Thermophiles

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 from the extremely thermophilic dissimilatory sulphate-reducing archaeon Archaeoglobus fulgidus . In common with other dissimilatory sulphite reductases thus far characterized, the enzyme has an alpha 2 beta 2-structure and contains sirohaem, non-haem iron atoms and acid labile sulphide . The oxidized enzyme exhibited absorption maxima at 281, 394, 545 and 593 nm with a weak band around 715 nm . We have cloned and sequenced the genes for the alpha and beta subunits of this enzyme, which we designate dsrA and dsrB, respectively . They are contiguous in the order dsrA dsrB and probably comprise an operon, since dsrA is preceded by sequences characteristic of promoters in methanogenic archaea, and dsrB is followed by a sequence resembling termination signals in extremely thermophilic sulphur-dependent archaea . dsrA and dsrB encode 47.4 kDa and 41.7 kDa peptides, which have 25.6% amino acid sequence identity, indicating that they may have arisen by duplication of an ancestral gene . Each deduced peptide contains cysteine clusters resembling those postulated to bind sirohaem-{Fe4S4} complexes in sulphite reductases and nitrite reductases from other species . The dsrB encoded peptide lacks a single cysteine residue in one of the two clusters, suggesting that only the alpha subunit binds a sirohaem-{Fe4S4} complex, and chemical analyses showed the presence of only two sirohaems per alpha 2 beta 2 enzyme molecule . Both deduced peptides also contain an arrangement of cysteine residues characteristic of {Fe4S4} ferredoxins, and chemical analyses were consistent with the presence of six {Fe4S4} clusters per alpha 2 beta 2 enzyme molecule, two of which would be expected to be associated with sirohaem while the other four could bind to the ferredoxin-like sites.

J Bacteriol, 1993 Aug, 175(15), 4772 - 9
Phylogenetic analysis of anaerobic thermophilic bacteria: aid for their reclassification; Rainey FA et al.; Small subunit rDNA sequences were determined for 20 species of the genera Acetogenium, Clostridium, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobium, and Thermobacteroides, 3 non-validly described species, and 5 isolates of anaerobic thermophilic bacteria, providing a basis for a phylogenetic analysis of these organisms . Several species contain a version of the molecule significantly longer than that of Escherichia coli because of the presence of inserts . On the basis of normal evolutionary distances, the phylogenetic tree indicates that all bacteria investigated in this study with a maximum growth temperature above 65 degrees C form a supercluster within the subphylum of gram-positive bacteria that also contains Clostridium thermosaccharolyticum and Clostridium thermoaceticum, which have been previously sequenced . This supercluster appears to be equivalent in its phylogenetic depth to the supercluster of mesophilic clostridia and their nonspore-forming relatives . Several phylogenetically and phenotypically coherent clusters that are defined by sets of signature nucleotides emerge within the supercluster of thermophiles . Clostridium thermobutyricum and Clostridium thermopalmarium are members of Clostridium group I . A phylogenetic tree derived from transversion distances demonstrated the artificial clustering of some organisms with high rDNA G+C moles percent, i.e., Clostridium fervidus and the thermophilic, cellulolytic members of the genus Clostridium . The results of this study can be used as an aid for future taxonomic restructuring of anaerobic sporogenous and asporogenous thermophillic, gram-positive bacteria.

J Biol Chem, 1993 Jul 25, 268(21), 15368 - 73
Chemical modification and mutagenesis studies on zinc binding of aminoacyl-tRNA synthetases; Nureki O et al.; Thermus thermophilus methionyl-tRNA synthetase consists of two identical subunits with a potential Zn(2+)-binding sequence of Cys-X2-Cys-X13-Cys-X2-His (Nureki, O., Muramatsu, T., Suzuki, K., Kohda, D., Matsuzawa, H., Ohta, T . Miyazawa, T., and Yokoyama, S . (1991) J . Biol . Chem . 266, 3268-3277) . Upon chemical modification of the 3 Cys residues of T . thermophilus MetRS with sodium p-(hydroxymercuri)phenylsulfonate, one Zn2+ ion was released from one subunit of the molecule, as monitored with 4-(2-pyridylazo)resorcinol . Site-directed mutagenesis of Cys and His residues in the Zn(2+)-binding sequence reduced the aminoacylation activity; the kcat value was markedly decreased, and the Km values for L-methionine and tRNAf(Met) were increased . Similarly, Cys modification released two Zn2+ ions from T . thermophilus and Escherichia coli isoleucyl-tRNA synthetases and E . coli threonyl-tRNA synthetase, which have Zn(2+)-binding motifs, and impaired their activities . By contrast, three other aminoacyl-tRNA synthetases that lack Zn(2+)-binding motif neither released Zn2+ ion nor lost their activities upon Cys modification . These results indicate that the Zn(2+)-binding sequences are important for catalysis and recognition in the aminoacylation reactions of a subgroup of aminoacyl-tRNA synthetases.

Biochim Biophys Acta, 1993 Jul 18, 1174(1), 95 - 8
Nucleotide sequence, transcription and phylogeny of the gene encoding the superoxide dismutase of Sulfolobus acidocaldarius; Klenk HP et al.; The gene encoding the superoxide dismutase (SOD) of the thermophilic archaeon Sulfolobus acidocaldarius has been isolated and sequenced . Both the start site and the termination sites of the corresponding transcript were mapped . The deduced amino acid sequence of the protein is very similar to the sequence of manganese- or iron-containing SODs . Phylogenetic sequence analysis corroborated the monophyletic nature of the archaeal domain.

Eur J Biochem, 1993 Jul 15, 215(2), 473 - 9
Affinity labeling of c-H-ras p21 consensus elements with periodate-oxidized GDP and GTP; Low A et al.; The amino acid sequence motifs of human c-H-ras p21 involved in the interaction with guanosine nucleotides were cross-linked to in situ periodate-oxidized {alpha-32P}GDP or {alpha-32P}GTP . Site-specific reaction was achieved by cross-linking conserved lysine residues close to the G-nucleotide binding site of p21 with the 2',3'-dialdehyde derivatives of GDP or GTP under kinetically controlled conditions . After endoproteinase Asp-N digestion, HPLC separation of 32P-labeled peptides and N-terminal microsequence analysis, two single lysine residues, namely, K117 and K147, which are parts of the N-K-X-D and S-A-K/L consensus elements of ras proteins, respectively, were identified . No significant divergences in the position and extent of covalent modification could be detected between p21.GDP and p21.GTP . This is in contrast to Thermus thermophilus EF-Tu.GDP and EF-Tu.GTP, which were investigated with the same technique {Peter, M . E., Wittmann-Liebold, B . & Sprinzl, M . (1988) Biochemistry 27, 9132-9139} and which exhibited considerable differences in cross-linking efficiency in the GTP form as compared to the GDP form of the protein . The described affinity labeling technique of cross-linking {alpha-32P}GTP with GTP-binding proteins can be used as a general analytical method for the detection and identification of consensus elements in GTPases from different organisms.

Biochim Biophys Acta, 1993 Jul 10, 1164(2), 179 - 88
S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus: purification, physico-chemical and immunological properties; Porcelli M et al.; S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity . The specific activity of the homogeneous enzyme is 161 nmol of S-adenosylhomocysteine formed per min per mg of protein, and the overall yield, by immunoaffinity purification, is 51% . The enzyme has a molecular mass of 190 kDa, is composed of four identical subunits (subunit mass 47 kDa), and contains four molecules of tightly-bound NAD+ per tetramer of which about 40% is in the reduced form . Physico-chemical features, including amino-acid composition and secondary structure, are reported . The pure protein, used to raise specific rabbit antisera, shows immunological properties different from other S-adenosylhomocysteine-metabolizing enzymes . The enzyme is thermophilic with an optimum temperature of 75 degrees C, and shows an apparent melting temperature of 95 degrees C by measuring its residual activity after 10 min incubation at increasing temperatures.

J Mol Biol, 1993 Jul 5, 232(1), 312 - 3
Purification, crystallization and preliminary X-ray analysis of inorganic pyrophosphatase from Thermus thermophilus; Obmolova G et al.; High yields of inorganic pyrophosphatase from Thermus thermophilus HB8 have been purified to homogeneity using anion exchange and hydrophobic chromatography . Crystals suitable for X-ray analysis were obtained by vapour diffusion using ammonium sulphate as precipitant . They belong to the rhombohedral space group R32, with unit cell dimensions a = b = 110.3 A and c = 82.0 A, with one subunit per asymmetric unit . The crystals diffract to 2.0 A resolution.

J Mol Biol, 1993 Jul 5, 232(1), 308 - 9
Crystallization and preliminary crystallographic study of citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum; Russell RJ et al.; Single crystals of citrate synthase from the Archaeon Thermoplasma acidophilum were obtained in two forms using the hanging drop vapour diffusion method and polyethylene glycol 3350 as precipitant . Type 1 crystals belong to the orthorhombic space group P222(1), with unit cell dimensions a = 80.9 A, b = 103.8 A, c = 98.3 A and one dimer in the asymmetric unit . Type 2 crystals belong to the monoclinic space group P2(1), with unit cell dimensions a = 53.8 A, b = 173.8 A, c = 86.7 A and beta = 97.1 degrees and two dimers in the asymmetric unit.

FEBS Lett, 1993 Jul 5, 325(3), 183 - 6
Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus . Implications for the structure of prokaryotic aspartyl-tRNA synthetases; Poterszman A et al.; The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strain VK-1 and HB8, have been cloned and sequenced . Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases . This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids) . A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones . When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E . coli . It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography . Single crystals of the pure protein, diffracting at least to 2.2 A resolution (space group P2(1)2(1)2(1), a = 61.4 A, b = 156.1 A, c = 177.3 A) are routinely obtained . The same crystals have previously been described as crystals of threonyl-tRNA synthetase {1}.

Mol Microbiol, 1993 Jul, 9(1), 65 - 75
S-layer protein from Thermus thermophilus HB8 assembles into porin-like structures; Caston JR et al.; The cells of the extreme thermophile Thermus thermophilus are surrounded by a regular layer (S-layer) built up by a protein with an apparent molecular mass of 100 kDa (P100) . From purified membrane fractions, three different class of two-dimensional crystals can be obtained by following alternative extractive procedures . One of these crystals, with p6 symmetry, clearly represents the native S-layer detected by freeze etching on whole cells, while the other two, showing p2 and p3 symmetries respectively, closely resemble aggregates of bacterial porins . We demonstrate here by limited proteolysis and Western blotting the surprising fact that the protein component of the three crystals is the P100 protein . Our biochemical data also show how this protein forms Ca(2+)-stabilized trimers in each crystal, which support the structural analysis that points to p3 units as the common structural block in all of them, and again resembles the situation found in bacterial porins.

Mol Gen Genet, 1993 Jul, 240(1), 81 - 91
Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains; Nolling J et al.; The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFV1 and pFZ1 from M . thermoformicicum strains THF and Z-245, respectively . Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains . Multiple copies of the pFV1-specific element FR-I were detected in the M . thermoformicicum strains CSM3, FF1, FF3 and M . thermoautotrophicum delta H . Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and delta H . Comparison of the FR-I elements from these strains with that from pFV1 revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable . Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT . These results suggest that FR-I represents a mobile element . FR-II was located on both plasmids pFV1 and pFZ1, and on the chromosome of M . thermoformicicum strains THF, CSM3 and HN4 . Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5-3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.

Plasmid, 1993 Jul, 30(1), 67 - 78
Shuttle vectors developed from Streptococcus thermophilus native plasmid; Solaiman DK et al.; pER8 (2.2 kb), a native plasmid of Streptococcus thermophilus ST108, was used to develop pMEU-series shuttle vectors . In addition to the replication function of the pER8, these vectors contain origin of replication and beta-lactamase gene (bla) of Escherichia coli vector pUC18/19, the cat gene of pC194, and the pPV141-borne erm determinant of Staphylococcus hyicus ssp . chromogenes 3688 . pMEU5a, pMEU5b, pMEU6a, and pMEU6b (all 5.7 kb in size) contain bla and erm markers, are capable of transforming E . coli and S . thermophilus at frequencies in the order of 10(5)-10(6) and 10(3) colony forming units (CFU)/microgram DNA, respectively, and are highly stable in the two host systems . pMEU9 and pMEU10 (both 6.9 kb) contain the cat marker in addition to the DNA elements found in pMEU5-6 . These plasmids are also highly effective in transforming E . coli (at ca . 6 x 10(5) CFU/microgram DNA) and S . thermophilus (ca . 10(3) CFU/microgram DNA) . Although expression of the resistance markers is not completely consistent, pMEU9 and pMEU10 remain important shuttle vectors for clonal selection by an insertional inactivation method.

