilic eubacteria belonging to a very slowly evolving branch in the eubacterial tree . In this report, we describe the purification and characterization of an ATP-independent DNA topoisomerase from the Thermotogale, Fervidobacterium islandicum . The enzyme, a monomer of about 75 kDa, is a type I DNA topoisomerase sharing many properties with the other bacterial topoisomerases I: it absolutely requires Mg2+ for activity, relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA and spermidine . A feature of the F . islandicum ATP-independent DNA topoisomerase I is its thermophily . The optimal temperature for the enzymatic activity is 75 degrees C . Studies about thermostability show that the enzyme is more stable when incubated undiluted in the storage buffer . In these conditions, 60% activity was retained after a 30 min preincubation at 75 degrees C. J Biol Chem, 1993 Nov 15, 268(32), 24402 - 7 Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus . Cloning and sequencing of the gene and expression in Escherichia coli; Laderman KA et al.; A gene encoding a highly thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus was cloned and expressed in Escherichia coli . The nucleotide sequence of the gene predicts a 649-amino acid protein with a calculated molecular mass of 76.3 kDa, which corresponds well with the value obtained from purified enzyme using denaturing polyacrylamide gel electrophoresis . The NH2 terminus of the deduced amino acid sequence corresponds precisely to that obtained from the purified enzyme, excluding the NH2-terminal methionine . The amylase expressed in E . coli exhibits temperature-dependent activation characteristic of of the original enzyme from P . furiosus, but has a higher apparent molecular weight which is attributed to the improper formation of the native quaternary structure . No homology was found with previously characterized promotor or termination sequences . The deduced amino acid sequence displayed strong homology to the alpha-amylase A of Dictyoglomus thermophilum, an obligately anaerobic, extremely thermophilic bacterium . Evolutionary implications of this homology are discussed. FEBS Lett, 1993 Nov 8, 334(1), 79 - 82 Trimeric forms of the photosystem I reaction center complex pre-exist in the membranes of the cyanobacterium Spirulina platensis; Shubin VV et al.; Oligomeric and monomeric forms of chlorophyll-protein complexes of photosystem I (PSI) have been isolated from the mesophilic cyanobacterium Spirulina {(1992) FEBS Lett . 309, 340-342} . Electron microscopic analysis of the complexes showed that the oligomeric form is a trimer of the shape and dimensions similar to those of the trimer from thermophilic cyanobacteria . The chlorophyl ratio in the isolated trimer and monomer was found to be 7:3 . The trimeric form of PSI complex in contrast to the monomeric one contains the chlorophyll emitting at 760 nm (77K), which is also found in Spirulina membranes and therefore could be used as an intrinsic probe for the trimeric complex . The 77K circular dichroism spectrum of the trimeric form is much more similar to that of Spirulina membranes than the spectrum of the monomer . Thus, the trimeric PSI complexes exist and dominate in the Spirulina membranes. J Mol Biol, 1993 Nov 5, 234(1), 222 - 33 Refined crystal structure of the seryl-tRNA synthetase from Thermus thermophilus at 2.5 A resolution; Fujinaga M et al.; The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution . The final model consists of a dimer of 421 residues each and 190 water molecules . The R-factor is 18.4% for all the data between 10 and 2.5 A resolution . The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s . difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent . The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile. J Biol Chem, 1993 Nov 5, 268(31), 23172 - 8 Transcriptional regulation of the carbon monoxide dehydrogenase gene (cdhA) in Methanosarcina thermophila; Sowers KR et al.; The mechanisms of gene regulation in the phylogenetic domain Archaea are not yet understood . To examine the expression of a gene encoding a highly regulated catabolic enzyme from the methanogenic archaea, a Methanosarcina thermophila lambda gt11 chromosomal library was probed with antiserum prepared against the 89-kDa subunit of carbon monoxide dehydrogenase, an enzyme which is required for growth and methanogenesis from acetate . A 2.3-kilobase DNA fragment was isolated that encoded 300 bases of the 5'-end of cdhA, the gene which encodes the 89-kDa subunit, and 2 kilobases upstream of cdhA that included an upstream open reading frame (ORF1) . Primer extension analyses determined that cdhA and ORF1 each had a single transcriptional initiation site located 370 and 9 nucleotides, respectively, 5' of the putative translation initiation codons for cdhA and ORF1 . Each promoter element had sequence similarity to a consensus archaeal promoter sequence . Three discrete mRNA cdhA transcripts of 9.5, 5.6, and 4.8 kilobases and one mRNA ORF1 transcript of < 2 kilobases were identified . All four transcripts were optimally expressed in cells grown with acetate, while growth with the more energetically favorable substrates methanol and trimethylamine caused a significant reduction in levels of the cdhA and ORF1 mRNA's . The half-lives of the 5' ends of the three cdhA transcripts and entire ORF1 mRNA transcript were approximately 2 min upon addition of methanol to cells growing exponentially in medium that contained acetate . Results of this study demonstrate that transcription of both cdhA and ORF1 is highly regulated in response to substrate by this methanogenic archaeon. Biochim Biophys Acta, 1993 Nov 2, 1183(1), 130 - 8 The genes in the thermophilic cyanobacterium Synechococcus vulcanus encoding cytochrome-c oxidase; Sone N et al.; It is still controversial whether cyanobacteria (blue-green algae) contain an aa3-type cytochrome-c oxidase . We have approached this problem using DNA analysis . Using a DNA probe coding for the most conserved part of subunit I of the Bacillus enzymes, structural genes for the oxidase of a thermophilic cyanobacterium Synechococcus vulcanus were cloned and sequenced . We found genes for subunits II, I, III and IV of this order like those of the Bacillus enzymes, and a terminator structure after the gene for subunit IV . The deduced protein sequences for the subunits II, I and III showed consensus amino-acid residues atevery important portion, suggesting that these genes are operating . However, the S . vulcanus oxidase lacked a cytochrome-c-moiety fused to subunit II, the 13th and 14th hydrophobic segments of subunit I which are lacking in the Paracoccus enzyme, and the 1st and 2nd ones of subunit III which are lacking in the Bacillus enzyme, were not found . A gene homologous to ctaB gene, which locates at the 5'-upstream region of the gene for subunit II and co-transcribed in Bacillus subtilis, was not found . Comparison of protein sequences showed that S . vulcanus cytochrome oxidase is closer to Bacillus cytochrome oxidases than the mitochondrial and Paracoccus enzymes, or quinol oxidases from B . subtilis and Escherichia coli. Chromosoma, 1993 Nov, 102(9), 637 - 47 Phosphorylation of linker histones by cAMP-dependent protein kinase in mitotic micronuclei of Tetrahymena; Sweet MT et al.; Linker histones (LHs) in transcriptionally inactive, mitotically dividing micronuclei of Tetrahymena thermophila, alpha, beta, gamma and delta, are highly phosphorylated in vivo . Analysis of the derived sequences of these LHs suggests that none of these polypeptides contain sites of phosphorylation by p34cdc2, the kinase thought to play an essential role governing the entry of all cells into mitosis . Surprisingly alpha, beta, gamma and delta each contain sites for phosphorylation by cyclic AMP-dependent kinase (PKA) . p34cdc2 kinase phosphorylases H1 in vitro but fails to phosphorylate alpha, beta, gamma and delta . Conversely, PKA phosphorylates each of the micronuclear LHs but is unable to phosphorylate macronuclear H1 . Micronuclear LHs labeled in vivo with {32P}phosphate were purified by reverse phase HPLC . Phosphoamino acid analysis showed that all four micronuclear LHs are phosphorylated exclusively on serine residues in vitro . Cyanogen bromide mapping of alpha, beta, gamma and delta labeled in vivo or in vitro by PKA indicates that each LH is phosphorylated only on peptides that contain either optimum (RR/KXS) or less optimum (RXXS) PKA sequences . This study suggests that PKA or a PKA-like activity(ies), but not p34cdc2 kinase, is(are) responsible for the in vivo phosphorylation of LHs in the mitotic micronucleus of Tetrahymena . We suggest that, at least in Tetrahymena, PKA-driven phosphorylation or dephosphorylation plays a significant role in the control of mitotic processes such as chromosome condensation. Bioorg Khim, 1993 Nov, 19(11), 1073 - 6 {Isolation and properties of site-specific endonuclease BspTS514I from the thermophilic bacteria Bacillus species TS514}; Kovalevskaia NP et al.; New site-specific endonucleases BspBS31I, BstBS32I, BspIS41, BstTS5I, BspTS514I were isolated from five thermophilic soil bacteria Bacillus sp . BS31, B . stearothermophilus BS32, Bacillus sp . IS4, B . stearothermophilus TS5, Bacillus sp . TS514 . The enzymes are isoschizomers of the restriction endonuclease BbvII . Endonuclease BspTS514I was obtained pure from interfering contaminations by two consecutive chromatographies on blue agarose and hydroxyapatite . The enzyme exhibits a maximal activity at 55 degrees C in 10 mM tris-HCl (pH 9.2), 10 mM MgCl2 and 50 mM NaCl. J Bacteriol, 1993 Nov, 175(21), 6822 - 9 Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila; Latimer MT et al.; The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced . Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene . Consensus archaeal promoter sequences are found upstream of the pta coding region . The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively . A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptides; however, no membrane-spanning domains are evident . Comparison of the amino acid sequences deduced from the M . thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity) . The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues . Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E . coli acetate kinase . The pta and ack genes were hyperexpressed in E . coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M . thermophila . The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively . The activity of the acetate kinase is optimal at 65 degrees C and is protected from thermal inactivation by ATP . Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis (2-nitrobenzoic acid) and N-ethylmaleimide were ineffective. Eur J Epidemiol, 1993 Nov, 9(6), 645 - 9 Identification of Rickettsia prowazekii using the polymerase chain reaction; Aniskovich LP et al.; Polymerase chain reaction (PCR) was used to identify Rickettsia prowazekii, the etiologic agent of epidemic typhus . For the PCR, Thermus thermophilus thermostable DNA polymerase was applied with buffer containing a relatively low Mg2+ concentration (1.5-2 mM with dNTP's at 250 microM each) . A primer pair used to amplify a 448-base-pair (bp) fragment of R . prowazekii genome was synthesized on the basis of the DNA sequence of gene rpa14/16, coding for a precursor of the mature polypeptides of molecular weight (Mr) 14,000 and/or 16,000 (16kD) from R . prowazekii strain E . For determining the specificity of the primer pair, purified genomic DNAs of 16 rickettsial and 10 other bacterial strains were used. J Biochem (Tokyo), 1993 Nov, 114(5), 732 - 4 Effects of polyamines on a continuous cell-free protein synthesis system of an extreme thermophile, Thermus thermophilus; Uzawa T et al.; A continuous cell-free protein synthesis system of an extremely thermophilic eubacterium, Thermus thermophilus HB27, was constructed . This system produced MS2 phage RNA translation products at a rate of more than 5 micrograms per hour per 1.9 mg of ribosomes at 65 degrees C and the production continued linearly for at least 340 min . When no polyamine was added, the system did not produce the proteins . The highest activity was recorded when 0.1 mM tetrakis(3-aminopropyl)ammonium and 1.0 mM spermine were added simultaneously. Int J Pept Protein Res, 1993 Nov, 42(5), 490 - 5 Stereospecificity of hydrogen transfer by the NADP-linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii; Peretz M et al.; Class A and class B NAD(H)/NADP(H) coenzyme-dependent dehydrogenases distinguish between the diastereotopic hydrogens pro-R and pro-S at position 4 of the cofactor . We investigated the stereochemistry of hydride transfer in reactions catalyzed by an unusual thermophilic, zinc-containing, NADP-linked enzyme Thermoanaerobium brockii alcohol dehydrogenase (TBAD) . Using proton NMR spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers the pro-R hydrogen from the pyridine 4 position of the reduced coenzyme . This stereospecificity is stable over (a) a broad range of temperatures up to 70 degrees C, (b) different concentrations of the coenzyme (catalytic or stoichiometric) and (c) a wide scope of substrates . Although NAD+ is not an effective coenzyme for TBAD, NADP+ and its synthetic analogs, 3-acetylpyridine-ADP+ and thio-NADP+, can be used successfully. Biochemistry, 1993 Oct 26, 32(42), 11390 - 6 The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity; Liu J et al.; The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc . This metal ion is strongly bound, as it is not removed by 8 M urea . Slow removal of the zinc at 4 degrees C in the presence of the specific chelating agent, 1,10-phenanthroline, is proportional to the loss of aminoacylation activity and to the presence of a more open conformer of the enzyme . This conformer migrates more slowly than the native enzyme during gel electrophoresis under nondenaturing conditions and binds tRNA(Glu) . Infrared spectroscopy measurements show that it differs from the native enzyme by a lower alpha-helix content and a higher proportion of beta-sheet and unordered structures . ATP protects the enzyme against 1,10-phenanthroline-mediated zinc removal, suggesting that the zinc-binding region is closely associated with the catalytic site . Additional support for this conclusion comes from the presence of zinc in the 27-kDa N-terminal half of the enzyme and in a 10-kDa fragment . The latter is homologous to the tRNA acceptor helix binding domain of E . coli glutaminyl-tRNA synthetase . The presence of the conserved CYC motif in this domain of the zinc-containing glutamyl-tRNA synthetases of E . coli and Bacillus subtilis, and its absence in that of Thermus thermophilus and the E . coli glutaminyl-tRNA synthetase which do not contain zinc, suggest that the cysteines of this motif and the C- and H-rich 125CRHSHEHHX5C138 segment present in the 10-kDa zinc-binding fragment are involved in zinc binding by the glutamyl-tRNA synthetase of E . coli. Nucleic Acids Res, 1993 Oct 25, 21(21), 4954 - 60 Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE); Liu X et al.; A procedure is described for mapping the ends of RNAs . Using T4 RNA ligase, a DNA (3' end) or RNA (5' end) oligonucleotide is ligated to RNA ends followed by cDNA synthesis, PCR amplification, cloning and sequencing . This method determines 5' ends, 3' polyadenylation sites and the size of poly(A) tails, and should be applicable to non-polyadenylated mRNAs and to non-message RNAs . Analysis of four Tetrahymena thermophila histone mRNAs revealed multiple, closely spaced 5' ends consistent with those determined by other methods . Except for a 'CCAAT' box in either orientation 100-200 nucleotides upstream of the transcription start site, no conserved sequence elements were observed in the untranslated 5' region or in sequences immediately flanking the transcription start site . Analysis of the 3' ends of mRNAs encoding four histones, two tubulins and the Tetrahymena TATA binding protein confirmed the observations that Tetrahymena histone messages are polyadenylated and that poly(A) tails in this organism are short (approximately 50 nt) . No canonical poly(A) addition signal was identified . The four histone messages analyzed have contained three sequence elements, TGTGT-TAA-AAGTATT, not found in non-histone messages . Two non-histone messages contained GCATT(N)15ATACC near the poly(A) addition site. J Mol Biol, 1993 Oct 20, 233(4), 629 - 43 A shortened form of the Tetrahymena thermophila group I intron can catalyze the complete splicing reaction in trans; Sargueil B et al.; The group I intron from Tetrahymena thermophila is able to catalyze its own excision from a precursor RNA . The intron recognizes the splice sites through an intron-encoded sequence called the internal guide sequence, or IGS . The 5' and 3' exons are thought to align on the IGS and form a pseudoknot structure consisting of two stems (P1 and P10) . We created a shortened form of the intron that lacks the exon sequences and the entire IGS . This RNA is unable to react upon itself . It can catalyze a sequential two-step transesterification reaction on a P1P10 substrate added in trans that completely mimics splicing . The reaction works for different substrates that contain a U.G base-pair preceding the 5' cleavage site and a guanosine base preceding the 3' cleavage site, but that are otherwise unrelated in sequence . The ribozyme uses primarily the correct 5' and 3' splice sites even in the presence of potential cryptic splice sites, and therefore it must rely on the structure of the substrate (formation of the P1 and P10 helices) for correct splice site recognition . A C-G base-pair after the 5' splice site in P1 decreases activity while a U.G or G.U base-pair enhances activity . The relative position in P1 of the U.G base-pair preceding the 5' splice site is an important determinant . The ability of the intron to recognize primarily a specific structure, rather than a sequence, has ramifications for splice-site selection, for molecular modeling of the group I intron, and for ribozyme-based gene targeting. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9295 - 9 Transformation of Tetrahymena thermophila by microinjection of a foreign gene; Kahn RW et al.; Tetrahymena thermophila has been transformed to paromomycin-resistant phenotypes by microinjection of an aminoglycoside 3'-phosphotransferase (neo) gene under the control of the T . thermophila histone H4-I promoter . This chimeric neo gene, by itself or on a vector containing a rRNA-encoding DNA (rDNA) origin of replication, transforms T . thermophila . In cells transformed with the rDNA origin vector, the neo gene is usually found integrated into the endogenous rDNA molecules and is present in high copy number . In transformants obtained by microinjecting only the linear chimeric gene, the neo gene is found to have replaced the histone H4-I gene or is found integrated into the 5' flanking region of the H4-I gene . The relative transcript levels of the neo gene in T . thermophila transformed by the linear chimeric gene are much higher than in cells transformed with the vector . The neo gene provides an effective selectable marker for transformation of T . thermophila. J Biol Chem, 1993 Oct 15, 268(29), 21701 - 5 (S)-geranylgeranylglyceryl phosphate synthase . Purification and characterization of the first pathway-specific enzyme in archaebacterial membrane lipid biosynthesis; Chen A et al.; The first pathway-specific step in the biosynthesis of the core membrane diether lipids in archaebacteria is the alkylation of the primary hydroxyl group in (S)-glyceryl phosphate by geranylgeranyl diphosphate . The reaction is catalyzed by (S)-3-O-geranylgeranylglyceryl phosphate ((S)-GGGP) synthase . The cytosolic enzyme was purified to homogeneity from the moderately thermophilic archaebacterium Methanobacterium thermoautotrophicum by a combination of ammonium sulfate precipitation, four chromatographic steps (DE52, Q-Sepharose, phenyl-Superose, and Protein Pak), and native polyacrylamide gel electrophoresis . SDS-polyacrylamide gel electrophoresis of gel-purified GGGP synthase gave a single band at 29 kDa . The enzyme requires Mg2+ for optimal activity, although prenyltransfer is also seen in buffers containing Mn2+ or Zn2+ . A well defined pH optimum occurs between 6.0 and 7.5 . Maximal activity is seen at 50-65 degrees C . The Michaelis constants for GGGP synthase are Vmax = 4.1 +/- 0.5 mumol min-1 mg-1, KMGGPP = 4.1 +/- 1.1 microM, and KMGP = 41 +/- 5 microM. FEMS Microbiol Lett, 1993 Oct 15, 113(2), 125 - 8 16S rDNA analysis reveals phylogenetic diversity among the polysaccharolytic clostridia; Rainey FA et al.; Small subunit rDNA sequences were determined for 13 mesophilic, polysaccharolytic, mainly cellulolytic species of the genus Clostridium and one cellulolytic Eubacterium species . Sequences were compared to those of 36 representatives of mesophilic and thermophilic clostridia, including those of nine thermophilic polysaccharolytic species published previously . The majority of strains group with 23S rRNA clusters I and III, while the others group with the thermophilic polysaccharolytic clostridia, i.e . C . stercorarium, C . thermolacticum and C . thermocellum . Lack of close genetic relationships between the various polysaccharolytic species is unexpected and may indicate that these biotechnologically important organisms differ with respect to the enzymology of polysaccharolytic degradation as well. FEBS Lett, 1993 Oct 11, 332(1-2), 37 - 8 Tyr-139 in Thermus thermophilus 3-isopropylmalate dehydrogenase is involved in catalytic function; Miyazaki K et al.; The role of Tyr-139, which is thought to be located at the active site of Thermus thermophilus HB8 3-isopropylmalate dehydrogenase, has been investigated by site-specific replacement with phenylalanine . The replacement scarcely affected the Michaelis constant (Km) for 3-isopropylmalate, but caused a 13-fold decrease of that for NAD . The catalytic constant (kcat) showed a 14-fold decrease . Accordingly, the catalytic efficiency (kcat/Km) decreased for 3-isopropylmalate but not for NAD . The results suggest that Tyr-139 is involved in the catalytic function through interaction with 3-isopropylmalate. Biochim Biophys Acta, 1993 Oct 6, 1202(2), 244 - 50 Glutamate dehydrogenase from the extremely thermophilic archaebacterial isolate AN1; Hudson RC et al.; Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase, deaminating and transaminating, EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic archaebacterial