Int J Biochem, 1993 Jul, 25(7), 1029 - 33
Effects of Mg2+ and Ca2+ on Fe2+ uptake by Bifidobacterium thermophilum; Kot E et al.; Ferrous iron uptake was investigated in Bifidobacterium thermophilum (B . thermophilum) in the presence of Mg2+ and Ca2+ with the following findings: 1 . Mg2+ inhibited Fe2+ accumulation in the cells in a dose-dependent manner at 37 degrees, but not at 0 degrees . Removal of Mg2+ from the medium resulted in a resumption of rapid iron uptake . 2 . Mg2+ had no effect on the binding of Fe2+ by B . thermophilum protoplasts, its cellular particulate fraction, or distribution between the particulate and soluble fractions . 3 . Ca2+ exerted a stimulatory effect on iron uptake by B . thermophilum, but was not able to reverse the inhibitory effects of Mg2+ . 4 . It was concluded that Mg2+ has no effect on the binding of iron on the surface or interior of B . thermophilum and that it affected the Fe2+ transport mechanism (permease) in a reversible manner . It is possible that iron and magnesium share the same permease in this microorganism.

FEMS Microbiol Rev, 1993 Jul, 11(1-3), 19 - 30
Amplification of ribulose biphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) gene fragments from Thiobacillus ferrooxidans and a moderate thermophile using polymerase chain reaction; Holden PJ et al.; Southern blot analysis of DNA from an iron-oxidising moderate thermophile NMW-6 and from Thiobacillus ferrooxidans strain TFI-35 demonstrated sequences homologous to the RuBisCO LSU gene of Synechococcus . DNA fragments (457 bp) encoding part of the RuBisCO LSU gene (amino acids 73-200) were amplified from the genomic DNA of Thiobacillus ferrooxidans and the moderate thermophile NMW-6 using the polymerase chain reaction (PCR) technique (Saiki et al . (1985) Science 233, 1350-1354) . A comparison with the LSU sequences from T . ferrooxidans, Alcaligenes eutrophus, Chromatium vinosum, Synechococcus and Spinacea oleracea, which all have RuBisCOs with a hexadecameric structure, showed that the RuBisCO LSU gene sequence from NMW-6 appeared to be most closely related to that of the hydrogen bacterium A . eutrophus which showed 71.9% homology at the amino acid level . Despite its physiological similarity, T . ferrooxidans showed only 64.1% homology to the amino acid sequence from NMW-6 and had the lowest DNA homology (60.9%) of the hexadecameric type RuBisCOs . In the region sequenced, T . ferrooxidans and the RuBisCOs of the phototrophs C . vinosum, Synechococcus and S . oleracea, had 17 residues that were completely conserved which were substituted in both NMW-6 and A . eutrophus, 11 of these being identical substitutions . Comparison of the nucleotide and derived amino acid sequences of the RuBisCO LSU fragment from T . ferrooxidans with other RuBisCO sequences indicated a closer relationship to the hexadecameric type LSU genes of photosynthetic origin than to that of A . eutrophus . The T . ferrooxidans amino acid sequence showed 93.8%, 78.9% and 77.3% homology, respectively, to the C . vinosum, Synechococcus and S . oleracea (spinach) sequences but only 56.2% to A . eutrophus . The DNA sequence from Rhodospirillum rubrum, which has the atypical large subunit dimer RuBisCO structure with no small subunit, showed 39.2% and 42.7% homology, respectively, with the sequences of NMW-6 and T . ferrooxidans, and 25.0% and 29.7% amino acid homology, indicating that the DNA homology was substantially random in nature . PCR fragments (126 bp) that overlaped the last 15 codons of the fragments above were also amplified and sequenced . They showed incomplete homology with the larger fragments, supporting evidence obtained from Southern hybridizations that T . ferrooxidans and the moderate thermophile NMW-6 have multiple copies of RuBisCO LSU genes.

J Bacteriol, 1993 Jul, 175(14), 4315 - 24
Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus; Mollet B et al.; Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes . Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration . Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid . Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures . By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion . In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon . The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly . Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene . As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose . The methodology represents a stable system in which to express and regulate foreign genes in S . thermophilus, which could qualify in the future for an application with food.

J Eukaryot Microbiol, 1993 Jul-Aug, 40(4), 515 - 20
Processing and secretion of lysosomal acid alpha-glucosidase in Tetrahymena wild type and secretion-deficient mutant cells; Banno Y et al.; The proteolytic processing and secretion of a lysosomal enzyme, acid alpha-glucosidase, was studied by pulse-chase labeling with {35S}methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride . This cell secreted a large amount of acid alpha-glucosidase into the cultured medium during starvation . The secretion was found to be repressed by addition of ammonium chloride (NH4Cl) . Acid alpha-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min . This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH4Cl-treated cells . NH4Cl did not affect the processing of the precursor acid alpha-glucosidase . Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion . Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid alpha-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties . They contained no mannose 6-phosphate residues in N-linked oligosaccharides . These results suggested that unlike mammalian cells, Tetrahymena acid alpha-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.

Mikrobiologiia, 1993 Jul-Aug, 62(4), 761 - 72
{Methane fermentation of cattle manure at low temperature}; Kotsiurbenko OR et al.; The acclimated microbial association fermenting cattle manure at 6 degrees C with methane production was obtained by a long-time (2.5 years) autoselection . It is shown that the basic barrier to carry out this process at low temperature is long-time period of adaptation of microbial community . It is removed by addition of the psychrophilic barm obtained . After cultivating of this psychrophilic methanogenic association under mesophilic (35 degrees) and thermophilic (55 degrees) conditions its activity decreased to 2 and 10 times, respectively . Based on the kinetic estimations it was proposed, that the essential changes of properties of the anaerobic microflora investigated was occurred during the acclimation to low temperature . The psychrotrophic microorganisms played the main role in the process of organic matter degradation at low temperature in the studied system . The pure cultures of saccharolitic and acetogenic psychrotrophic bacteria those are capable to grow in the wide range of temperature, from 0 degrees to 35 degrees C were isolated . Methanosarcina and methanothrix ix were detected in the enrichment cultures growing with acetate at 6 degrees C.

Biol Trace Elem Res, 1993 Jul, 38(1), 1 - 12
Iron uptake by Bifidobacterium thermophilum protoplasts; Kot E et al.; Protoplasts of Bifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out . Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface . This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process . It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium . Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies . The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only . Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact . The amount of iron so bound was dose-dependent on the amount of iron entering the cell . The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO4 . It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.

J Mol Biol, 1993 Jun 20, 231(4), 960 - 81
Studies of the hyperthermophile Thermotoga maritima by random sequencing of cDNA and genomic libraries . Identification and sequencing of the trpEG (D) operon; Kim CW et al.; Random sequencing of cDNA and genomic libraries has been used to study the genome of the hyperthermophile Thermotoga maritima . To date, 175 unique clones have been analyzed by comparing short sequence tags with known proteins in the PIR and GenBank databases . We find that a significant proportion of sequences can be matched to previously identified protein from non-Thermotoga sources . A high match rate was obtained from an oligo(dT)-primed cDNA library, where one-third of all unique sequences analyzed (21/65) shared high amino acid sequence similarity with proteins in the PIR and GenBank databases . Also, approximately one-third of the unique sequences from a second cDNA library (28/89), constructed with random oligo primers, could be matched to sequences in PIR and GenBank . Identification of genes from the oligo(dT)-primed cDNA library indicates that some Thermotoga mRNAs are polyadenylated . Genes have also been identified from a 1 to 2 kb genomic DNA library . Here, (3/21) of genomic sequences analyzed could be matched to protein in PIR and GenBank . One of the genomic clones had high sequence similarity to the tryptophan synthesis gene anthranilate synthase component I (trpE) . Using this sequence tag, the Thermotoga trp operon was isolated and sequenced . The Thermotoga maritima trp operon is arranged with trpE forming an overlapping transcript with a second protein consisting of a fusion of anthranilate synthase component II (trpG) and anthranilate phosphoribosyltransferse (trpD) . With regard to the fusion, the operon organization is similar to Escherichia coli and Salmonella typhimurium, but lacks the classic attenuation system of enteric bacteria . Amino acid sequence comparison with 19 trpE, 18 trpG and 14 trpD genes from other organisms suggest that the Thermotoga trp genes resemble corresponding genes from other thermophiles more closely than expected.

Eur J Biochem, 1993 Jun 15, 214(3), 917 - 25
Energy-transducing properties of primary proton pumps reconstituted into archaeal bipolar lipid vesicles; Elferink MG et al.; Archaeal lipids differ considerably from eubacterial and eukaryotic lipids in their structure and physical properties . From the membranes of the extreme thermophilic archaea Sulfolobus acidocaldarius a tetraether lipid fraction was isolated, which can form closed and stable monolayer liposomes in aqueous media . The function of three different primary proton pumps originating from archaeal, bacterial and eukaryotic lipid sources have been studied after reconstitution in these liposomes: bacteriorhodopsin from the archaea Halobacterium halobium; cytochrome-c oxidase from the thermophilic bacterium Bacillus stearothermophilus and cytochrome-c oxidase from beef heart mitochondria . Liposomes composed of tetraether lipids form a competent matrix for all three exogenous proton pumps . Bacteriorhodopsin was inserted inside-out in these liposomes, as normally observed in bilayer-forming lipid . The activities of the two oxidases were inhibited at high tetraether-lipid concentration, probably due to the low fluidity of these membranes . Only bacteriorhodopsin, which originates from diether archaeal lipids is fully functional in the tetraether membranes.

Nucleic Acids Res, 1993 Jun 11, 21(11), 2641 - 7
Identification of the gene encoding the mitochondrial elongation factor G in mammals; Barker C et al.; Protein synthesis in cytosolic and rough endoplasmic reticulum associated ribosomes is directed by factors, many of which have been well characterized . Although these factors have been the subject of intense study, most of the corresponding factors regulating protein synthesis in the mitochondrial ribosomes remain unknown . In this report we present the cloning and initial characterization of the gene encoding the rat mitochondrial elongation factor-G (rEF-Gmt) . The rat gene encoding EF-Gmt (rMef-g) maps to rat chromosome 2 and it is expressed in all tissues with highest levels in liver, thymus and brain . Its DNA sequence predicts a 752 amino acid protein exhibiting 72% homology to the yeast Saccharomyces cerevisiae mitochondrial elongation factor-G (YMEF-G), 62% and 61% homology to the Thermus thermophilus and E . coli elongation factor-G (EF-G) respectively and 52% homology to the rat elongation factor-2 (EF-2) . The deduced amino acid sequence of EF-G contains characteristic motifs shared by all GTP binding proteins . Therefore, similarly to other elongation factors, the enzymatic function of EF-Gmt is predicted to depend on GTP binding and hydrolysis . EF-Gmt differs from its cytoplasmic homolog, EF-2, in that it contains an aspartic acid residue at amino acid position 621 which corresponds to the EF-2 histidine residue at position 715 . Since this histidine residue, following posttranslational modification into diphthamide, appears to be the sole cellular target of diphtheria toxin and Pseudomonas aeruginosa endotoxin A, we conclude that EF-Gmt will not be inactivated by these toxins . The severe effects of these toxins on protein elongation in tissues expressing EF-Gmt suggest that EF-Gmt and EF-2 exhibit nonoverlapping functions . The cloning and characterization of the mammalian mitochondrial elongation factor G will permit us to address its role in the regulation of normal mitochondrial function and in disease states attributed to mitochondrial dysfunction.

J Mol Biol, 1993 Jun 5, 231(3), 927 - 9
Crystals of the phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 complexed with tRNA(Phe); Reshetnikova L et al.; Phenylalanyl-tRNA synthetase (EC 6.1.1.20) from the extreme thermophile Thermus thermophilus HB8 has been crystallized with its cognate tRNA . Compared with the native crystals, the crystals of the complex are more stable to radiation damage and diffract to 3.0 A resolution . They are of space group P3(2)21, with a = b = 175 A, c = 142.1 A, gamma = 120 degrees, almost identical with the crystal parameters of the native synthetase.