isolate AN1 (a member of the Thermococcales) . The enzyme comprised a large proportion of the soluble cell protein (11%) and was purified in high yield . The molecular mass of the native enzyme was 204 kDa, while the subunit molecular mass was 47 kDa, indicating a tetrameric structure . The enzyme is specific for NADP(H) rather than NAD(H) by a factor of greater than 1000, as judged by Vmax/Km . Glutamate synthase activity was about 50% of the glutamate dehydrogenase activity . Activity was markedly enhanced by calcium, magnesium and manganese ions . The enzyme was highly thermostable with t1/2 values of 12.5 h and 47 min at 90 degrees C and 103 degrees C, respectively. Biochim Biophys Acta, 1993 Oct 6, 1202(2), 235 - 43 The identification of a lysine residue reactive to pyridoxal-5-phosphate in the glycerol dehydrogenase from the thermophile Bacillus stearothermophilus; Paine LJ et al.; The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is inactivated by incubation with pyridoxal-5-phosphate (PALP) . The complex formed between the two can be trapped by reduction with sodium borohydride to yield a protein with an absorbance band at 325 nm and a fluorescence emission band at 430 nm, typical of trapped pyridoxal-5-phosphate moieties . Total loss of catalytic activity of the enzyme is associated with the modification of approximately one equivalent of the reagent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenzyme . Peptides derived from the labelled protein have been sequenced and have identified Lys-97 as the reactive residue . Site-directed mutagenesis had been used to replace Lys-97 by a His residue . This mutated enzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH. FEBS Lett, 1993 Oct 4, 331(3), 291 - 5 Inhibition of an archaeal protein phosphatase activity by okadaic acid, microcystin-LR, or calyculin A; Oxenrider KA et al.; Soluble extracts of the methanogenic archaeon, Methanosarcina thermophila TM-1, contained a divalent metal ion-stimulated protein-serine phosphatase activity . This activity was sensitive to micromolar concentrations of okadaic acid, microcystin-LR, or calyculin A, three compounds thought to be highly specific inhibitors of the type 1/2A/2B genetic superfamily of eukaryotic protein-serine/threonine phosphatases . The observation that each of these three chemically unrelated compounds inhibited this archaeal protein phosphatase activity suggests the existence of structural homology, and perhaps even common genetic ancestry, with the type 1/2A/2B superfamily of protein-serine/threonine phosphatases found in eukaryotic organisms. Mol Cell Biol, 1993 Oct, 13(10), 6586 - 99 Sequence-specific DNA primer effects on telomerase polymerization activity; Lee MS et al.; The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme . Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila . This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA . The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added . Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction . These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo. Biol Pharm Bull, 1993 Oct, 16(10), 973 - 7 Studies on thermophile products . VI . Activation of mouse peritoneal macrophages by bis(2-hydroxyethyl) trisulfide; Kohama Y et al.; The biological effects of a cytotoxic substance (BS-1), isolated from Bacillus stearothermophilus UK563 and identified as bis(2-hydroxyethyl) trisulfide, on elicited mouse peritoneal macrophages induced by a casein injection, were investigated in vitro . BALB/c mouse macrophages treated or pretreated with BS-1 (1-10 micrograms/ml) showed cytotoxicity against syngeneic DBA/2 mouse P815 mastocytoma . BS-1 also showed weak cytotoxicity directly against P815 in the absence of macrophages . BS-1 significantly increased the glucose consumption of macrophages without producing cytotoxicity . This trisulfide compound increased nitric oxide formation, interleukin-1 production and prostaglandin E2 release in macrophages . It did not, however, increase the production of active oxygen species in macrophages, but it reduced cytochrome c in the presence of phagocytes . These results indicate that BS-1 activates macrophages to the cytolytic stage. J Biochem (Tokyo), 1993 Oct, 114(4), 478 - 86 Effects of novel polyamines on cell-free polypeptide synthesis catalyzed by Thermus thermophilus HB8 extract; Uzawa T et al.; Effects of novel, naturally occurring polyamines on protein synthesis catalyzed by Thermus thermophilus cell-free extract were investigated . The results revealed the physiological importance of a branched quaternary polyamine, tetrakis(3-aminopropyl) ammonium, in thermophile protein biosynthesis . Longer polyamines than triamine supported the polypeptide synthesis at high temperature, though both the activity and the optimum temperature varied depending on polyamines added . The highest activity was found when tetrakis(3-aminopropyl)ammonium and a tetraamine were simultaneously present . The optimum temperature of the reaction supported by the combination of the branched polyamine and spermine was the highest and in accord with the optimum temperature of the bacterial growth . These results suggested an essential role of the quaternary amine in protein synthesis in vivo . This amine effectively stabilized the ternary complex between ribosomes, the messenger, and phenylalanyl-tRNA, and this stabilization may account, at least in part, for its action on the present reaction . In contrast, another branched polyamine, tris(3-aminopropyl)amine supported the activity only moderately even in the presence of another polyamine, though the tris amine stabilized the ternary complex as effectively as the quaternary amine . This result suggests the presence of another essential site for polyamine action in the thermophile polypeptide synthesis, in addition to the stabilization of the ternary complex . The effects of polyamines on MS2 RNA directed reaction resembled those on poly(U) directed polypeptide synthesis, indicating that polyamines are essential in protein biosynthesis directed by natural messengers in vivo . The quaternary amine inhibited the aminoacylation of tRNA(Phe), and the inhibition was canceled by the addition of another polyamine.(ABSTRACT TRUNCATED AT 250 WORDS) Curr Opin Genet Dev, 1993 Oct, 3(5), 730 - 5 Developmentally regulated processing and replication of the Tetrahymena rDNA minichromosome; Kapler GM; The ribosomal DNA locus of Tetrahymena thermophila undergoes a dramatic series of developmentally regulated processing events to generate the amplified rDNA minichromosome during formation of the somatic macronucleus . DNA transformation and classical genetic approaches have identified cis-acting elements that regulate rDNA processing in the developing macronucleus and subsequent vegetative rDNA maintenance. FEMS Microbiol Lett, 1993 Oct 1, 113(1), 81 - 6 Phylogenetic analysis of Desulfotomaculum thermobenzoicum using polymerase chain reaction-amplified 16S rRNA-specific DNA; Redburn AC et al.; The 16S rRNA gene of the thermophilic sulfate-reducing bacterium Desulfotomaculum thermobenzoicum was amplified by polymerase chain reaction using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16S rRNA gene, cloned, and sequenced . Phylogenetic analysis revealed that D . thermobenzoicum belongs to the Gram-positive (low G + C content) branch and is more related to the thermophilic sulfate-reducing bacterium, D . australicum than the moderate thermophile D . nigrificans, or the mesophiles D . orientis, and D . ruminis . This relationship is further strengthened by the presence of an unusual idiosyncrasy in helix 6 of the 16S rRNA gene of D . thermobenzoicum resembling that of D . australicum but not found in other desulfotomacula species and in any other bacteria sequenced to date. Plant Mol Biol, 1993 Oct, 23(1), 67 - 76 Organization of plastid-encoded ATPase genes and flanking regions including homologues of infB and tsf in the thermophilic red alga Galdieria sulphuraria; Kostrzewa M et al.; We have cloned and sequenced the plastid ATPase operons (atp1 and atp2) and flanking regions from the unicellular red alga Galdieria sulphuraria (Cyanidium caldarium) . Six genes (5 atpI, H, G, F, D and A 3) are linked in atp1 encoding ATPase subunits a, c, b, b, delta and alpha, respectively . The atpF gene does not contain an intron and overlaps atpD by 1 bp . As in the genome of chloroplasts from land plants, the cluster is located downstream of rps2, but between this gene and atp1 we found the gene for the prokaryotic translation elongation factor TS . Downstream of atpA, we detected two open reading frames, one encoding a putative transport protein . The genes atpB and atpE, encoding ATPase subunits beta and epsilon, respectively, are linked in atp2, separated by a 2 bp spacer . Upstream of atpB, an uninterrupted orf167 was detected which is homologous to an intron-containing open reading frame in land plant chloroplasts . This orf167 is preceded on the opposite DNA strand by a homologue to initiation factor 2 in prokaryotes . The arrangement of atp1 and atp2 is the same as observed in the multicellular red alga Antithamnion sp., indicating a conserved genome arrangement in the red algal plastid genome . Differences compared to green chloroplast genomes suggest a large phylogenetic distance between red algae and green plants, while similarities in arrangement and sequence to chromophytic ATPase operons support a red algal origin of chlorophyll a/c-containing plastids or alternatively point to a common prokaryotic endosymbiont. Res Microbiol, 1993 Oct, 144(8), 657 - 60 Physiology of some actinomycete genera; Ensign JC et al.; Actinomycetes are widespread in the environment and are mainly organotrophic . Studies of their ecology have been primarily focussed on their detection and isolation, with comparatively little attention to the control mechanisms that determine their occurrence and behaviour in their natural environments . This session provided some diverse examples of approaches to this problem . Several actinomycete genera produce motile spores . The significance of flagella proteins and factors influencing spore motility and germination are considered . The genus Frankia forms nitrogen-fixing associations with non-leguminous plants . Molecular techniques have been used to clarify the endophyte-host relationships . Micromonospora species are common in the environment . The growth and physiology of a gentamicin-producing strain are described . Thermophilic actinomycetes in the genus Thermoactinomyces are common in composts and other self-heating environments . Novel isolates from acid soil, which grow and produce enzymes active at high temperatures and in acidic conditions, are discussed. Biosci Biotechnol Biochem, 1993 Oct, 57(10), 1704 - 7 Purification of protein disulfide isomerase from a thermophilic fungus; Sugiyama H et al.; A protein disulfide isomerase (PDI) was purified to homogeneity from the thermophilic fungus Humicola insolens by a rapid three-step procedure, anion-exchange chromatography, concanavalin A-affinity chromatography, and reverse phase high performance liquid chromatography . Forty-one micrograms of PDI was obtained from 100 g of wet mycelium . Concanavalin A-Sepharose chromatography is available for purification of the fungal PDI, indicating that the enzyme is also glycosylated like the yeast PDI . The fungal PDI exists as a dimer (2 x 60 kDa), has a pI of 3.5, and is fairly heat-stable . The amino acid composition of the PDI is similar to those of yeast and bovine liver PDI, and the high content of acidic amino acid residues agrees with the lower acidic pI. Biosci Biotechnol Biochem, 1993 Oct, 57(10), 1646 - 9 Identification of the replication region of Streptococcus thermophilus No . 