Epidemiol Infect, 1993 Jun, 110(3), 633 - 9
Arcobacter cryaerophilus and thermophilic campylobacters in a sewage treatment plant in Italy: two secondary treatments compared; Stampi S et al.; Microaerophilic organisms were monitored in sewage effluent undergoing two secondary treatments: air and oxygen-activated sludge . The mean numbers of Arcobacter cryaerophilus and thermophilic campylobacters detected in incoming sewage were 5639/100 ml and 1720/100 ml respectively . Secondary treatment in air tanks reduced the population of A . cryaerophilus by 97.1% and of thermophilic campylobacters by 99.08%, whereas treatment in oxygen tanks reduced the bacteria 97.8% and 99.63% respectively, showing that oxygen-activated sludge treatment was more effective . Subsequent tertiary treatment with 2 p.p.m . chlorine dioxide evidenced the removal of A . cryaerophilus to 99.9% and eliminated thermophilic campylobacters . Campylobacter jejuni and C . coli constituted 54.1% and 45.9% of 74 thermophilic campylobacter strains isolated . In air-activated sludge effluent C . jejuni was found more often, thus appearing more sensitive to oxygen . The most probable number assay used for detection of campylobacters, blood medium for enrichment and blood-free medium for plating, also appeared to be fit for A . cryaerophilus, the high density of which in urban sewage may be due to inflows from slaughterhouses.

J Bacteriol, 1993 Jun, 175(11), 3661 - 3
Structures of the modified folates in the extremely thermophilic archaebacterium Thermococcus litoralis; White RH; The chemical structures of the two modified folates present in Thermococcus litoralis were established . These compounds, each containing a core structure of 1-{4-{{1-(2-amino-7-methyl- 4-oxo-6-pteridinyl)-ethyl}amino}phenyl}-1-deoxy-{1-alpha-D- ribofuranosyl}-ribitol, were characterized . The five position of the ribose in this core structure was beta-linked to the C-1 of a poly-beta (1-->4)N-acetylglucosamine having a chain length of four or five N-acetylglucosamine residues . Thus, these compounds are N-acetylglucosamine homologs of the modified folates found in Pyrococcus furiosus.

Zentralbl Mikrobiol, 1993 Jun, 148(4), 253 - 64
Mycoflora and natural occurrence of mycotoxins in tobacco from cigarettes in Egypt; el-Maghraby OM et al.; Forty-two species and 4 varieties belonging to 21 genera were collected from 40 tobacco samples on glucose- and cellulose-Czapek's agar at 28 degrees C and 45 degrees C . The most common mesophiles (at 28 degrees C) in tobacco on the two types of media were: Aspergillus flavus, A . flavus var . columnaris, A . fumigatus, A . niger, Penicillium chrysogenum and P . funiculosum . Two samples were heavily contaminated with members of Fusarium (F . moniliforme, F . oxysporum, F . solani) . Some fungi were encountered only on plates of cellulose agar as Chaetomium globosum, Stachybotrys atra var . microspora and S . chartarum . At 45 degrees C the most prevalent fungus was A . fumigatus . Truely thermophiles were also collected: Humicola grisea var . thermoidae, Rhizomucor pusillus and Thermoascus aurantiacus . Based on biological assays (brine shrimp larvae (Artemia salina L.) and Bacillus megatherium test) and chemical analysis of chloroform extraction of tobacco (TLC and UV spectrophotometric), four samples (out of 40) had toxicity and four compounds of mycotoxins were detected namely; aflatoxins B1 & B2 (2 samples; 15.5 and 20.7 micrograms/kg), zearalenone (1 sample, 5.5 micrograms) and T-2 toxin (1 sample, 2.8 micrograms) . For studying the tracing of aflatoxins in smoking cigarettes, three doses (10, 20 and 50 micrograms) of aflatoxins B1 and B2 (w/w, 1:1) were injected each in ten cigarettes . All extracts of cigarettes smoke proved to be non-toxic and mycotoxins not detected . However, aflatoxins were detected in topping filter (2.8, 3.5 and 8.8 micrograms/the three doses, respectively).

J Biomol Struct Dyn, 1993 Jun, 10(6), 945 - 72
Modeling study on the cleavage step of the self-splicing reaction in group I introns; Setlik RF et al.; A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step . Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E . J . Mol . Biol . 216, 585-610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9 . Next, an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space . A lowering of P1 helix relative to surrounding helices provided the additional space required . Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al . (Pyle, A . M., Murphy, F . L., Cech, T . R . Nature 358, 123-128 . (1992)), based on mutational studies involving the J8/7 segment . Two hydrated Mg2+ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A . EMBO J . 10, 25-33 (1991)) as a guide . The presence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg2+ in catalysis . The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg2+ complexes in catalysis is further explored using molecular orbital calculations.

Biol Chem Hoppe Seyler, 1993 Jun, 374(6), 377 - 83
Isolation and characterization of a new ribosomal protein from the thermophilic eubacteria, Thermus thermophilus, T . aquaticus and T . flavus; Choli T et al.; A ribosomal protein, showing no homology with other known prokaryotic ribosomal proteins, was isolated and characterized from the thermophilic eubacteria, Thermus thermophilus, T . aquaticus and T . flavus . This small (26 amino acids) and strongly basic (1 acidic and 13 basic residues) protein displayed the same primary structure from all three sources . Interestingly, it shows about 65% homology with a ribosomal protein from spinach chloroplasts (J . Schmidt, personal communication).

Res Microbiol, 1993 Jun, 144(5), 381 - 7
Biotyping of Streptococcus thermophilus strains by DNA fingerprinting; Salzano G et al.; DNA of 33 strains of Streptococcus thermophilus (7 isolated from natural whey cultures utilized as starter, 25 from commercial yogurts and 1 reference strain) was analysed by restriction endonuclease digestion and both conventional and pulsed-field agarose gel electrophoresis . The restriction patterns after digestion of total DNA with HindIII, EcoRI and BamHI enabled 10 different groups to be distinguished by conventional electrophoresis . Digesting genomic DNA with SmaI or NotI and separating it by pulsed-field gel electrophoresis, the 33 strains could be classified into 5 groups . The same result was obtained after digestion with HaeIII and conventional electrophoresis . The 33 isolates could be divided into 13 groups when diversities among patterns obtained after both the first and second method were considered . A combination of the two techniques turned out to be the most reliable methodological approach for characterizing strains of S . thermophilus for scientific, industrial and legal purposes.

Zentralbl Veterinarmed B, 1993 Jun, 40(4), 245 - 52
NaCl-tolerance of Campylobacter isolates from birds and Campylobacter type strains and variation of their serological behaviour; Glunder G; Growth on media containing 1.5% NaCl is one of the criteria for phenotypical differentiation of Campylobacter laridis from other thermophilic Campylobacter spp . Campylobacter isolates from birds and Campylobacter type strains could be adapted to growth at 3% NaCl within 19 to 72 subsequent passages on nutrient agar with increasing salt contents . The acquisition of salt-tolerance was stable after ten passages on media without salt and did not induce changes in other phenotypical characteristics . The results of slide agglutination demonstrate changes in the antigenic pattern of the Campylobacter strains after growth in salt . Heat-labile and heat-stable antigens of the salt-tolerant variants of Campylobacter type strains differed from those of the parent strains.

Appl Microbiol Biotechnol, 1993 Jun, 39(3), 341 - 6
Cloning, nucleotide sequence, and overexpression in Escherichia coli of the beta-tyrosinase gene from an obligately symbiotic thermophile, Symbiobacterium thermophilum; Hirahara T et al.; Symbiobacterium thermophilum is an obligately symbiotic thermophile that can grow only in coculture with a specific Bacillus strain . The amino acid sequences of fragments obtained by cyanogen bromide decomposition of the thermostable beta-tyrosinase (tyrosine phenol-lyase, E.C . 4.1.99.2) from this organism resembled that of the tryptophanase produced by the same organism . DNA-probing with the tryptophanase gene as the hybridization probe led to cloning in Escherichia coli of the beta-tyrosinase (tpl) gene . The nucleotide sequence revealed that the beta-tyrosinase of 458 amino acids (relative molecular mass, 52269) showed significant similarity in amino acid sequence to the tryptophanase over the entire sequence . DNA manipulation of the cloned tpl gene in E . coli led to production of 375 times as much beta-tyrosinase as that produced by the original S . thermophilum strain.

Nucleic Acids Res, 1993 May 25, 21(10), 2409 - 14
A micronucleus-specific sequence exists in the 5'-upstream region of calmodulin gene in Tetrahymena thermophila; Katoh M et al.; Tetrahymena thermophila possesses a transcriptionally inactive micronucleus and an active macronucleus . Both nuclei are developed from micronucleus-derived germ nuclei during conjugation . Extensive DNA rearrangement and transcriptional activation are known to be involved in macronuclear development, but little has been known about these processes in a particular functional gene . Therefore the micro- and macronuclear genomic DNAs for calmodulin gene were analyzed . A 1,384 bp micronucleus-specific sequence located about 3.5 kb upstream of calmodulin gene has been found, suggesting DNA rearrangement during macronuclear development . The micronucleus-specific sequence had 85% A + T, no extensive ORF, ATTAs at both ends, and two palindromic structures just outside of both ends . Interestingly, the micronucleus-specific sequence included a T-rich tract, T16CT5, in the middle, and a nearly complementary A-rich tract, A5TA10GA5, existed 7 bp upstream from the initiation codon . In addition, there was a 20 bp repetitive sequence TAAT(TAAC)4 about 100 bp upstream of the micronucleus-specific sequence and also in the promoter region of calmodulin gene . Although the functional significance of the micronucleus-specific sequence remains unclear, T16CT5 and TAAT(TAAC)4 elements might exert an influence on transcription of the calmodulin gene . Stringent Southern hybridization revealed that this micronucleus-specific sequence or very similar sequence(s) were abundant in the Tetrahymena micronuclear genome.

J Biol Chem, 1993 May 25, 268(15), 10813 - 9
A novel cloning strategy reveals the gene for the yeast homologue to Escherichia coli ribosomal protein S12; Alksne LE et al.; Using a novel technique designed to identify genes of Saccharomyces cerevisiae which carry introns, we have cloned two genes encoding ribosomal protein S28 . Although the genes differ by 15 nucleotides within their coding regions, they are predicted to encode identical proteins of 145 amino acids . The predicted amino acid sequence of S28 contains significant homology to ribosomal protein S25 of Tetrahymena thermophila and to ribosomal protein S12 of several archaebacteria, suggesting a relationship to S12 of Escherichia coli . Dot matrix analysis confirmed that regions of S12, especially those implicated in the accuracy of translation, have been conserved in S28 of S . cerevisiae . Either RPS28A or RPS28B alone can support growth, but heterozygous disruption of both genes abolishes the ability to sporulate . Haploids harboring a disruption of both genes cannot survive without an intact gene on a plasmid . RPS28A maps to the right arm of chromosome VII and RPS28B to the right arm of chromosome XVI.

Biochemistry, 1993 May 25, 32(20), 5291 - 300
An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme; Murphy FL et al.; The Tetrahymena thermophila pre-rRNA contains a 413-nucleotide self-splicing group I intron . This intron has been converted into a sequence-specific endonuclease or ribozyme . A 160-nucleotide portion of the ribozyme consisting of both highly conserved sequence elements (P4 and P6) and nonconserved peripheral extensions (P5abc and P6ab) was synthesized as a separate molecule . Solvent-based Fe(II)-EDTA, a probe that monitors higher-order RNA structure, revealed a protection pattern that was a large subset of that observed in the whole ribozyme . Data from dimethyl sulfate modification and partial digestion with nucleases were also consistent with maintenance of the proper secondary and tertiary structure in the shortened RNA molecule . Thus, this 160-nucleotide molecule (P4-P6 RNA) is an independently folding domain of RNA tertiary structure . A series of mutations and deletions were made within the P4-P6 domain to further dissect its tertiary structure . Fe(II)-EDTA and dimethyl sulfate analysis of these mutants revealed that the domain consists of two substructures, a localized subdomain involving the characteristic adenosine-rich bulge in P5a, and a subdomain-stabilized structure involving long-range interactions . Therefore, like some proteins, the intron RNA is modular, containing a separable domain and subdomain of tertiary structure.

Biochemistry, 1993 May 18, 32(19), 5247 - 56
Binding of guanosine and 3' splice site analogues to a group I ribozyme: interactions with functional groups of guanosine and with additional nucleotides; Moran S et al.; Dissociation constants, Kd, were measured by equilibrium dialysis at 5 degrees C for a series of substrates binding to the L-21 ScaI ribozyme derived from the Tetrahymena thermophila self-splicing large subunit (LSU) ribosomal RNA intron . These substrates are analogues for the 3' exon splice site, the cyclization site, and the exogenous G that initiates group I splicing . UCG has a Kd of 17 microM . Lengthening the substrate to GUCG and GGUCG enhances binding but by less than expected from potential base pairing . Functional groups on the 3'-terminal G of GUCG were replaced with H to test their effect on binding . GUC(2'dG) binds slightly tighter than the all-ribose molecule but shows no reactivity as a substrate . GUC(3'dG) binds weaker than GUCG . Inosine and 2-aminopurine ribonucleoside at the 3' position weaken binding by 16- and 26-fold, respectively, but both tetramers are reactive . Thus hydrogen bonds to Watson-Crick pairing positions of the 3'G of GUCG contribute 1-2 kcal/mol to the free energy change for binding . Similar results are found in comparisons of UCG with UC(2'dG), UC(3'dG), and UCI . The nonreactive substrate GUCdGA includes a phosphodiester bond 3' to the guanosine that is the site of chemistry for the all-ribose substrate GUCGA; GUCdGA binds 50 times more weakly than GUCdG . A similar result is obtained for GUCdGU . Competition experiments show that guanosine and guanosine 5'-monophosphate bind with dissociation constants of about 0.9 mM . The monomers 2'dG and 3'dG have Kd's of 0.5 and > or = 3 mM, respectively . This suggests that sugar pucker and/or interactions with hydroxyl groups affect binding . Implications for ribozyme catalysis, splicing, cyclization, and design of antisense oligomers are discussed.

Eur J Biochem, 1993 May 15, 214(1), 37 - 42
Dye-linked L-malate dehydrogenase from thermophilic Bacillus species DSM 465 . Purification and characterization; Ohshima T et al.; The distribution of dye-linked L-malate dehydrogenase (L-malate: acceptor oxidoreductase, EC 1.1.99.16) was investigated in many thermophilic bacteria . The enzyme occurred widely in thermophilic spore-forming bacteria like bacilli and thermoactinomycetes . The enzyme was purified to homogeneity from a thermophile, Bacillus sp . DSM 465, with a 2.7% overall recovery by DEAE-Toyopearl column chromatography, Sephacryl S-400 column chromatography and preparative slab PAGE . The enzyme had a molecular mass of about 660 kDa and consisted of about ten subunits all with a molecular mass of 66 kDa . The enzyme retained its full activity upon heating at 55 degrees C for at least 60 min and with incubation at pH 5.0-10.0, 55 degrees C, for 10 min . The enzyme exclusively catalyzed L-malate dehydrogenation in the presence of an electron acceptor such as 2,6-dichloroindophenol . The Michaelis constants for L-malate and 2,6-dichloroindophenol were determined to be 1.67 mM and 0.050 mM, respectively . FAD was identified as a prosthetic group of the enzyme by HPLC.

Gene, 1993 May 15, 127(1), 71 - 8
Genes encoding eleven subunits of photosystem I from the thermophilic cyanobacterium Synechococcus sp; Muhlenhoff U et al.; We have isolated the genes encoding 11 photosystem I (PSI) subunits from Synechococcus sp., from which this reaction center has been crystallized . The recombinant DNAs, including psaA, psaB, psaC, psaD, psaE, psaF, psaI, psaJ, psaK and psaL, were obtained by heterologous hybridization with probes from appropriate cDNAs or genes from spinach and Synechocystis sp . PCC 6803, or with synthetic oligodeoxyribonucleotides . Genes psaA/psaB, psaF/psaJ and psaL/psaI are each closely linked . The open reading frames predict polypeptides of 83 kDa (subunits Ia and Ib, encoded by genes psaA and psaB, respectively), 15.4 kDa (II, psaD), 17.7 kDa (III, psaF), 8.4 kDa (IV, psaE), 8.8 kDa (VII, psaC), 4.6 kDa (VIII, psaI), 4.8 kDa (IX, psaJ), 8.5 kDa (X, psaK) and 15.5 kDa (XI, psaL) . A novel subunit (XII, psaM) was also identified . Subunits II, III, IV and VII seem to be peripheral, while the others seem to be intrinsic components of the reaction center . These data imply a striking similarity of cyanobacterial and eukaryotic PSI . All subunits studied are encoded by single-copy genes which seem to be transcribed into monocistronic (psaC, psaD, psaC, psaK) or dicistronic (psaA/psaB, psaF/psaJ, psaL/psaI) RNA species . Subunit III is translated as a 17.7-kDa precursor, including a transit peptide of 23 amino acid residues . This is consistent with its location in the thylakoid lumen.

Gene, 1993 May 15, 127(1), 133 - 7
Sequence of the Streptomyces thermoviolaceus CUB74 alpha-amylase-encoding gene and its transcription analysis in Streptomyces lividans; Bahri SM et al.; The alpha-amylase (Amy)-encoding gene (amy) of Streptomyces thermoviolaceus CUB74, previously cloned in Escherichia coli and S . lividans and localised on a 1.7-kb BamHI-SphI genomic DNA fragment, has been sequenced . A single open reading frame of 1380 bp, which could encode an Amy protein of 460 amino acids (aa), was identified . The deduced aa sequence of the thermophilic Amy is similar (up to 69.5%) to the mesophilic Amy of S . griseus, S . limosus, S . venezuelae and S . hygroscopicus . A 40% sequence similarity was found between the extracellular forms of the S . thermoviolaceus and the pig pancreatic Amy . In addition, the activity of the S . thermoviolaceus Amy is strongly inhibited by tendamistat, a potent inhibitor of mammalian Amy . The nucleotide sequence at the 5' end of amy was able to initiate transcription in S . lividans and contains a promoter whose sequence is identical to the promoters of the S . limosus, S . venezuelae and S . griseus amy.

Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4753 - 7
Reverse gyrase: a helicase-like domain and a type I topoisomerase in the same polypeptide; Confalonieri F et al.; Reverse gyrase is a type I DNA topoisomerase able to positively supercoil DNA and is found in thermophilic archaebacteria and eubacteria . The gene coding for this protein was cloned from Sulfolobus acidocaldarius DSM 639 . Analysis of the 1247-amino acid sequence and comparison of it with available sequence data suggest that reverse gyrase is constituted of two distinct domains: (i) a C-terminal domain of approximately 630 amino acids clearly related to eubacterial topoisomerase I (Escherichia coli topA and topB gene products) and to Saccharomyces cerevisiae top3; (ii) an N-terminal domain without any similarity to other known topoisomerases but containing several helicase motifs, including an ATP-binding site . These results are consistent with those from our previous mechanistic studies of reverse gyrase and suggest a model in which positive supercoiling is driven by the concerted action of helicase and topoisomerase in the same polypeptide: this constitutes an example of a composite gene formed by a helicase domain and a topoisomerase domain.

Biochemistry, 1993 May 11, 32(18), 4912 - 24
The dimanganese(III,IV) oxidation state of catalase from Thermus thermophilus: electron nuclear double resonance analysis of water and protein ligands in the active site; Khangulov S et al.; The 1H hyperfine tensors of the dimanganese(III,IV) oxidation state of the non-heme-type catalase enzyme from the thermophilic bacterium Thermus thermophilus have been measured by electron nuclear double resonance (ENDOR) spectroscopy at pH 6.5-9 . These were compared to model dimanganese(III,IV) complexes possessing six-coordinate N4O2, N3O3, and O6 atom donor sets to each Mn and mu-oxo and mu-carboxylato bridging ligands . The lack of 14N hyperfine couplings in the enzyme suggests either O6 or O5N ligand donors to each Mn . Moreover, the two sigma coordination sites on Mn(III) directed at the dz2 orbital cannot be occupied by N ligands . The 1H ENDOR spectrum revealed two types of anisotropic tensors, attributable to two D2O-exchangeable protons on the basis of the magnitude of the electron paramagnetic resonance (EPR) line narrowing in D2O . All six of the 1H hyperfine couplings are proposed to arise from a single displaceable water molecule in the active site, on the basis of their reversible disappearance, upon incubation in D2O or by precipitation from ammonium sulfate, and by simulation of the 1H ENDOR spectrum . The Mn ions are coordinated predominantly by nonmagnetic O atoms lacking covalently bound protons in both alpha and beta positions . This implicates predominantly carboxylato-type ligands (Asp and Glu) and possibly a di-mu-oxo bridge between Mn ions . The latter is supported also by the presence of strong antiferromagnetic coupling . Comparison to other dimetalloproteins also possessing the four-helix bundle structural motif shows that the polyoxo(carboxylato) coordination in catalase differs significantly from the polyhistidine coordination adopted by the diiron(II,II) site in the O2-binding protein myohemerythrin, but resembles the polycarboxylato ligation adopted by the diiron(III,III) site of ribonucleotide reductase . The catalase 1H ENDOR spectrum is essentially identical to that for the exchangeable protons in the active site of the diiron(II,III) state of uteroferrin, an acid phosphatase {Doi et al . (1988) J . Biol . Chem . 263, 5757-5763}, and also for a polycarboxylato complex possessing the Mn2(mu-O)2 core with H-bonded water ligands . The 1H ENDOR line shape in catalase could be simulated using a theoretical model suitable for multispin clusters . It treats the two Mn spins as point dipoles which are exchange-coupled . It includes both dipolar and isotropic ligand hyperfine couplings . Using this model, the position of the proton with the largest interaction could be located with respect to the Mn-Mn vector because of the extreme sensitivity of line shape to position.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1993 May 4, 32(17), 4475 - 80
Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena; Christian EL et al.; We have used nucleoside phosphorothioates (NTP alpha S) and a substitution-interference method to identify phosphate oxygens that appear to be important to guanosine cofactor addition in the self-splicing group I intron from Tetrahymena thermophila . For the majority of these phosphate oxygens, however, the effect of NTP alpha S substitution is significantly reduced in reactions containing the added presence of manganese ion (Mn2+) relative to magnesium ion (Mg2+) alone . The observed "rescue" of the NTP alpha S effect at these positions is thought to be due to the larger affinity of Mn2+ for sulfur . These data suggest the direct coordination of divalent metal ions within the highly conserved catalytic core of the Tetrahymena intron . Because many of these metal binding sites appear to be in positions of close backbone-backbone approach, and adjacent to the guanosine binding site the splice junction, we suggest roles for the corresponding ions in stabilizing tertiary structure and substrate recognition and as participants in catalysis.

Clin Infect Dis, 1993 May, 16(5), 640 - 5
Infection due to Rhizomucor pusillus: report of four cases in patients with leukemia and review; St-Germain G et al.; Rhizomucor pusillus, a thermophilic fungus of the order Mucorales, is a rare cause of human infection . A search of the literature has produced only seven reports describing nine cases of infection caused by this organism . Recently, over a period of 17 months, four cases of R . pusillus infection in patients with leukemia were diagnosed: a cluster of three cases in a Montreal hospital and one isolated case from Quebec City . All four cases were proven both by histopathologic examination and by culture of tissues . In three cases, pulmonary involvement was confirmed following lung surgery, and in one case, disseminated infection was observed at autopsy . All patients received amphotericin B, and two underwent surgical debridement; however, none of the patients survived.

Protein Sci, 1993 May, 2(5), 814 - 25
Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction; Wagner UG et al.; The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model . This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J . Mol . Biol . 206, 787-788) . Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters . The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T . thermophilus . In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme . As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T . thermophilus, and the dimers interdigitate . The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T . thermophilus MnSOD.

Bioorg Khim, 1993 May, 19(5), 583 - 5
{New type II and IIs restriction endonucleases from thermophilic bacteria of the genus Bacillus}; Repin VE et al.; Restriction endonucleases have been isolated from 26 strains of thermophilic strains of the Bacillus genus, their recognition sequences were determined, and for 15 of them cleavage sites identified . The enzymes proved to be isoschizomers of known endonucleases BstNI, EarI, HaeIII, HpaII, Cfr10I, BsiYI, BclI, BbvII, BbvI, BstEII, BsaBI, BsrI, FspI, ClaI, SfeI.