29 plasmid pST1; Hashiba H et al.; The replication region of the 2.77-kilobases (kb) plasmid pST1 from Streptococcus thermophilus No . 29 was identified . Deletion derivatives of pST1 were introduced into plasmid-free S . thermophilus No . 29 and examined for their ability to replicate autonomously . The nucleotide sequence had one open reading frame encoded for a 315 amino acid protein (Rep) . Comparisons with proteins encoded by other Gram-positive bacterial plasmids strongly suggest that the deduced protein (RepS) encoded by pST1 has a replicative role . pST1 also contains a DNA sequence similar to the origin nick sequences of pLP1 or pLAB1000, which initiate plasmid replication at the plus origin . In a maxicell system, E . coli CSR603 carrying pSTUC4 produced a protein that was considered to correspond to the product of the RepS gene . These results strongly suggest that pST1 is replicated following a rolling-circle mechanism via single-stranded DNA intermediates. Biochemistry, 1993 Sep 28, 32(38), 9906 - 16 Crystal structure of the catalytic domain of a thermophilic endocellulase; Spezio M et al.; One way to improve the economic feasibility of biomass conversion is to enhance the catalytic efficiency of cellulases through protein engineering . This requires that high-resolution structures of cellulases be available . Here we present the structure of E2cd, the catalytic domain of the thermophilic endocellulase E2 from Thermomonospora fusca, as determined by X-ray crystallography . The structure was solved by multiple isomorphous replacement at 2.6-A resolution and has been refined at 1.8-A resolution to an R-value of 18.4% for all reflections between 10- and 1.8-A resolution . The fold of E2cd is based on an unusual parallel beta-barrel and is equivalent to the fold determined for the catalytic domain of cellobiohydrolase II, an exocellulase from Trichoderma reesei {Rouvinen et al . (1990) Science 249, 380-385} . The active site cleft of the enzyme, approximately 11 A deep and running the entire length of the molecule, is seen to be completely free for ligand binding in the crystal . A 2.2-A resolution analysis of crystals of E2cd complexed with cellobiose, an inhibitor, shows how cellobiose binds in the active site and interacts with several residues which line the cleft . Catalytic roles are suggested for three aspartic acid residues at the active site . A comparison of the E2cd and CBHIIcd structures reveals a large difference in their active site accessibilities and supports the hypothesis that the main difference between endo- and exocellulases is the degree to which their active sites are accessible to substrate. FEBS Lett, 1993 Sep 27, 331(1-2), 81 - 5 Physical map of the extremely thermophilic bacterium Thermus thermophilus HB27 chromosome; Tabata K et al.; The physical map of the chromosome of Thermus thermophilus HB27 was constructed using three restriction enzymes; EcoRI, SspI and MunI by applying pulsed-field gel electrophoresis techniques . Although the genome size of 1.82 Mb was almost the same as that (1.74 Mb) reported for T . thermophilus HB8 {Borges, K.M., and P.L . Bergquist . (1993) J . Bacteriol . 175, 103-110}, the MunI cleavage maps were different . A 240 kb plasmid was detected in HB27, and its physical map was also constructed . In addition, several genes were located on the chromosomal physical map. J Biol Chem, 1993 Sep 25, 268(27), 20408 - 13 Cellular localization of cytochrome c550 . Its specific association with cyanobacterial photosystem II; Shen JR et al.; Cellular localization of cytochrome (cyt) c550, a low potential, c-type monoheme cytochrome, in a thermophilic cyanobacterium Synechococcus vulcanus was investigated by systematic fractionation of the cells followed by its enzymatic and immunological detection . While cyt c-553, a soluble cyt that donates an electron to P700, was detected in the supernatant of osmotic disruption of lysozyme-treated cells, cyt c550 was detected only in the thylakoid membrane fraction, being tightly bound to thylakoids, and its removal required sonication in the presence of 1 M CaCl2 . Upon further fractionation of the thylakoids into photosystem (PS) I and PSII by lauryl dimethylamine N-oxide solubilization, cyt c550 was exclusively concentrated in the crude PSII fraction together with cyt f, with no significant amount being detected in any of the soluble and PSI fractions . Upon further fractionation of the crude PSII by n-dodecyl beta-D-maltoside solubilization followed by column chromatography, cyt c550 was detected exclusively in the purified PSII core complex fraction but not in any other fractions . A 12-kDa protein, one of the extrinsic components of cyanobacterial PSII, exhibited completely the same behavior as that of cyt c550 during these fractionation procedures . These results, coupled with our previous results that cyt c550 binds stoichiometrically to the cyanobacterial PSII core complex and enhances O2 evolution (Shen, J.-R., and Inoue, Y . (1993) Biochemistry 32, 1825-1832), indicate that there is only one species of cyt c550 in cyanobacterial cells and that this cyt is exclusively associated with PSII as a functional component for O2 evolution. Nucleic Acids Res, 1993 Sep 25, 21(19), 4610 - 4 Retroviral-type zinc fingers and glycine-rich repeats in a protein encoded by cnjB, a Tetrahymena gene active during meiosis; Taylor FM et al.; We have determined the nucleotide sequence of the cnjB gene from the ciliate Tetrahymena thermophila . This gene is transcriptionally active only during early conjugation, peaking in meiotic prophase . It contains 13 introns, four transcription start points and codes for a putative polypeptide (CnjB) of 1748 amino acids with a calculated molecular weight of 200 kilodaltons and a pl of 7.9 . The coding region of cnjB has a low GC content (32% GC) and unusual codon usage . The C-terminal one-third of CnjB consists of three repetitive domains . Introns were absent in this region of cnjB . One of the repetitive domains consists of seven CCHC or retroviral-type zinc fingers, a motif found in one or two copies in retroviral nucleocapsid proteins . This motif has also been found recently in seven copies in the human nucleic-acid binding protein CNBP, in an apparent CNBP homologue in Schizosaccharomyces pombe and in one copy in a Xenopus gene active in early embryos . The other two domains are on either side of the zinc finger domain and contain a repeated glycine-rich motif seen in the heterogeneous nuclear ribonuclear proteins A1 and A2/B1 as well as other proteins . Both CCHC zinc fingers and glycine-rich repeats have been found in proteins with single-stranded nucleic acid-binding activity as well as strand-annealing activity . CnjB is, to our knowledge, the first protein found to contain both types of motifs. Eur J Biochem, 1993 Sep 15, 216(3), 709 - 15 The L-lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in Escherichia coli; Ostendorp R et al.; The gene for a L(+)-lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was cloned by complementation of an Escherichia coli pfl . Idh mutant . The gene is part of a 4.5 kb SauIIIA fragment obtained by partial digestion of the Thermotoga genome . The DNA fragment was physically mapped and the putative Shine-Dalgarno sequence within the non-coding region determined . The gene contains 960 bp, including the stop codon, corresponding to 319 amino acids/subunit of the homotetrameric enzyme . Part of the amino acid sequence was confirmed by Edman degradation of peptides obtained from nanomolar quantities of the purified enzyme by tryptic digestion . A comparison of the amino acid sequence with those of known prokaryotic L-lactate dehydrogenases reveals a high similarity, especially with the enzyme from thermophilic sources, where up to 48% identity is found . The gene was expressed as an active enzyme in a heterologous host. Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8357 - 61 Cooperative and anticooperative binding to a ribozyme; Bevilacqua PC et al.; The effects of guanosine 5'-monophosphate and 2'-deoxyguanosine 5'-monophosphate on the thermodynamics and kinetics of pyrene-labeled 5' exon mimic (pyCUCU) binding to the catalytic RNA (ribozyme) from Tetrahymena thermophila have been determined by fluorescence titration and kinetics experiments at 15 degrees C . pyCUCU binding to L-21 Sca I-truncated ribozyme is weaker by a factor of 5 in the presence of saturating guanosine 5'-monophosphate, whereas it is 4-fold stronger in the presence of saturating 2'-deoxyguanosine 5'-monophosphate . Results from kinetics experiments suggest that anticooperative effects in the presence of guanosine 5'-monophosphate arise primarily from slower formation of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex formed by pyCUCU and GGAGGG of the ribozyme . Conversely, cooperative effects in the presence of 2'-deoxyguanosine 5'-monophosphate arise primarily from slower disruption of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex . Additional experiments suggest that these cooperative and anticooperative effects are not a function of the pyrene label, are not caused by a salt effect, and are not specific to one renaturation procedure for the ribozyme. Biochem J, 1993 Sep 15, 294 ( Pt 3), 705 - 9 Modulation of the proton-translocation stoichiometry of H(+)-ATP synthases in two phototrophic prokaryotes by external pH; Krenn BE et al.; The stoichiometry between proton translocation and ATP synthesis/hydrolysis was studied in two different photosynthetic prokaryotes, the thermophilic cyanobacterium Synechococcus 6716 and the purple bacterium Rhodospirillum rubrum . The H+/ATP ratio was determined by acid-base transitions as a function of the external pH . The H+/ATP ratio of the Synechococcus 6716 ATP synthase was found to increase with increasing pH . In contrast, in R . rubrum this ratio decreased with increasing pH . These results were qualitatively supported by experiments using the fluorescence probe 9-aminoacridine . The degree of coupling between the H+ flux and the ATP synthesis/hydrolysis reaction is apparently modulated by the conditions under which the proton pump has to work . Such modulation of the H+/ATP ratio may be of physiological significance for an organism, for example when ATP synthesis is necessary at low proton-electrochemical potential difference (delta mu H+ levels) . The different pH dependencies of the H+/ATP ratios in these organisms are considered in relation to the differences in the charged amino acids that are present in the F0 subunits a and c. Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8362 - 6 Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding; McConnell TS et al.; The L-21 Sca I ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA catalyzes an endonuclease reaction analogous to the first step of self-splicing . Guanosine (G) is bound by the ribozyme, and its 3'-hydroxyl group acts as the nucleophile . Here, we provide evidence that Km for G in several single-turnover reactions is equal to the equilibrium dissociation constant for G . This evidence includes the observation that removal of the 2'-hydroxyl group at the cleavage site of the oligoribonucleotide substrate {from CCCUCUA to CCCUC(dU)A} decreases the rate of cleavage approximately 1000-fold but has no effect on either the Km for G (0.17 mM) or for guanosine 5'-monophosphate (pG) (0.09 mM) . In the course of this study, it was observed that Km for G or pG was lower by a factor of 5 for reactions with the ribozyme-CCCUC(dU)A complex compared with the free ribozyme, indicating a modest amount of thermodynamic coupled binding of the two substrates . The decrease in the rate of oligonucleotide dissociation upon addition of saturating pG provides independent support for this coupling . Coupling is lost with a substrate that cannot make the normal tertiary interactions with the ribozyme, providing evidence that coupled binding requires docking of the substrate into the catalytic core . Surprisingly, the binding of product CCCUCU and G is slightly anticooperative, indicating that the cleaved pA is important for coupling with substrate . Coupled binding suggests a splicing model in which the intron binds G tightly to promote the first step of reaction, after which its binding is an order of magnitude weaker, thereby facilitating the second step. Biochem Biophys Res Commun, 1993 Sep 15, 195(2), 776 - 84 Chromosomal gene integration and enhanced xylanase production in an alkalophilic thermophilic Bacillus sp . (NCIM 59); Shendye A et al.; Chromosomal integration and xylanase gene amplification were demonstrated for the first time in an alkalophilic thermophilic Bacillus sp . (NCIM 59) . The integrants were characterized by larger zone of xylan clearance than the parent culture and hybridization with vector (pUC8) DNA . Repeated transformation strategy was used for further amplification of the xylanase gene . The results of Southern blot analysis indicated the occurrence of homologous recombination in the 6.5 kb xylanase gene region of the genomic DNA and suggested a non campbell mode of recombination . The integrants were checked for xylanase production up to ten subcultures and consistently showed two fold higher xylanase activity than the parent strain with the maximum xylanase productivity (U/ml/h) at 16 h. Biochim Biophys Acta, 1993 Sep 13, 1144(2), 213 - 9 Cytochrome c-551 of the thermophilic bacterium PS3, DNA sequence and analysis of the mature cytochrome; Fujiwara Y et al.; The structural gene for cytochrome c-551 was isolated from genomic DNA of the thermophilic bacterium PS3 . The amino acid sequence of cytochrome c-551 as deduced from the DNA sequence consists of 111 amino acid residues and contains one heme c-binding site (-CASCH-) located approximately in the middle of the polypeptide . The N-terminus of isolated cytochrome c-551 was blocked, but treatment with Rhizopus lipase and molecular weight measurement of the mature and lipase-treated forms by ion spray mass spectroscopy suggest that the mature c-551 may have 93 or 94 amino acid residues with a diacylated glycerol-cysteine at the N-terminal region . The first 17 or 18 amino acid residues in the N-terminal region of the nascent polypeptide, rich in hydrophobic and basic amino acid residues, may be a signal peptide to translocate the major portion of cytochrome c-551 to the extracellular surface and to be processed . Similarity of amino acid sequence of this protein is discussed in relation to other c-type cytochromes of bacilli as well as bacterial small cytochromes c such as Pseudomonas aeruginosa cytochrome c-551 and cytochrome c6 of cyanobacteria. Nucleic Acids Res, 1993 Sep 11, 21(18), 4356 - 62 rseB, a chromosomal locus that affects the stability of a temperature-specific surface protein mRNA in Tetrahymena thermophila; McMillan PJ et al.; In Tetrahymena thermophila, the expression of a temperature-specific surface protein known as SerH3 is primarily controlled by a temperature-dependent change in the stability of the mRNA that encodes this protein . At 30 degrees C the SerH3 mRNA displays a half-life of 60 minutes while at 40 degrees C the half-life decreases to only 3 minutes . We used a Tetrahymena mutant cell line (rseB) defective in expression of SerH3 at 30 degrees C to explore the mechanisms involved in temperature-dependent mRNA stability . The results of in vitro nuclear run-off assays and Northern and slot blot analysis of cytoplasmic and nuclear RNAs show that the rseB locus encodes a temperature-sensitive product that has no effect on SerH3 gene transcription or the steady-state levels of SerH3 nuclear RNA . However, the product of this locus does have a dramatic effect on cytoplasmic levels of the SerH3 mRNA at 30 degrees C, indicating that SerH3 gene expression is affected post-transcriptionally within the cytoplasm . To explore the possibility that the rseB locus controls SerH3 mRNA stability we developed an in vitro mRNA decay assay . This assay successfully duplicates the differential decay of the SerH3 mRNA observed in wild-type cells grown at different temperatures . The apparent half-life of the SerH3 mRNA in cytoplasmic extracts derived from cells grown at 30 degrees C is approximately 45 minutes while in cytoplasmic extracts derived from cells grown at 40 degrees C it is only 6 minutes . When similar experiments are performed using extracts prepared from the Tetrahymena rseB cell line, we find that the SerH3 mRNA is only stable in extract prepared from cells grown under conditions in which the mRNA accumulates to detectable levels in the cytoplasm . These results indicate that the product of the rseB locus is a trans-acting cytoplasmic factor that exerts its effect on SerH3 gene expression by regulating SerH3 mRNA stability. Nature, 1993 Sep 9, 365(6442), 126 - 32 Crystal structure of active elongation factor Tu reveals major domain rearrangements; Berchtold H et al.; The crystal structure of intact elongation factor Tu (EF-Tu) from Thermus thermophilus has been determined and refined at an effective resolution of 1.7 A, with incorporation of data extending to 1.45 A . The effector region, including interaction sites for the ribosome and for transfer RNA, is well defined . Molecular mechanisms are proposed for transduction and amplification of the signal induced by GTP binding as well as for the intrinsic and effector-enhanced GTPase activity of EF-Tu . Comparison of the structure with that of EF-Tu-GDP reveals major mutual rearrangements of the three domains of the molecule. FEBS Lett, 1993 Sep 6, 330(1), 46 - 8 Ribosomal proteins, TL4 and TL5, from Thermus thermophilus form hybrid complexes with 5 S ribosomal RNA from different microorganisms; Gongadze GM et al.; Hybrid complexes of the ribosomal proteins, TL4 and TL5, from Thermus thermophilus with 5 S ribosomal RNA from Escherichia coli and Bacillus stearothermophilus have been prepared . There was no competition between the two proteins for the binding sites . Stoichiometry of 5 S RNA binding for both proteins was 1:1 (protein/RNA) . The TL4 protein competed with the E . coli ribosomal L5 protein, and the TL5 protein competed with the E . coli ribosomal proteins, L18 and L25, for binding with 5 S RNA. J Biol Chem, 1993 Sep 5, 268(25), 19044 - 54 Structure of the alpha subunit of F1-ATPase probed by limited proteolysis; Tozawa K et al.; The structure of the isolated alpha subunit of F1-ATPase from the thermophilic Bacillus strain PS3 was probed using limited proteolysis by four different proteases, and the following results were obtained . 1) Distribution of 21 protease-cleaved sites is similar to that of the beta subunit of F1-ATPase (Tozawa, K., Odaka, M., Date, T., and Yoshida, M . (1992) J . Biol . Chem . 267, 16484-16490), thus providing experimental evidence for similar folding topology of the two subunits, and the locations of 11 water-exposed loop regions in the tertiary structure are predicted . 2) Most proteolytic peptides remain associated to maintain the gross structure of the alpha subunit and can reassociate each other after denaturing urea treatment . 3) However, the carboxyl-terminal peptides comprising approximately 80 residues (C1 peptides) are released from other peptide(s) during proteolysis, and those comprising approximately 105 residues (C2 peptides) are released during native polyacrylamide gel electrophoresis after proteolysis . 4) Inclusion of Mg-ATP in the native electrophoretic system prevents the release of the C2 peptide . Addition of Mg-ATP to the proteolysis mixtures results in an increase of the C2 peptide population and a decrease of the C1 peptide population . Thus, Mg-ATP induces a conformational change at the regions of C1 and C2 peptides of the alpha subunit . 5) Except for the trypsin-treated one, protease-treated alpha subunits are reconstitutable with the native beta subunit into the form of alpha 3 beta 3 complexes, which show significantly higher ATPase activities than the intact alpha 3 beta 3 complex . This activation is attributable to the cleavage of a peptide bond that produces C2 peptides . The carboxyl-terminal region of the alpha subunit is likely to be involved in the regulation of ATPase activity in F1-ATPase. J Eukaryot Microbiol, 1993 Sep-Oct, 40(5), 668 - 76 The dcc mutation affects ciliary length in Tetrahymena thermophila; Gitz DL et al.; We have characterized ciliogenesis in a mutant Tetrahymena thermophila that both fails to regain motility following deciliation and that fails to complete cytokinesis . Scanning electron microscopic (SEM) observations revealed that starved deciliated cells regenerated fewer, shorter cilia at the restrictive temperature than similarly treated cells incubated at the permissive temperature . Transmission electron microscopic evaluation of isolated, regenerated cilia revealed no structural abnormalities . Incorporation of S-35 methionine was similar during ciliary regeneration at both the restrictive and permissive temperatures, indicating the mutant phenotype was not due to a simple failure in translation or transcription . Mutant cells incubated in growth medium at the restrictive temperature arrested in cytokinesis and assembled a large number of abnormally short cilia . These cells also developed irregular surface projections that were not visible on wild-type cells . These observations suggest that ciliogenesis can be initiated in growing cells as well as in starved deciliated cells but that elongation is inhibited before cilia reach full length . The mutation was named dcc for defective in ciliogenesis and cytokinesis. J Eukaryot Microbiol, 1993 Sep-Oct, 40(5), 650 - 60 Biochemical analysis of a mutant Tetrahymena lacking outer dynein arms; Ludmann SA et al.; Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature, and axonemes isolated from nonmotile mutants lack approximately 90% of their outer dynein arms . Electrophoretic analyses of axonemes isolated from nonmotile mutants (oad axonemes) indicate they contain significantly fewer of the 22 S dynein heavy chains that axonemes isolated from wild-type cells (wild-type axonemes) contain . The 22 S dynein heavy chains