Mol Gen Mikrobiol Virusol, 1993 May-Jun, (3), 22 - 5
{Isolation and properties of BstBSI restriction endonuclease from the thermophilic soil bacteria Bacillus stearothermophilus BS}; Kovalevskaia NP et al.; A new restriction endonuclease BstBSI was isolated and purified from the thermophilic soil bacterium Bacillus stearothermophilus BS by the blue sepharose and hydroxyapatite chromatographies . The enzyme is an isoschizomer of SnaI from Sphaerotilus natans C . It recognizes the hexanucleotide GTATAC and cleaves DNA in the center of the sequence . The maximal catalytic activity of the endonuclease is registered in 50 mM tris-HCl (pH 9.0) buffer with the high ionic strength (100 mM NaCl) in the presence of 10 mM MgCl2 at 45 degrees C . Glucosylated DNA of the phage T4 is not cleaved by the enzyme.

Appl Microbiol Biotechnol, 1993 May, 39(2), 216 - 20
Restriction/modification in Streptococcus thermophilus: isolation and characterization of a type II restriction endonuclease Sth455I; Guimont C et al.; Streptococcus thermophilus strain CNRZ 455 produces a type II restriction endonuclease designated Sth455I . This enzyme was isolated from cell extracts by anionic and cationic exchange chromatography . This yielded an enzyme preparation free of non-specific nucleases . The optimal reaction conditions for Sth455I are: MgCl2, 30 mM; pH range, 8-9; incubation temperature, 37-42 degrees C; and a high NaCl concentration, 100-200 mM . The results of single- and double-digestion experiments indicates that Sth455I is an isoschizomer of BstNI and EcoRII showing different sensitivity to methylation . The enzyme exhibits restriction activity on the DNA of three bacteriophages of S . thermophilus and no activity on the phage lytic for strain CNRZ 455 . The restriction/modification system associated with this strain is discussed.

Biochim Biophys Acta, 1993 Apr 29, 1173(1), 75 - 8
Primary structure of Tetrahymena hemoglobins; Takagi T et al.; The primary structure of hemoglobins of Tetrahymena thermophila and T . pyriformis was determined by peptide and cDNA sequence analysis . Their sequences were 83.5% identical to each other and homologous to other protozoan and cyanobacteria hemoglobins, but not to proteins of the globin family . An intron was not present in the T . thermophila hemoglobin gene.

J Mol Biol, 1993 Apr 20, 230(4), 1309 - 10
Crystals of protein L30 from the 50 S ribosomal subunit of Thermus thermophilus . Preliminary crystallographic data; Shikaeva OS et al.; Crystals have been obtained of protein L30 from the large ribosomal subunit of an extreme thermophile, Thermus thermophilus, using ammonium sulphate as a precipitant . The crystals belong to space group P3(1)12 with cell parameters a = b = 64.2 A, c = 78.3 A . They diffract X-rays to at least 2.3 A resolution.

Biochemistry, 1993 Apr 20, 32(15), 3913 - 22
Determinants of protein thermostability observed in the 1.9-A crystal structure of malate dehydrogenase from the thermophilic bacterium Thermus flavus; Kelly CA et al.; A binary complex of malate dehydrogenase from the thermophilic bacterium Thermus flavus (tMDH) with NADH has been crystallized from poly(ethylene glycol) 3500, pH 8.5, yielding diffraction-quality crystals in space group P2(1)2(1)2(1) . The structure was solved at 1.9-A resolution using molecular replacement and refined to an R factor of 15.8% with good geometry . The primary sequence of tMDH is 55% identical to that of cytoplasmic malate dehydrogenase (cMDH) {Birktoft, J . J., Rhodes, G., & Banaszak, L . J . (1989) Biochemistry 28, 6065-6081}, and overall their three-dimensional structures are very similar . Like cMDH, tMDH crystallized as a dimer with one coenzyme bound per subunit . The coenzyme binds in the extended conformation, and most of the interactions with enzyme are similar to those in cMDH . In tMDH, small local conformational changes are caused by the replacement of a glutamic acid for the aspartic acid involved in hydrogen bonding to the adenine ribose of NADH . Comparison of tMDH with cMDH reveals that both tMDH subunits more closely resemble the B subunit of cMDH which therefore is the more likely representative of the solution conformation . While cMDH is inactivated at temperatures above about 50 degrees C, tMDH is fully active at 90 degrees C . On the basis of the X-ray crystal structure, a number of factors have been identified which are likely to contribute to the relative thermostability of tMDH compared to cMDH . The most striking of the differences involves the introduction of four ion pairs per monomer . All of these ion pairs are solvent-accessible . Three of these ion pairs are located in the dimer interface, Glu27-Lys31, Glu57-Lys168, and Glu57-Arg229, and one ion pair, Glu275-Arg149, is at the domain interface within each subunit . Additionally, we observe incorporation of additional alanines into alpha-helices of tMDH and, in one instance, incorporation of an aspartate that functions as a counterchange to an alpha-helix dipole . The possible contributions of these and other factors to protein thermostability in tMDH are discussed.

Biochemistry, 1993 Apr 20, 32(15), 3981 - 90
Purification, cloning, and cofactor independence of glutamate racemase from Lactobacillus; Gallo KA et al.; Glutamate racemase has been purified more than 12,000-fold from Lactobacillus fermenti . The racemase gene has been cloned using standard hybridization techniques combined with a novel selection for in vivo glutamate racemase activity, and the racemase has been expressed in Escherichia coli as 20-25% of the total soluble cell protein . The cloned gene product is indistinguishable from that purified from Lactobacillus and is a monomer of M(r) 28,300 . Both a coupled enzymatic assay and a circular dichroism assay show that the enzyme follows Michaelis-Menten kinetics, with a Km of 0.3 mM and a kcat of 70 s-1 in each reaction direction . Investigations into the cofactor dependence of glutamate racemase indicate that the enzyme employs neither pyridoxal phosphate nor a pyruvoyl group in the labilization of the proton at the stereogenic center of glutamate . Furthermore, the racemase activity is unaffected by the presence of the metal-chelating reagent EDTA . The gene sequence of the racemase is 24% identical to that of aspartate racemase from Streptococcus thermophilus and 30% identical to that of an unidentified open reading frame in the rrnB ribosomal RNA operon of E . coli . Because the two cysteine residues in glutamate racemase and their surrounding regions are well-conserved in both of these sequences, and since glutamate racemase is stabilized by the presence of reduced thiols, these residues are possible candidates for the enzymic bases that deprotonate glutamate at C-2.

FEBS Lett, 1993 Apr 19, 321(1), 46 - 50
AT(D)PMg-induced dissociation of the alpha 3 beta 3 complex of the F1-ATPase from a thermophilic Bacillus PS3 into alpha 1 beta 1 heterodimers is prevented by mutation beta (Y341C); Kaibara C et al.; AT(D)PMg induces dissociation of the alpha 3 beta 3 complex of F1-ATPase from a thermophilic Bacillus strain . PS3, into the alpha 1 beta 1 heterodimers {(1991) Biochim . Biophys . Acta 1056, 279-284} but the location of the AT(D)PMg binding site responsible is not known . From the analysis of AT(D)PMg binding properties of the isolated mutant beta subunit, beta(Y341C), and the stability of the alpha 3 beta(Y341C)3 complex in the presence of AT(D)PMg, we conclude that binding of AT(D)PMg to the Tyr-341 site of the beta subunit(s) in the alpha 3 beta 3 complex triggers the dissociation of the alpha 3 beta 3 complex into the alpha 1 beta 1 heterodimers.

FEMS Microbiol Lett, 1993 Apr 15, 108(3), 297 - 302
Cloning and extracellular expression in Escherichia coli of xylanases from an alkaliphilic thermophilic Bacillus sp . NCIM 59; Shendye A et al.; A genomic DNA library of an alkaliphilic thermophilic Bacillus was constructed in Escherichia coli with pUC 8 vector and was screened using a Congo red xylan plate clearance assay . Six xylanase positive transformants having identical inserts showed immunological reactivity towards polyclonal antibodies raised against purified xylanase (M(r) 15,800) from the Bacillus . A 4.5-kb HindIII-EcoRI subfragment was found to code for two xylanases of M(r) 14,500 and 35,000 . Equivalent amounts of xylanase activity were detected from IPTG induced and noninduced recombinants irrespective of the orientation of the 4.5-kb insert with respect to the lac promoter, indicating that xylanase gene expression was under the control of its own promoter . 95% of the xylanase activity (2 U/ml) was found in the extracellular culture filtrate . The hydrolysis of xylan by the recombinant xylanases yielded mainly xylobiose.

J Mol Biol, 1993 Apr 5, 230(3), 1086 - 8
Crystallization and preliminary X-ray diffraction studies of a NADH oxidase from Thermus thermophilus HB8; Erdmann H et al.; The thermophile NADH oxidase from Thermus thermophilus, cloned and expressed in Escherichia coli, has been purified to homogeneity and crystallized . Three different crystal forms were found to be suitable for X-ray diffraction analysis . Crystals of the tetragonal form, grown in the presence of 25% polyethylene glycol 4000 and 0.25 M-NaCl at pH 6.6, were chosen for further analysis . These crystals belong to the space group P4(1)(3)2(1)2 with refined lattice constants of a = 94.8 A and c = 49.0 A, indicating a cell content of one monomer per asymmetric unit of the crystal . The crystals diffract to a resolution of 2.2 A.

Biochem Mol Biol Int, 1993 Apr, 29(5), 867 - 73
Purification of aspartate aminotransferase from Thermus aquaticus; Walker JM et al.; A method is described for the purification of the enzyme aspartate aminotransferase from the thermophile Thermus aquaticus . The enzyme has been characterized with respect to its molecular weight on SDS PAGE and by amino acid analysis . Attempts to obtain N-terminal sequence data was unsuccessful, presumably due to a blocked N-terminus . The purified enzyme has been shown to be highly thermostable, having a half life of inactivation of about 6 hours at 100 degrees C, and to have a temperature optimum greater than 95 degrees C.

J Appl Bacteriol, 1993 Apr, 74(4), 460 - 6
Isolation and characterization of a Bacillus strain capable of degrading the extracellular glucan from Cellulomonas flavigena strain KU; Bertram PA et al.; Bacteria capable of utilizing the water-insoluble purified extracellular (1-->3)-beta-D-glucan (curdlan) from Cellulomonas flavigena strain KU by extracellular enzymes, were isolated and characterized . Enrichment cultures from a Winogradsky column were incubated anaerobically at 55 degrees C with curdlan as the sole source of carbon . Colonies surrounded by zones of clearing were selected from subcultures on solid curdlan media . One of the isolates was chosen for further study and identified by conventional methods, API-tests with calculation of similarity coefficients and ID-scores, estimation of mol% (G+C) and DNA-DNA liquid hybridization . The isolate is a facultatively anaerobic, facultatively thermophilic Bacillus sp . Identification at the species-level was not achieved . The isolate was characterized by some rare traits among bacilli, but it remains unresolved whether it defines a new taxon.

Biotechnol Appl Biochem, 1993 Apr, 17 ( Pt 2), 239 - 50
Exo-glucosidase activity and substrate specificity of the beta-glycosidase isolated from the extreme thermophile Sulfolobus solfataricus; Nucci R et al.; The enzyme with beta-galactosidase activity from Sulfolobus solfataricus strain MT-4, like other enzymes of this type isolated from thermophilic sources, has broad specificity for beta-D-gluco-, fuco- and galacto-sides . The beta-galactosidase activity was purified by a new procedure that improved yields (44%) and final specific activity (182 units mg-1 at 75 degrees C using chromogenic beta-D-galactoside as substrate) . The enzyme hydrolysed a large number of beta-linked glycoside dimers and oligomers; chromogenic beta-glucosides and beta-fucosides are the preferred substrates, and kinetic analysis indicated that they bind to a common catalytic site . The order of catalytic efficiency was beta 1-3 > beta 1-4 > beta 1-6 and cellotetraose > cellotriose > cellobiose for glucose dimers and oliogomers respectively . The cleavage occurred at the non-reducing end of the oligosaccharide, and the enzyme showed noticeable specificity also for the aglycone part of substrates . From these results the enzyme from S . solfataricus strain MT-4 is defined as a true glycosyl hydrolase with remarkable exo-glucosidase activity and it is designated S beta-gly.

Exp Cell Res, 1993 Apr, 205(2), 286 - 92
Three pools of lysosomal enzymes in Tetrahymena thermophila; Kiy T et al.; Secretion of lysosomal enzymes into the extracellular surroundings has been observed in many eukaryotic cells . We studied the activity of lysosomal enzymes in different subcellular fractions of Tetrahymena thermophila to get more insight into this general phenomenon . By density gradient centrifugation a light and a dense fraction of lysosomal particles were found . Electron microscopy revealed that the light fraction mainly consists of cell surface membranes . By immunostaining a lysosomal enzyme (beta-hexosaminidase) was detected on the plasma membrane . The Triton X-114 assay showed that the light fraction as well as purified cilia (an enriched source of plasma membrane) contain lysosomal enzymes predominantly covalently bound to the membrane . The dense fraction contains both membrane-bound and soluble forms of lysosomal enzymes . By labeling phagosomes/phagolysosomes with magnetic particles the dense fraction can be subdivided into two lysosomal vesicle populations: phagolysosomes and a further population of lysosomal vesicles which can not be labeled . The relationship between membrane-bound and soluble enzyme forms in phagolysosomes and this unlabeled vesicle population is different: In phagolysosomes 80% of the acid phosphatase and 20% of the beta-hexosaminidase are membrane-bound, whereas in the unlabeled vesicles 42% of the acid phosphatase and 8% of the beta-hexosaminidase are bound to the membrane . Furthermore, we present results suggesting that the unlabeled vesicle population of the dense fraction is the source of secreted lysosomal enzymes . A working model summarizing our present knowledge about the connection of the three pools of lysosomal enzymes in Tetrahymena is presented.

Appl Environ Microbiol, 1993 Apr, 59(4), 1168 - 75
Some characteristics of a proteinase from a thermophilic Bacillus sp . expressed in Escherichia coli: comparison with the native enzyme and its processing in E . coli and in vitro; Peek K et al.; Proteinase Ak.1 was produced during the stationary phase of Bacillus sp . Ak.1 cultures . It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The enzyme was stable at 60 and 70 degrees C, with half-lives of 13 h and 19 min at 80 and 90 degrees C, respectively . Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro-Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide) . Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln-4-His-5, and Glu-13-Ala-14 . The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75 degrees C . The enzyme is expressed in E . coli as an inactive proproteinase at 37 degrees C and is converted to the mature enzyme by heating the cell-free media to 60 degrees C or above . The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa . The NH2-terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point . Heating the proenzyme resulted in the production of active proteinase with an NH2-terminal sequence identical to that of the native enzyme . The characteristics of the cloned proteinase were identical to those of the native enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1993 Apr, 59(4), 1003 - 11
Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures; Van Lier JB et al.; The effects of acetate, propionate, and butyrate on the anaerobic thermophilic conversion of propionate by methanogenic sludge and by enriched propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum delta H were studied . The methanogenic sludge was cultivated in an upflow anaerobic sludge bed (UASB) reactor fed with propionate (35 mM) as the sole substrate for a period of 80 days . Propionate degradation was shown to be severely inhibited by the addition of 50 mM acetate to the influent of the UASB reactor . The inhibitory effect remained even when the acetate concentration in the effluent was below the level of detection . Recovery of propionate oxidation occurred only when acetate was omitted from the influent medium . Propionate degradation by the methanogenic sludge in the UASB reactor was not affected by the addition of an equimolar concentration (35 mM) of butyrate to the influent . However, butyrate had a strong inhibitory effect on the growth of the propionate-oxidizing enrichment culture . In that case, the conversion of propionate was almost completely inhibited at a butyrate concentration of 10 mM . However, addition of a butyrate-oxidizing enrichment culture abolished the inhibitory effect, and propionate oxidation was even stimulated . All experiments were conducted at pH 7.0 to 7.7 . The thermophilic syntrophic culture showed a sensitivity to acetate and propionate similar to that of mesophilic cultures described in the literature . Additions of butyrate or acetate to the propionate medium had no effect on the hydrogen partial pressure in the biogas of an UASB reactor, nor was the hydrogen partial pressure in propionate-degrading cultures affected by the two acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiol Infect, 1993 Apr, 110(2), 273 - 8
The role of management systems in the epidemiology of thermophilic campylobacters among poultry in eastern zone of Tanzania; Kazwala RR et al.; A total of 255 samples of droppings collected from a total of 22 different poultry units were examined for the presence of thermophilic campylobacters and the isolates biotyped using Skirrow's protocol . The organisms were isolated from 90 (35.3%) of all samples . Among the 22 units investigated, 13 (59%) were found to have unsatisfactory management systems, while 7 (32%) and 2 (9%) were found to have unsatisfactory and good systems respectively . Significantly large numbers of isolations, 68 of 147 (46.2%), were made from samples collected from poultry units with poor management (P < 0.005), compared with 19 out of 84 (22.6%) samples which were collected from satisfactory units and 3 out of 24 (12.5%) samples collected from units exercising particularly good management . Nineteen of 72 (26.4%) samples collected from broilers, 32 out of 132 (24.2%) samples collected from layers and 39 out of 51 (76.49%) samples collected from indigenous free range poultry were positive for campylobacters . Among the 90 strains isolated from various units, 64 (70.1%) were Campylobacter jejuni, 25 (27.7%) were C . coli, and only 1 (2.2%) was C . laridis.

Int J Biochem, 1993 Apr, 25(4), 609 - 17
Purification and properties of a beta-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli; Schofield LR et al.; 1 . An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile "Caldocellum saccharolyticum" strain Tp8T6.3.3.1 . It was found to have an M(r) of 42,000 and an isoelectric point of approx . 5.0 . 2 . The enzyme showed optimum activity at pH 5.0-7.7 and had an activation energy of 44 kJ mol-1 . It was stable at room temperature at pH 4.5-11.5 in the presence of 0.5 mg ml-1 bovine serum albumin . The half-life of the enzyme at 75 degrees C was 20 min at pH 6.0 in the presence of 0.5 mg ml-1 bovine serum albumin . 3 . The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose . The Km for oat spelts xylan was 0.021% (w/v) at pH 6.0 . 4 . The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl beta-D-cellobioside and p-nitrophenyl beta-D-cellobioside, but could not hydrolyse xylobiose . 5 . It showed transferase activity on p-nitrophenyl beta-D-xylopyranoside . Xylose did not inhibit the enzyme.

Genetika, 1993 Apr, 29(4), 556 - 70
{N-acetylglutamate-5-phosphotransferase of the thermophilic bacterium Bacillus stearothermophilus: nucleotide sequence of the gene and enzyme characterization}; Sakanian VA et al.; A nucleotide sequence of the argB gene of strain Bacillus stearothermophilus NCIB 8224 was determined . The argB gene codes for N-acetylglutamate-5-phosphotransferase of 258 amino acids with a molecular weight of 26918 D . This value is in good agreement with the SDS-PAG electrophoresis gata for identification of the heat stable B . stearothermophilus argB product synthesized in mesophilic Escherichia coli host cells . The substrates MgATP and N-acetyl-L-glutamate efficiently protect the enzyme against temperature denaturation . Amino acid sequences of bacterial (B . stearothermophilus and E . coli) and yeast (Saccharomyces cerevisiae and S . pombe) N-acetylglutamate-5-phosphotransferases share homologous conservative sites which can be responsible for MgATP binding and other structural and functional features of the enzymes of evolutionary distant microorganisms . Gel-filtration followed by K-phosphate buffer/N-acetyl-L-glutamate elution points out that the enzyme should have a molecular weight of 55,000 D . This predicts a dimeric form of the enzyme in physiological conditions . N-acetylglutamate-5-phosphotransferase activity is not inhibited by arginine-the end product of the biosynthetic pathway . The enzyme synthesis is repressed 4-fold in B . stearothermophilus by adding arginine to a growth medium . On the contrary, in E . coli hosts independent of their argR status, bacillary enzyme synthesis is not influenced by arginine . The plasmid-cloned B . stearothermophilus argB gene is well expressed in heterologous host cells (N-acetylglutamate-5-phosphotransferase activity was more than 150 and 600-fold higher in comparison with the plasmidless E . coli and B . stearothermophilus hosts, respectively) . This is a result of efficient utilization of bacillary transcriptional and translational signals, convenient codon usage of the argB gene in E . coli and the absence of any repressive action of arginine and E . coli ArgR repressor on mRNA synthesis.

J Bioenerg Biomembr, 1993 Apr, 25(2), 103 - 14
Cytochrome caa3 from the thermophilic bacterium Thermus thermophilus: a member of the heme-copper oxidase superfamily; Fee JA et al.; The subject of this short review is the cytochrome c oxidase (caa3) from the thermophilic bacterium Thermus thermophilus . First, some of the extensive physical and enzymological results obtained with this enzyme are reviewed, and two experiments are described, involving isotope substitutions in combination with Mossbauer and ENDOR spectroscopies, which have provided novel insight into the active sites of the enzyme . Second, we summarize recent molecular genetic work showing that Thermus cytochrome caa3 is a bona fide member of the superfamily of heme-copper oxidases . Finally, we present a rough three-dimensional model and speculate about certain features of the metal-binding sites.

J Clin Microbiol, 1993 Apr, 31(4), 882 - 6
Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay; Young KK et al.; Amplification of RNA by the polymerase chain reaction (PCR) is normally a two-step process requiring separate enzymes and buffer conditions . We describe a combined reverse transcription-PCR (RT-PCR) assay for hepatitis C virus (HCV) RNA amplification in which a single enzyme and buffer condition are used . In this assay, both the RT and PCR steps are carried out with the thermoactive DNA polymerase of Thermus thermophilus . A transcription vector containing HCV sequences has also been constructed to generate quantifiable HCV RNA templates that can be used to optimize reaction conditions and to assess the efficiency of amplification . Amplification from < or = 100 copies of RNA was detected reproducibly by gel electrophoresis . The assay sensitivity was increased to 10 RNA copies by hybridization to a probe . The patterns of viremia in three individuals infected with HCV were examined by amplification of HCV RNA from plasma samples collected serially over a period of 1 year . These results were correlated with the times of seroconversion and the onset of rise in levels of alanine aminotransferase in serum . In all three subjects, HCV RNA was detected prior to seroconversion and the initial rise in levels of alanine aminotransferase in serum . Upon seroconversion, HCV RNA fell to a level below the detection limit of the assay . This pattern of transient viremia appears to be characteristic of acute, resolving HCV infections . The combined RT-PCR assay is a sensitive method which circumvents the problems associated with PCR amplification of RNA . Using this assay, we demonstrated that three donors infected by the same index case all have similar patterns of viremia.

Mycopathologia, 1993 Apr, 122(1), 21 - 8
Mushroom worker's lung: serologic reactions to thermophilic actinomycetes present in the air of compost tunnels; Van den Bogart HG et al.; Vast numbers of spores of the thermophilic actinomycetes Excellospora flexuosa, Thermomonospora alba, T . curvata and T . fusca were collected from the air in fermentation tunnels during the spawning of mushroom compost, i.e . over 10(9) CFU m-3 of air . Five different genera of fungi, namely, Aspergillus, Aureobasidium, Cladosporium, Penicillium and Scytalidium, were found at only 10(3) CFU m-3 of air . Agaricus bisporus, used for spawning, was absent . Sera of 10 mushroom growers affected by Mushroom Worker's Lung (MWL) were tested by a qualitative dot-ELISA for antibodies against the spores of these micro-organisms . All 10 were positive for one or more of the actinomycetes E . flexuosa, T . alba, T . curvata and T . fusca . No antibodies were found against Streptomyces thermovulgaris, Thermoactinomyces vulgaris and T . sacchari nor against the fungi Aspergillus fumigatus, Penicillium brevicompactum, P . chrysogenum, Scytalidium thermophilum and Trichoderma viride . Sera of 11 of 14 workers engaged in routine spawning of compost in tunnels reacted positively with 1 or more of the actinomycetes . Their 10log serum titres increased with the duration of employment to an upper limit of 2.5 . The sera of 19 non-exposed individuals were negative . Because high numbers of spores of E . flexuosa, T . alba, T . curvata and T . fusca were present in the air that was used for successful inhalation provocation of mushroom workers with MWL and because of the elevated serum titres of these workers, we presume these organisms to contribute in the occurrence of MWL.