that remain in axonemes isolated from nonmotile, oad mutants are assembled into 22 S dynein particles that exhibit wild-type levels of ATPase activity . Two-dimensional gel electrophoresis of oad axonemes show that they are deficient in no proteins other than those proteins thought to be components of 22 S dynein . This report is the first formal proof that outer dynein arms in Tetrahymena cilia are composed of 22 S dynein. J Bacteriol, 1993 Sep, 175(18), 5945 - 52 Transcription of the ileS operon in the archaeon Methanobacterium thermoautotrophicum Marburg; Jenal U et al.; In the thermophilic archaeon Methanobacterium thermoautotrophicum Marburg, the structural gene for isoleucyl-tRNA synthetase (ileS) is flanked upstream by orf401 and downstream by purL . orf401 encodes a 43.5-kDa protein with an unknown function . Northern (RNA) hybridization and S1 nuclease protection experiments showed that the orf401, ileS, and purL genes are cotranscribed from an archael consensus promoter in front of orf401 . The corresponding transcript was about eightfold increased in cells that had been exposed to pseudomonic acid A, a specific inhibitor of isoleucyl-tRNA synthetase . Growth inhibition by puromycin, tryptophan starvation, or starvation for hydrogen did not affect the level of this transcript . The level of a trpE transcript, however, was drastically elevated upon tryptophan starvation, while inhibition by pseudomonic acid A had no effect on the level of this transcript . Expression of ileS thus appears to be controlled by a regulatory mechanism which specifically responds to the availability of isoleucyl-tRNA . Extensive decay of the orf401-ileS-purL message was observed . Degradation occurred, presumably by endonucleolytic cleavage, within the orf401 region. Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 7975 - 9 Energy transduction in the thermophilic anaerobic bacterium Clostridium fervidus is exclusively coupled to sodium ions; Speelmans G et al.; The thermophilic, peptidolytic, anaerobic bacterium Clostridium fervidus is unable to generate a pH gradient in the range of 5.5-8.0, which limits growth of the organism to a narrow pH range (6.3-7.7) . A significant membrane potential (delta psi approximately -60 mV) and chemical gradient of Na+ (-Z delta pNa approximately -60 mV) are formed in the presence of metabolizable substrates . Energy-dependent Na+ efflux is inhibited by the Na+/H+ ionophore monensin but is stimulated by uncouplers, suggesting that the Na+ gradient is formed by a primary pumping mechanism rather than by secondary Na+/H+ antiport . This primary sodium pump was found to be an ATPase that has been characterized in inside-out membrane vesicles and in proteoliposomes in which solubilized ATPase was reconstituted . The enzyme is stimulated by Na+, resistant to vanadate, and sensitive to nitrate, which is indicative of an F/V-type Na(+)-ATPase . In the proteoliposomes Na+ accumulation depends on the presence of ATP, is inhibited by the ATPase inhibitor nitrate, and is completely prevented by the ionophore monensin but is stimulated by protonophores and valinomycin . These and previous observations, which indicated that secondary amino acid transport uses solely Na+ as coupling ion, demonstrate that energy transduction at the membrane in C . fervidus is exclusively dependent on a Na+ cycle. J Bacteriol, 1993 Sep, 175(17), 5344 - 9 Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus; Branny P et al.; A fragment of 1,185 bp containing the gene coding for phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase; EC 2.7.1.11) in Lactobacillus bulgaricus has been cloned, sequenced, and expressed in Escherichia coli . The amino acid sequence of this enzyme was homologous to those of the ATP-dependent phosphofructokinases from E . coli, Thermus thermophilus, Spiroplasma citri, and Bacillus stearothermophilus, suggesting that these enzymes have closely related structures despite their different regulatory properties . The recombinant protein had the same structural and functional properties as did the original enzyme . The 3' end of the 1,185-bp fragment showed the presence of an open reading frame corresponding to the N-terminal amino acid sequence of the pyruvate kinase from L . bulgaricus . This gene organization, the same as that in S . citri (C . Chevalier, C . Saillard, and J . M . Bove, J . Bacteriol . 172:2693-2703, 1990) and B . stearothermophilus (D . Walker, W . N . Chia, and H . Muirhead, J . Mol . Biol . 228:265-276, 1992; H . Sakai and T . Ohta, Eur . J . Biochem . 311:851-859, 1993) but different from that in E . coli (H . W . Hellinga and P . R . Evans, Eur . J . Biochem . 149:363-373, 1985), indicated that the same transcription unit apparently contained the genes for phosphofructokinase and pyruvate kinase, the two key enzymes of glycolysis . The possibility that these genes could be transcribed at the same time suggested that in L . bulgaricus, the coordinated regulation of phosphofructokinase and pyruvate kinase occurs at the levels of both biosynthesis and enzymatic activity. J Biochem (Tokyo), 1993 Sep, 114(3), 370 - 7 3-Isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans: DNA sequence, enzyme purification, and characterization; Kawaguchi H et al.; 3-Isopropylmalate dehydrogenase encoded by the Thiobacillus ferrooxidans leuB gene was purified to homogeneity from Escherichia coli cells harboring a recombinant plasmid containing the leuB gene . The native enzyme molecule is a dimer of molecular weight 38,000 . The Km value for 3-isopropylmalate was estimated to be 26 microM and that for NAD+ 0.8 mM . The presence of K+ or NH4+ is essential for the enzyme reaction . The enzyme is activated about 4-fold by the addition of 1.0 mM Mg2+ or Co2+ . The optimum pH and temperature for the activity are 9.0 and 60 degrees C, respectively . The properties of the enzyme are similar to those of the Salmonella typhimurium and Thermus thermophilus enzymes, except for substrate specificity . T . ferrooxidans 3-isopropylmalate dehydrogenase is able to utilize D- and L-malate as substrates in addition to 3-isopropylmalate . Sequencing of subcloned DNA revealed that the leuB gene consists of a 1,074 bp open reading frame and encodes 358 amino acid residues corresponding to the subunit (38,462 Da) . The amino acid sequence of 3-isopropylmalate dehydrogenase from T . ferrooxidans and those of some heterotrophic microorganisms have high homology. Z Naturforsch {C}, 1993 Sep-Oct, 48(9-10), 799 - 802 Complete sequence of one copy of the psbA gene from the thermophilic cyanobacterium Synechococcus elongatus; Kloos R et al.; One copy of the psbA gene which codes for the photosystem II reaction center D-1 protein from the thermophilic cyanobacterium Synechococcus elongatus has been sequenced . It is feasible that a disulfide bridge between D-1 Cys212 and D-2 Cys212 is responsible for the thermostability of the photosystem II reaction center from Synechococcus elongatus. Am J Vet Res, 1993 Sep, 54(9), 1471 - 5 Increase of mannose residues, as Salmonella typhimurium-adhering factor, on the cecal mucosa of germ-free chickens infected with Eimeria tenella; Baba E et al.; To study increase of the Salmonella population in the cecum of chickens infected with Eimeria tenella, quantitative changes in mannose residues on the cecal mucosa were investigated . Inhibition of S typhimurium adherence to the cecum by a 2% carbohydrate (D-mannose, D-galactose, L-fucose, alpha-methyl-D-glucoside) in phosphate-buffered saline solution was examined . Only D-mannose had inhibitory effects . Whereas, D-galactose had somewhat enhancing effects on adherence of S typhimurium to the cecal mucosa of uninfected germ-free chickens . In infected and uninfected chickens, D-mannose inhibited adherence of S typhimurium . D-Mannose significantly (P < 0.05) increased adherence of Bacteroides sp . In infected and uninfected chickens, D-mannose did not have any effect on adherence of Clostridium perfringens and Bifidobacterium thermophilum . Under microscopic observation, only concanavalin A and Lens culinaris agglutinin, of 8 lectins examined, were recognized as lectin-positive staining lines or spots in the cecal mucosa, indicating presence of mannose residues on the cecal mucosa . In E tenella-infected chickens, lectin-positive staining was seen strongly on the coarse surface of damaged cells and at the bottom of the crypts . These results indicate that coccidial infection may induce increase of mannose residues on the intestinal surface and allow adhesion of more salmonellae to the intestine. Appl Environ Microbiol, 1993 Sep, 59(9), 3150 - 3 Molecular cloning and sequence analysis of the crtB gene of Thermus thermophilus HB27, an extreme thermophile producing carotenoid pigments; Hoshino T et al.; We have cloned and sequenced a 1.5-kb chromosomal fragment of Thermus thermophilus which promoted the overproduction of carotenoids in T . thermophilus . An open reading frame (ORF-A) coding for a polypeptide with 289 amino acids was responsible for carotenoid overproduction . The putative ORF-A protein showed significant homology with the amino acid sequences of crtB gene products (phytoene syntheses) of other microorganisms . The clone containing the ORF-A on a multicopy plasmid produced about three times as much carotenoid as that produced by the host strain, suggesting that the crtB gene product is a rate-limiting enzyme for carotenoid biosynthesis in T . thermophilus. J Biol Chem, 1993 Aug 25, 268(24), 17767 - 74 Characterization, cloning, and in vitro expression of the extremely thermostable glutamate dehydrogenase from the hyperthermophilic Archaeon, ES4; DiRuggiero J et al.; Glutamate dehydrogenase (GDH) from the hyperthermophilic Archaeon ES4 (optimal growth temperature 98 degrees C and maximum growth temperature 110 degrees C) was purified to homogeneity . The purified native enzyme had an M(r) of 270,000 +/- 5,000 and was shown by gel filtration and SDS-polyacrylamide gel electrophoresis to be a hexamer with identical subunits of M(r) = 46,000 +/- 3,000 . The hexameric subunit composition was also evident from electron micrographs, which show a triangular antiprism structure very similar to that of bovine GDH . The enzyme is exceptionally thermostable, with a half-time of inactivation of 3.5 h at 105 degrees C . Differential scanning calorimetry revealed a tm for denaturation of 113 degrees C, and a tm for activation at 60 degrees C . Antigenic cross-reaction with ES4 GDH was observed with the purified GDH from the thermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis as well as with bovine and yeast GDHs . The genome of ES4 was shown to contain a single copy of the gdhA gene, and this was cloned and sequenced . The deduced amino acid sequence of the GDH from ES4 corresponded to the NH2-terminal amino acid sequence obtained from the pure protein . From the nucleotide sequence the ES4 protein is composed of 420 residues . It has a relatively high hydrophobicity and a low number of sulfur-containing residues compared with mesophilic GDHs . Relatively high homology (52%) exists between the deduced amino acid sequence of ES4 GDH and Clostridium difficile GDH . Of the two distinct families of GDH sequences known, ES4 GDH belongs to the same family as vertebrates, C . difficile, and other