J Bacteriol, 1993 Apr, 175(7), 2060 - 6
Amino acid transport in the thermophilic anaerobe Clostridium fervidus is driven by an electrochemical sodium gradient; Speelmans G et al.; Amino acid transport was studied in membranes of the peptidolytic, thermophilic, anaerobic bacterium Clostridium fervidus . Uptake of the negatively charged amino acid L-glutamate, the neutral amino acid L-serine, and the positively charged amino acid L-arginine was examined in membrane vesicles fused with cytochrome c-containing liposomes . Artificial ion diffusion gradients were also applied to establish the specific driving forces for the individual amino acid transport systems . Each amino acid was driven by the delta psi and delta mu Na+/F and not by the Z delta pH . The Na+ stoichiometry was estimated from the amino acid-dependent 22Na+ efflux and Na(+)-dependent 3H-amino acid efflux . Serine and arginine were symported with 1 Na+ and glutamate with 2 Na+ . C . fervidus membranes contain Na+/Na+ exchange activity, but Na+/H+ exchange activity could not be demonstrated.

J Bacteriol, 1993 Apr, 175(7), 1919 - 28
Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for symbiotic nitrogen fixation is atypical; Watson RJ et al.; In Rhizobium meliloti, an aspartate aminotransferase (AspAT) encoded within a 7.3-kb HindIII fragment was previously shown to be required for symbiotic nitrogen fixation and aspartate catabolism (V . K . Rastogi and R.J . Watson, J . Bacteriol . 173:2879-2887, 1991) . A gene coding for an aromatic aminotransferase located within an 11-kb HindIII fragment was found to complement the AspAT deficiency when overexpressed . The genes encoding these two aminotransferases, designated aatA and tatA, respectively, have been localized by subcloning and transposon Tn5 mutagenesis . Sequencing of the tatA gene revealed that it encodes a protein homologous to an Escherichia coli aromatic aminotransferase and most of the known AspAT enzymes . However, sequencing of the aatA gene region revealed two overlapping open reading frames, neither of which encoded an enzyme with homology to the typical AspATs . Polymerase chain reaction was used to selectively generate one of the candidate sequences for subcloning . The cloned fragment complemented the original nitrogen fixation and aspartate catabolism defects and was shown to encode an AspAT with the expected properties . Sequence analysis showed that the aatA protein has homology to AspATs from two thermophilic bacteria and the eukaryotic tyrosine aminotransferases . These aminotransferases form a distinct class in which only 13 amino acids are conserved in comparison with the well-known AspAT family . DNA homologous to the aatA gene was found to be present in Agrobacterium tumefaciens and other rhizobia but not in Klebsiella pneumoniae or E . coli.

Enzyme Microb Technol, 1993 Apr, 15(4), 286 - 92
Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents; Liao HH; A series of variants of kanamycin nucleotidyltransferase (KNTase), isolated previously on the basis of enhanced thermostability by cloning and selection for enzymatic activity in the thermophile Bacillus stearothermophilus, was used to systematically test the hypothesis that thermostable enzymes would also be more resistant to other forms of protein denaturation . The purified KNTases were treated with proteinase K or assayed at 37 degrees C in the presence of urea, N-lauroylsarcosine, Triton X-100, tetrahydrofuran, ethanol, or dimethylformamide . With all these agents, the KNTases displayed increasing resistance to denaturation in the order: wild type, mutant TK9 (with a Thr130-->Lys substitution), TK1 (Asp80-->Tyr), and TK101 (both substitutions) . This is the same order in which their thermostability increases, indicating that the structural mechanism(s) whereby the mutations yield enhanced resistance to heat denaturation also yield stabilization towards chemical forms of enzymatic inactivation . These results suggest that selection in thermophiles is a useful method to obtain enzyme variants with increased overall stability, even at nonthermophilic temperatures.

Appl Microbiol Biotechnol, 1993 Apr, 39(1), 31 - 5
The high maltose-producing alpha-amylase of the thermophilic actinomycete, Thermomonospora curvata; Collins BS et al.; The alpha-amylase of Thermomonospora curvata catalyses the formation of very high levels of maltose from starch (73%, w/w) without the attendant production of glucose . The enzyme was produced extracellularly in high yield during batch fermentation in a 5-1 fermentor . Purification was achieved by ammonium sulphate fractionation, Superose-12 gel filtration and DEAE-Sephacel ion-exchange chromatography . The enzyme exhibited maxima for activity at pH 6.0 and 65 degrees C, had a relative molecular mass of 60,900-62,000 and an isoelectric point at 6.2 . The exceptionally high levels of maltose produced and the unique action pattern exhibited on starch and related substrates indicate a very unusual maltogenic system . The predominance of maltose as the final end-product may be explained by the participation of reactions other than simple hydrolysis and the preferential cleavage of maltotriose from higher maltooligosaccharides . The enzyme exhibits very low affinity for maltotriose (Km = 7.7 x 10(-3) M) and its conversion to maltose is achieved by synthetic followed by hydrolytic events, which result in the very high levels of maltose observed and preclude glucose formation . This system is distinguished from other very high maltose-producing amylases by virtue of its high temperature maximum, very low affinity for maltotriose and the absence of glucose in the final saccharide mixture.

Philos Trans R Soc Lond B Biol Sci, 1993 Mar 29, 339(1289), 305 - 12
A chaperonin from a thermophilic bacterium, Thermus thermophilus; Yoshida M et al.; Unlike Escherichia coli chaperonins, a chaperonin (cpn) from a thermophilic bacterium, Thermus thermophilus, consisting of homologues to GroEL (cpn 60) and GroES (cpn 10) is co-purified as a large complex . Thermus chaperonin shows a bullet-like shape in the side view seen by electron microscopy, and antibody against cpn 10 binds only to the round side of the bullet . We conclude that a single cpn 60-heptamer ring with two stripes stacks into two layers and a cpn 10 oligomer binds to one side of the layers . The purified Thermus chaperonin contains endogenously bound ADP, and incubation with ATP causes a partial dissociation of chaperonin into cpn 60 monomers and a cpn 10 heptamer . The effect of Thermus chaperonin on protein refolding upon dilution from guanidine HC1 is different at three temperature ranges . At high temperatures above 55 degrees C, where the native proteins are stable but their spontaneous foldings fail, the chaperonin induces productive folding in an ATP-dependent manner . At middle temperatures (25-55 degrees C) where spontaneous foldings of the enzymes occur, the chaperonin slows down the rate of folding without changing the final yield of productive folding . At lower temperatures below 25 degrees C where spontaneous foldings also occur, the chaperonin arrests the folding even in the presence of ATP . When a solution of relatively heat labile protein is incubated at high temperatures, and then residual activity of the protein is measured at its optimal temperature after incubation with ATP, the temperature that causes irreversible heat denaturation of the protein is elevated about 10 degrees C by inclusion of Thermus chaperonin in the solution.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1993 Mar 20, 230(2), 664 - 6
Crystallization and preliminary X-ray analysis of a thermophillic Bacillus xylanase; Pickersgill RW et al.; A xylanase of M(r) 20,700 from the hyperproductive mutant D3 of the thermophillic Bacillus, strain XE has been purified and crystallized from 2-methyl-2,4-pentanediol . The unit cell is triclinic with a = 48.5 A, b = 51.5 A, c = 72.6 A, alpha = 90.4 degrees, beta = 95.4 degrees, gamma = 92.3 degrees (all +/- 0.2) . There are four molecules in the asymmetric unit related by 222 symmetry . These crystals diffract to at least 2.5 A using X-rays from a rotating anode generator.

Biochim Biophys Acta, 1993 Mar 20, 1172(3), 357 - 60
Nucleotide sequence of the Mn-stabilizing protein gene of the thermophilic cyanobacterium Synechococcus elongatus; Miura K et al.; The nucleotide sequence of the psbO gene encoding the extrinsic 33 kDa protein (the Mn-stabilizing protein) from the thermophilic cyanobacterium Synechococcus elongatus was determined . The deduced amino acid sequence consisted of 272 residues; 26 for the signal peptide and 246 for the mature protein . The amino acid sequences of nine proteolytic peptides from the isolated protein completely agreed with the deduced amino acid sequence . Several unique variations of amino acids were found in the primary structure, of which some may be related to the high thermostability of the protein.

J Mol Biol, 1993 Mar 20, 230(2), 529 - 42
Crystal structure of ribonuclease H from Thermus thermophilus HB8 refined at 2.8 A resolution; Ishikawa K et al.; The crystal structure of Thermus thermophilus RNase H was determined at 2.8 A resolution . The structure was solved by the molecular replacement method, based on the accurately refined structure of Escherichia coli RNase HI, which shows 52% amino acid sequence identity . Crystallographic refinement led to an R-factor of 0.205, with a 0.019 A root-mean-square deviation from ideal bond lengths and 0.048 A from ideal bond angle distances . Structural comparison shows a striking similarity in the overall folding of the thermophilic and mesophilic enzymes . The root-mean-square displacement is 0.95 A between equivalent alpha-carbon atoms from all elements of secondary structure (five alpha-helices and five beta-strands) . However, some notable differences, which account for the enhanced thermostability of T . thermophilus RNase H, are observed in loop structures and side-chain conformations . The substitution of Gly for the left-handed helical residue (Lys95) in the E . coli enzyme is proposed to substantially enhance the thermostability, due to the release of steric hindrance caused by the beta-carbon atom . Furthermore, it is likely that the expansion of an aromatic cluster, arising from the replacement of Ile78 in the mesophilic enzyme by Phe, and the increased number of salt-bridges additively contribute to the stability.

FEBS Lett, 1993 Mar 15, 319(1-2), 185 - 8
Participation of the overproduced elongation factor Tu from Thermus thermophilus in protein biosynthesis of Escherichia coli; Zeidler W et al.; The influence of the overproduced elongation factor Tu (EF-Tu) from Thermus thermophilus on the protein biosynthesis in Escherichia coli was investigated both in vivo and in vitro . A kirromycin-resistant E . coli strain became sensitive to this antibiotic upon the expression of the tuf A-gene of T . thermophilus present on a plasmid . In in vitro translation with components of the kirromycin-resistant E . coli strain the poly(Phe) synthesis stopped when minute amounts of the EF-Tu from T . thermophilus were added . Both results indicate the sensitivity of the T . thermophilus EF-Tu to kirromycin and its participation in the polypeptide synthesis of E . coli.

J Biol Chem, 1993 Mar 15, 268(8), 5395 - 408
Cytochrome oxidase genes from Thermus thermophilus . Nucleotide sequence of the fused gene and analysis of the deduced primary structures for subunits I and III of cytochrome caa3; Mather MW et al.; Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, has been purified and extensively characterized as a two-subunit enzyme containing the metal centers characteristic of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c (Fee, J . A., Kuila, D., Mather, M . W., and Yoshida, T . (1986) Biochim . Biophys . Acta 853, 153-185) . We have now cloned and sequenced the genes encoding the subunits of this enzyme . The smaller subunit consists of a typical oxidase subunit II sequence fused to a cytochrome c domain (Mather, M . W., Springer, P., and Fee, J . A . (1991) J . Biol . Chem . 266, 5025-5035) . The larger subunit, the A-protein, is encoded by a fusion gene lying immediately downstream of the subunit IIc gene . The 5' portion of this gene encodes an oxidase subunit I homolog, whereas the 3' portion is homologous to oxidase subunits III . The A-protein from the purified enzyme appears too small from SDS-polyacrylamide gel electrophoresis and quantitative amino acid analyses to be a complete subunit I/III fusion, but it is currently not known if proteolytic processing occurs . Analyses of the sequences of oxidase subunits are presented which clearly identify T . thermophilus cytochrome caa3 as a bona fide member of the greater family of heme- and copper-requiring oxidases . As one consequence, it is confirmed that the set of invariant histidine residues (potential ligands of the metal centers) in cytochrome c oxidase subunits I and II is reduced to 8 . Possible topological and helix packing models are developed based on considerations of homology, hydropathy, and variability.

Biochem Biophys Res Commun, 1993 Mar 15, 191(2), 550 - 7
Heat shock promoter of thermophilic chaperonin operon; Ohta T et al.; The translation of a heat shock protein (HSP), TGroEL, of thermophilic bacterium PS3 increased within 10 minutes when the culture temperature was raised from 60 degrees C to 70 degrees C . In contrast to hyperthermophilic HSPs such as Pyrodictium ATPase, TGroEL is homologous to GroEL of E . coli (Tamada et al . (1991) Biochem . Biophys . Res . Commun . 197, 565-571) . The promoter of the thermophilic heat shock gene (tgrE) was sequenced to analyze the heat shock response . The sequences of the promoter were CCTACTTGCA (-35 region) and GTTCATTAATA (-10 region), which are more heat labile than those of the sigma 32 recognition sites of GroEL . The expression of this homologous tgrE in E . coli may be lethal to the cells . The ATPase alpha subunit of this thermophile is also homologous to TGroEL.