Archaea . The gdhA gene of ES4 was expressed in vitro in a rabbit reticulocyte cell-free lysate, thus providing a system for structural studies of the mechanisms of thermostability in hyper-thermophilic proteins. Nature, 1993 Aug 19, 364(6439), 735 - 7 DNA topoisomerase V is a relative of eukaryotic topoisomerase I from a hyperthermophilic prokaryote; Slesarev AI et al.; The DNA topoisomerases are ubiquitous enzymes that fulfil vital roles in the replication, transcription and recombination of DNA by carrying out DNA-strand passage reactions . Here we characterize a prokaryotic counterpart to the eukaryotic topoisomerase I in the hyperthermophilic methanogen Methanopyrus kandleri . The new enzyme, called topoisomerase V, has the following properties in common with eukaryotic topoisomerase I, which distinguish it from all other known prokaryotic topoisomerases: (1) its activity is Mg(2+)-independent; (2) it relaxes both negatively and positively supercoiled DNA; (3) it makes a covalent complex with the 3' end of the broken DNA strand; and (4) it is recognized by antibody raised against human topoisomerase I . Eukaryotic-like enzymes have been discovered in some hyperthermophilic prokaryotes, namely the eocytes and the extremely thermophilic archaebacteria, and hyperthermophilic homologues of eukaryotic DNA polymerase-alpha, transcription factor IIB and DNA ligase have all been reported . Thus our findings support the idea that some essential parts of the eukaryotic transcription-translation and replication machineries were in place before the emergence of eukaryotes, and that the closest living relatives of eukaryotes may be hyperthermophiles. Biochim Biophys Acta, 1993 Aug 19, 1174(2), 187 - 90 Cloning, sequencing and expression of the gene encoding NADH oxidase from the extreme anaerobic thermophile Thermoanaerobium brockii; Liu XL et al.; The gene encoding the enzyme NADH oxidase from the extreme thermophile Thermoanaerobium brockii has been isolated from a recombinant library of genomic DNA and sequenced . An open reading frame corresponds to the 651 amino acids of the enzyme's subunit, which include characteristic FAD- and NADH-binding sequences, as well as cysteines which are involved in the FeS cluster present in the enzyme . The enzyme is expressed either from its own promoter or from vector promoters in Escherichia coli . After heat-treating the recombinant extracts at 70 degrees C, most of the host proteins are denatured, leaving the NADH oxidase 5- to 10-fold enriched. Biochemistry, 1993 Aug 17, 32(32), 8312 - 21 The importance of being ribose at the cleavage site in the Tetrahymena ribozyme reaction; Herschlag D et al.; The ribozyme derived from the intron of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides . The total transition-state stabilization by the ribozyme, encompassing the binding and chemical steps, is 4.8 kcal/mol greater with a single ribose at the cleavage site relative to the all-deoxyribose substrate . Here we show that this effect is specific to the chemical transition state, with a contribution of only approximately 0.7 kcal/mol toward binding . Substrates with a series of 2'-substituents, -OH(ribo), -F2 (2',2'-difluoro-2'-deoxyribo), F(2'-fluoro-2'-deoxyribo), and -H(deoxyribo), follow a linear free energy relationship between the rate of the chemical step of the ribozyme-catalyzed reaction and the pK(a) of the leaving group, with slope beta leaving group approximately -0.8 . Because proton donation to the 3'-oxygen atom from a general acid of the ribozyme would be expected to render the rate insensitive to the pK(a) of the leaving group, it is suggested that this ribozyme does not employ general acid catalysis . The 2'-OCH3 (2'-methoxy-2'-deoxyribo) substituent does not follow this correlation, apparently due to steric hindrance within the active site . The rate of cleavage of the 2'-substituted substrates by the ribozyme follows the order 2'-F2 > -F > -H, suggestive of an inductive effect, i.e., acceleration of the reaction by electron-withdrawing groups . The 2'-OH group provides the largest transition-state stabilization . Because of uncertainty in the relative effect of the 2'-OH and 2'-H substituents on the pK(a) of the neighboring 3'-oxygen leaving group, we do not discount the possibility of interactions between the 2'-hydroxyl group and the ribozyme that further enhance reactivity . Nevertheless, the 2'-OH effect can be explained at least partially by an intramolecular hydrogen bond to an incipient oxyanion at the neighboring 3'-position . This oxyanion is forming as the phosphodiester bond is breaking, explaining why the stabilization is specific to the transition state . Analogous differential hydrogen bonding might be widely used by enzymes to achieve selective transition-state stabilization. Biochemistry, 1993 Aug 17, 32(32), 8299 - 311 Contributions of 2'-hydroxyl groups of the RNA substrate to binding and catalysis by the Tetrahymena ribozyme . An energetic picture of an active site composed of RNA; Herschlag D et al.; The ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides: CCCUCUAAAAA + G<-->CCCUCU + GAAAAA . However, the RNA substrate (rS) binds approximately 10(4)-fold stronger than the DNA substrate (dS) and once bound reacts approximately 10(4)-fold faster . Here we have investigated the role of individual 2'-hydroxyl groups by comparing the binding and reactivity of "chimeric" oligonucleotide substrates, in which the 2'-substituents of the individual sugar residues have been varied . Chimeric substrates containing a single ribonucleotide at positions -6 to +3 (numbered from the cleavage site) were cleaved faster than dS by factors of 3.5, 3.5, 2.3, 65, 18, 1700, 7.8, 1.7, and 1.4 {(kcat/Km)chimeric S/(kcat/Km)dS} . The sum of the energetic contributions from the individual 2'-hydroxyl groups of 13.3 kcal/mol accounts for the 12.2 kcal/mol greater stabilization for RNA than for DNA in binding and cleavage (i.e., overall transition-state stabilization) . This observation and the significant energetic effects from single ribose substitutions at opositions-3 to +1 strongly suggest that local interactions, rather than overall helical differences, largely account for the different binding and reactivity of the DNA and RNA substrates . Each 2'-hydroxyl group was evaluated for its effect on each of three reaction steps leading to the chemical transition state: two binding steps (duplex formation and docking into tertiary interactions) and the chemical cleavage step . The 2'-hydroxyl groups at positions -3 and -2 stabilize docking, and this stabilization is maintained in the chemical step . This "uniform binding" indicates that these interactions contribute to catalysis by positioning the oligonucleotide substrate for reaction . The 2'-hydroxyl at position +1 has a small effect on the binding step and an additional small but significant effect on the chemical step . Thus, the ribozyme, like protein enzymes, can take advantage of interactions away from the site of chemistry to provide stabilization specifically in the transition state . The 2'-hydroxyl at position -1 exerts its large effect nearly exclusively on the chemical step {Herschlag, D., Eckstein, F., & Cech, T.R . (1993) Biochemistry (following paper in this issue)} . The energetic effects of other modifications of the 2'-substituents provide a crude picture of the active site . The 2'-OCH3 substituent at position -3 inhibits the reaction approximately 10-fold relative to 2'-H, suggesting than an unfavorable interaction cannot be avoided by an isoenergetic structural rearrangement.(ABSTRACT TRUNCATED AT 400 WORDS) Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1359 - 64 Cloning and expression in Escherichia coli of Thermotoga neapolitana genes coding for enzymes of carbohydrate substrate degradation; Dakhova ON et al.; A genomic library of thermophilic anaerobic eubacterium Thermatoga neapolitana was constructed in E . coli using the pTZ19R plasmid vector . Some groups of recombinant clones with different cellulase activities were revealed: clones carrying genes for an 1,4-beta-glucanases, 1,3-beta-glucanases, beta-xylanases, beta-glucosidases and beta-xylosidases . One clone possessing avicelase activity was obtained . Some clones were selected with amylolytic activities toward amylose, amylopectin and pullulan. Arch Biochem Biophys, 1993 Aug 15, 305(1), 186 - 92 Primary structure of Chromatium tepidum high-potential iron-sulfur protein in relation to thermal denaturation; Moulis JM et al.; A high-potential ferredoxin (HiPIP) has been purified from the thermophilic purple sulfur bacterium Chromatium tepidum . Most of the properties of this protein, including absorption and electron paramagnetic resonance spectra as well as redox potential, are identical to those of the similar protein isolated from the mesophilic organism Chromatium vinosum . The similarity extends to the amino acid sequences, which share 74 of the 83 residues composing the primary structure of C . tepidum HiPIP . The latter has been determined by sequencing overlapping peptides and precisely measuring the molecular mass of the holoprotein (9136 Da) by electrospray ionization mass spectrometry . The most significant difference between these sequences involves a stretch of 8 amino acids, which is shortened by two residues and notably changed in C . tepidum HiPIP . This region had been identified in the three-dimensional structure of C . vinosum HiPIP as both a link between two strands of a twisted beta sheet coordinating the {4Fe-4S} cluster and an area of strong interaction of the molecule with the solvent . These data have been used to discuss the molecular basis for the slightly improved thermal stability of C . tepidum HiPIP, as compared to C . vinosum HiPIP . Based on the physiological differences distinguishing C . tepidum from other small-sized Chromatiaceae, the presence of an abundant HiPIP in C . tepidum indicates that involvement as electron acceptor for the previously proposed thiosulfate oxidizing activity in C . vinosum may not be the sole function in all purple sulfur bacteria. Biochem J, 1993 Aug 15, 294 ( Pt 1), 239 - 51 Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716; Van Walraven HS et al.; The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined . The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha, beta, delta and epsilon, by determining their N-terminal sequences . They are arranged at three separate loci . Six of them are in one cluster in the order a: c: b': b: delta: alpha, and those for the beta and epsilon subunits form a second and separate cluster . The gene for the gamma-subunit is at a third site . As in other bacteria, the gene for subunit a is immediately preceded by a gene coding for a small hydrophobic protein of unknown function, known as uncI in Escherichia coli . The gene orders in Synechococcus 6716 are related to the orders of ATP synthase genes in the plastid genomes of higher plants, and particularly of a red alga and a diatom . The sequences of the subunits are similar to those of chloroplast ATP synthase, the alpha, beta and c subunits being particularly well conserved . Differences in the primary structures of the Synechococcus 6716 and chloroplast gamma subunits probably underlie different mechanisms of activation of ATP synthase . The nucleotide sequences that are presented also contain 12 other open reading frames . One of them encodes a protein sequence related to the E . coli DNA repair enzyme, photolyase, and another codes for a protein that contains internal repeats related to sequences in the myosin heavy chain. Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7829 - 33 Crystallization of ribozymes and small RNA motifs by a sparse matrix approach; Doudna JA et al.; The three-dimensional structures of RNA enzymes form catalytic centers that include specific substrate binding sites . High-resolution determination of these and other RNA structures is essential for a detailed understanding of the function of RNA in biological systems . The crystal structures of only a few RNA molecules are currently known . These include tRNAs, which were produced in vivo and contained modified bases, and short oligonucleotide duplexes lacking tertiary interactions . Here we report that a number of different RNA molecules of 4-50 kDa, all synthesized in vitro, have been crystallized . A highly successful method for the growth of RNA crystals based on previously reported conditions for tRNA crystallization is presented . This method is rapid and economical, typically requiring 1.1 mg of RNA to set up an experiment and 2 weeks to complete the observations . Using this technique, we have obtained crystals of 8 of 10 different RNA molecules tested, ranging in size from a dodecamer duplex to a 208-nucleotide catalytic intron . Several of these crystal forms diffract to high resolution; in one case, we have collected a 2.8-A native data set for a 160-nucleotide domain of the group I self-splicing intron from Tetrahymena thermophila . The solution of these RNA structures should reveal aspects of tertiary structure that relate to RNA function and catalytic mechanisms. J Mol Biol, 1993 Aug 5, 232(3), 987 - 8 Crystallization and preliminary X-ray diffraction analysis of crystals of Thermoascus aurantiacus xylanase; Viswamitra MA et al.; Crystals suitable for high resolution X-ray diffraction analysis have been grown of the 29,774-Da protein, xylanase (1,-4-beta-xylan xylanohydrolase EC 3.2.1.8) from the thermophilic fungus Thermoascus aurantiacus . This protein, an endoxylanase demonstrates the hydrolysis of beta-(1-4)-D-xylose linkage in xylans and crystallizes as monoclinic pinacoids in the presence of ammonium sulphate buffered at pH 6.5, and also with neutral polyethylene glycol 6000 . The crystals belong to space group P2(1) and have cell dimensions, a = 41.2 A, b = 67.76 A, c = 51.8 A; beta = 113.2 degrees. Photochem Photobiol, 1993 Aug, 58(2), 238 - 45 8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila . Distribution, repair and effect on rRNA synthesis; Fengquin X et al.; The distribution and repair of 8-methoxypsoralen-DNA interstrand cross-links in the ribosomal RNA genes (rDNA) in Tetrahymena thermophila have been studied in vivo by Southern blot analysis . It is found that the cross-links at a density of < or = 1/2 x 10(4) base pairs (bp) are distributed equally between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI) . It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis . Finally, it is shown that the cross-links in the rDNA molecules are repaired at equal rate in all three domains within 24 h and that RNA synthesis is partly restored during this repair period . The majority of the cells also go through one to two cell divisions in this period but do not survive. Appl Environ Microbiol, 1993 Aug, 59(8), 2546 - 51 Effects of hydrogen and formate on the degradation of propionate and butyrate in thermophilic granules from an upflow anaerobic sludge blanket reactor; Schmidt JE et al.; Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate, propionate, and butyrate as substrates was examined . The propionate and butyrate degradation rates in whole granules were 1.16 and 4.0 mumol/min/g of volatile solids, respectively, and the rates decreased 35 and 25%, respectively, after disintegration of the granules . The effect of adding different hydrogen-oxidizing bacteria (both sulfate reducers and methanogens), some of which used formate in addition to hydrogen, to disintegrated granules was tested . Addition of either Methanobacterium thermoautotrophicum delta H, a hydrogen-utilizing methanogen that does not use formate, or Methanobacterium sp . strain CB12, a hydrogen- and formate-utilizing methanogen, to disintegrated granules increased the degradation rate of both propionate and butyrate . Furthermore, addition of a thermophilic sulfate-reducing bacterium (a Desulfotomaculum sp . isolated in our laboratory) to disintegrated granules improved the degradation of both substrates even more than the addition of methanogens . By monitoring the hydrogen partial pressure in the cultures, a correlation between the hydrogen partial pressure and the degradation rate of propionate and butyrate was observed, showing a decrease in the degradation rate with increased hydrogen partial pressure . No significant differences in the stimulation of the degradation rates were observed when the disintegrated granules were supplied with methanogens that utilized hydrogen only or hydrogen and formate . This indicated that interspecies formate transfer was not important for stimulation of propionate and butyrate degradation. Appl Environ Microbiol, 1993 Aug, 59(8), 2538 - 45 Effect of medium composition and sludge removal on the production, composition, and architecture of thermophilic (55 degrees C) acetate-utilizing granules from an upflow anaerobic sludge blanket reactor; Ahring BK et al.; A thermophilic upflow anaerobic sludge blanket (UASB) reactor degrading acetate was started by applying published methods (W . M . Wiegant and A . W . A . de Man, Biotechnol . Bioeng . 28:718-77, 1986) for production of granules dominated by Methanothrix spp . The reactor was inoculated with thermophilic digested sludge . No granules were observed during the first 7 months of start-up of the UASB reactor . However, after the concentrations of potassium, phosphate, ammonium, and magnesium in the medium were gradually increased, granules developed, indicating that there was a critical concentration of one or more of the ions required for production of granules from the starting material . After several years of stable operation, the effect of removing 60% of the granular sludge was investigated . Immunologic qualitative and quantitative studies showed that removal of the granular sludge resulted in an increase in the number of the predominant methanogens, antigenically related to Methanosarcina thermophila TM-1 and Methanosarcina mazeii S-6, and Methanobacterium thermoautotrophicum delta H and GC1 . These changes were accompanied by modifications of the microanatomy of the granules, as demonstrated histochemically and immunohistochemically . The results indicated that different catabolic pathways dominated in different regions of the granules, i.e., acetate oxidation in the middle of the granules, where there is a low acetate concentration, and an aceticlastic reaction in the outer surfaces, with a high acetate concentration . The results also showed that removal of granules from a UASB reactor which has been under steady-state operation for a long period can improve the reactor's performance via formation of denser and larger granules with improved microbial activities. Mol Cell Biol, 1993 Aug, 13(8), 4814 - 25 Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila; Sweeney R et al.; Tetrahymena thermophila is an ideal organism with which to study functional aspects of the rRNAs in vivo since the somatic rRNA genes of T . thermophila can be totally replaced by cloned copies introduced via microinjection . In this study, we made small insertions into seven sites within the small subunit rRNA gene and observed their phenotypic effects on transformed cells . Two mutated genes coding for rRNA (rDNAs), both of which bear insertions in highly conserved sequences, failed to transform and are therefore believed to produce nonfunctional rRNAs . Three other altered rDNAs produce functional rRNAs that can substitute for most or all of the cellular rRNA . Two of these bear insertions in highly variable regions, and, surprisingly, the other has an insertion in a region that is well conserved for both sequence and secondary structure among eucaryotes . In addition, two other insertions appear to destabilize rRNAs that contain them . Our findings make predictions concerning the positions of some of these sites within the tertiary structure of the small ribosomal subunit and thus serve as an in vivo test of the existing tertiary structure models for the small subunit rRNA . Our results are in good agreement with expectations based on sequence comparison and in vitro work. Biokhimiia, 1993 Aug, 58(9), 1315 - 22 {Two site-specific endonucleases from the thermophilic strain Bacillus species LU11}; Zheleznaia LA et al.; Upon screening of natural strains of thermophilic bacteria, a strain has been found which contains two restriction endonucleases . One of those, BspLU11II, is an isoschizomer of XbaI, while the other one, BspLU11I, recognizes the new palindromic sequence 5'-A decreases CATGT-3' and cleaves it as indicated by the arrow . Functionally pure enzymes were obtained by stepwise chromatography with blueagarose, hydroxyapatite and heparin-Sepharose . The restriction endonuclease BspLU11I produces sticky ends identical to those produced by the restriction endonuclease NcoI; hence a combination of BspLU11I and NcoI can be used for enzymatic selection of recombinant DNA . The recognition sequence of BspLU11I contains the ATG codon and can be used to construct expression vectors for chemically synthesized genes. J Biotechnol, 1993 Aug, 30(2), 245 - 56 Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp; Fujiwara N et al.; Thermostable alkaline protease from an alkaliphilic thermophile Bacillus sp . B18' was purified by using DEAE- and CM-Toyopearl 650M column chromatographies . Molecular weights of the enzyme determined by SDS-PAGE and gel filtration were 30,000 and 28,000, respectively . The optimum pH and temperature toward the hydrolysis of casein were pH 12-13 and 85 degrees C, both of which are higher than those of a mesophilic alkaline protease from an alkaliphile, Bacillus sp . B21-2 . The enzyme was stable at pH 5.0-12.0 and about 60% of the initial enzymatic activity was retained after a 60 min incubation period at pH 10.0 and 70 degrees C . Thermostability of the enzyme was enhanced by Ca2+ . The enzyme activity was inhibited by DFP, suggesting that the enzyme is a serine protease . The NH2-terminal amino acid is Gln, which is that of many subtilisin-type proteases . The 20 residues of the NH2-terminal amino acid sequence have a comparative high homology with those of other alkaline proteases from alkaliphiles (40-50%), especially thermostable alkaline protease from Bacillus sp . No . AH-101 (95%) and Thermoactinomyces sp . HS682 (95%). J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1817 - 28 Dissimilatory sulphite reductase from Archaeoglobus fulgidus: physico-chemical properties of the enzyme and cloning, sequencing and analysis of the reductase genes; Dahl C et al.; A dissimilatory sulphite reductase was isolated fr