J Biol Chem, 1993 Mar 15, 268(8), 5371 - 5
Chaperonin from Thermus thermophilus can protect several enzymes from irreversible heat denaturation by capturing denaturation intermediate; Taguchi H et al.; Chaperonin isolated from Thermus thermophilus is stable up to 80 degrees C . Taking advantage of this heat stability, we have studied the effects of chaperonin on heat denaturation of several relatively heat labile enzymes . When the enzymes are incubated at their denaturating temperatures, the presence of T . thermophilus chaperonin in the solution has little effect on the rate of apparent inactivation of the enzyme . However, this inactivation is not irreversible since most activity is recovered when the solution is shifted to the second incubation at a moderate temperature with concomitant addition of MgATP . When the chaperonin is omitted from the solution, no recovery is observed . Recovery of the activity is also dependent on MgATP in the second incubation and 50% of recovery is attained at 5 microM MgATP . When the chaperonin is added after starting the incubation at a denaturing temperature, recovery of the activity becomes poorer as the delay of chaperonin addition increases . The critical temperature of the incubation at which irreversible denaturation occurs to the enzymes is elevated 8-15 degrees C by inclusion of T . thermophilus chaperonin in the solution . The heat stability of captured proteins by the chaperonin, assessed as retention of the ability to resume productive folding under optimal conditions, is measured more accurately using chemically produced folding intermediate-chaperonin complexes . The ability is lost at about 78 degrees C being irrespective of variable heat stability of individual enzymes . These results indicate that during heat denaturation proteins assume a common structure which is recognizable by the chaperonin . Once a protein with this structure is captured by T . thermophilus chaperonin, it retains the ability to resume productive folding even after exposure to the otherwise denaturing high temperature . Its heat stability seems to be limited solely by heat stability of chaperonin.

J Mol Biol, 1993 Mar 5, 230(1), 353 - 5
Crystal parameters of an alcohol dehydrogenase from the extreme thermophile Thermoanaerobium brockii; Zhang Z et al.; A bacterial thermophilic alcohol dehydrogenase which is stable and active at 85 degrees C, has been crystallized by vapor diffusion from solutions of polyethylene glycol . A monoclinic crystal form diffracts to 2.8 A resolution and belongs to space group C2 with unit cell dimensions a = 139.0 A, b = 137.4 A, c = 80.9 A and beta = 93.23 degrees . The asymmetric unit contains four molecules which exhibit 222 point symmetry . A second crystal form is orthohombic, space group P2(1)2(1)2 with unit cell dimensions a = 168.0 A, b = 123.0 A, c = 80.0 A, and it diffracts to 3.2 A resolution.

Biochem Mol Biol Int, 1993 Mar, 29(4), 729 - 37
The effect of cations on the thermophilic character of alkaline phosphatase from Thermoactinomyces vulgaris; Iny D et al.; The effect of cations on the thermophilic character of alkaline phosphatase from Thermoactinomyces vulgaris, is described . The optimal pH and temperature were 9.5 and 55 degrees C to 65 degrees C, respectively . The partial removal of cations with ethylene diamine tetraacetic acid converted the enzyme to mesophilic and susceptible to chemical denaturation . Their complete removal caused complete inhibition . The addition of 0.3mM cobalt and 10mM magnesium added before heating were found to be optimal for restoring its thermophilic character and its stability to a chemical denaturant.

J Biochem (Tokyo), 1993 Mar, 113(3), 401 - 10
Cloning of the DNA polymerase gene of Bacillus caldotenax and characterization of the gene product; Uemori T et al.; The pol gene of the thermophilic eubacterium Bacillus caldotenax was cloned in a plasmid and expressed in Escherichia coli . The PCR method was used to clone the gene with no previous knowledge of the gene or protein sequence . The 3,329-bp DNA fragment containing the structural gene for DNA polymerase was sequenced . DNA polymerase, as deduced from the DNA sequence, consisted of 877 amino acids, had a molecular weight of 99,452, and was structurally homologous to the DNA polymerases of the Pol I family (family A), which includes E . coli DNA polymerase I and T7 DNA polymerase . B . caldotenax DNA polymerase (Bca polymerase) purified from the recombinant E . coli strain was characterized . Like E . coli Pol I, Bca polymerase had 5'-->3' exonuclease activity . The degraded product with the molecular weight of 65,000 was also purified and found to have polymerase activity . To overproduce this Klenow-type fragment of Bca polymerase, a recombinant expression plasmid pUI205 with a deletion in the 5'-region of the pol structural gene was constructed . The DNA polymerase produced by pUI205 is more suitable for use in the dideoxy sequencing method than the other DNA polymerases that have been used for sequencing.

Zentralbl Mikrobiol, 1993 Mar, 148(2), 137 - 47
Mycoflora and mycotoxin of hazelnut (Corylus avellana L.) and walnut (Juglans regia L.) seeds in Egypt; Abdel-Hafez AI et al.; Fifty-one species and 3 varieties appertaining to 20 genera were collected from 20 samples of each of hazelnut and walnut seeds on glucose- and 40% (W/V) sucrose-Czapek's agar at 25 degrees C and 45 degrees C with the most common mesophiles were Aspergillus flavus, A . fumigatus, A . niger, Cladosporium cladosporioides, C . herbarum, Penicillium chrysogenum, P . citrinum and P . oxalicum . Fusarium (represented by F . equiseti, F . moniliforme and F . oxysporum) was recovered from walnut seeds in moderate frequency (on glucose-Czapek's agar) . Eurotium (E . amstelodami, E . chevalieri, E . repens and E . rubrum) was completely absent on glucose agar, but it was isolated in high frequency from the two types of seeds on 40% sucrose-Czapek's agar . Aspergillus fumigatus and Rhizomucor pusillus were the most common thermophilic fungi in hazelnut and walnut seeds on glucose agar at 45 degrees C . Humicola grisea var . themoidae and Thermoascus aurantiacus were encountered rarely from walnuts . The nuts samples were assayed for natural occurrence of aflatoxins B1, B2, G1 and G2, citrinin, ochratoxin A, patulin, sterigmatocystin, zearalenone, T-2 toxin and diacetoxyscirpenol by thin layer chromatography analysis . Aflatoxin was detected in 90% of hazelnut samples (25-175 micrograms/kg) and 75% of walnut samples (15-25 micrograms/kg) . Zearalenone was detected in one sample of walnut (125 micrograms/kg) . This is the first report for the presence of zearalenone in walnut . The other mycotoxins were not detected.

J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 393 - 402
Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase; Sakanyan V et al.; A 3.4 kb EcoRI fragment, cloned in E . coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands . The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD . The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918 . A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E . coli arg B mutants transformed with the 3.4 kb fragment on a plasmid . A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain . Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame . The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349 . The subcloned B . stearothermophilus argJ can complement a double argA argE E . coli mutant to prototrophy . Gel-filtration of a heat-treated extract of the complemented double mutant E . coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000 . Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine . These results suggest that both activities can be ascribed to a single protein.

Exp Lung Res, 1993 Mar-Apr, 19(2), 257 - 71
Cellular and cytokine profiles in spontaneous regression phase of hypersensitivity pneumonitis; Denis M et al.; The phase of spontaneous regression of hypersensitivity pneumonitis was evaluated using a mouse model . C57BL/6 mice were instilled intranasally with 150 micrograms of the thermophilic actinomycete Faeni rectivirgula 3 days a week so as to establish a mouse model of farmer's lungs . It was shown that instillation of mice for a period of more than 6 weeks was associated with a significant decrease in the lung inflammation, suggestive of the so-called spontaneous regression phase seen in this pathology . Indeed, the lung index was seen to decrease after more than 6 weeks of treatment (2.2 after 6 weeks vs . 1.7 at 12 weeks, p < .01) . There was also a significant decrease in lung hydroxyproline levels in animals given 12 weeks of treatment (175 micrograms/lung) compared to 6-week-treated animals (212 micrograms/lung, p < .05) . Treated mice did not show a significant decrease in the alveolitis after 9 weeks of treatment . Also, there was no evidence that there was a decrease in bronchoalveolar lavage macrophage or T lymphocyte activity in mice given more than 9 weeks of F . rectivirgula treatment, as judged by O2- release and antigen-driven proliferation . Conversely, it was shown that NK cell activity in the lung digest of mice given 9 to 12 weeks of instillation was significantly higher than that seen in mice given 6 weeks of treatment . Analysis of the lung cell cytokine profile seen after ConA mitogenesis showed that after 6 weeks of F . rectivirgula treatments, nonparenchymal cells secreted high levels of tumor necrosis factor alpha (TNF alpha) and granulocyte macrophage colony-stimulating-factor (GM-CSF), whereas similar cells from the lungs of mice given 9-12 weeks of treatment secreted larger amounts of interferon-gamma (IFN gamma) and interleukin-2 (IL-2) . Overall, these results suggest that the spontaneous regression phase is associated with changes in NK cell activity and lung cell lymphokine profile.

Proteins, 1993 Mar, 15(3), 283 - 9
The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent; Davies GJ et al.; The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast . The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures . The refinement residual, calculated using all the measured data between 8 and 1.65 A, is 0.18(1) . The stereo chemical deviations of the final model from ideality are 0.01 A for both bonds and planes . The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53 degrees C, respectively . Differential scanning calorimetry indicates that the energy difference (delta delta G) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol-1 at room temperature . The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better alpha-helix stability resulting from the removal of helix destabilizing residues and extra helix-dipole/helix side chain ionic interactions.

Biochem J, 1993 Mar 1, 290 ( Pt 2), 515 - 23
The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans; Tuohy MG et al.; Talaromyces emersonii, a thermophilic aerobic fungus, produces a complete xylan-degrading enzyme system when grown on appropriate substrates . In this paper we present the physicochemical and catalytic properties of three enzymes, xylosidase (Xyl) I (M(r) 181,000; pI 8.9), II (M(r) 131,000; pI 5.3) and III (M(r) 54,200; pI 4.2) . Xyl I and II appear to be dimeric and Xyl III is a single-subunit protein . All three enzymes catalyse the hydrolysis of aryl beta-D-xylosides and xylo-oligosaccharides . Xyl I is a classic beta-xylosidase (1,4-beta-D-xylan xylohydrolase; EC 3.2.1.37), and Xyl II and III are novel xylanases (endo-1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) which we believe have not hitherto been reported . In addition to the above substrates, they also catalyse the extensive hydrolysis of unsubstituted xylans, and may have considerable biotechnological potential . The hydrolysis product profiles and bond-cleavage frequencies with various substrates are presented.

Dev Dyn, 1993 Mar, 196(3), 195 - 204
Early encounters of the repetitive kind: a prelude to cell adhesion in conjugating Tetrahymena thermophila; Brown F et al.; The relationship between direct cell contacts and subsequent cell-cell adhesion was studied in the ciliated protozoan, Tetrahymena thermophila . During sexual reproduction, adhesion into pairs begins at approximately 1 hr after mixing starved complementary mating types . However, direct contacts between cells prior to pairing are known to be required for the development of adhesion-readiness . We find here that the initial contact interactions are necessary but not sufficient to drive the cells to adhesion-readiness . Secondary interactions are needed . Two distinct experimental strategies were used . First, we examined the effects of a mutant that is unable to pair but which can stimulate two different wild-type mating type cells to pair when mixed . We showed that stimulation by the mutant is only partial; in response to mutant cells, wild-type cells ceased forming food vacuoles but did not undergo tip transformation or concanavalin A (Con A)-receptor tipping . Further, kinetic analysis shows that when mixed together, pair-formation among partially stimulated wild-type cells is slightly delayed, allowing time for these pre-pairing processes to occur . This indicates that, beyond the initial contact interaction, mutant-stimulated wild-type cells require a subsequent interaction which cannot be fulfilled by the mutants . Secondly, we found that by blocking contact interactions between wild-type mating types at various time intervals after they were mixed, additional increase in tip transformation and Con A receptor tipping was prevented . Further, both processes underwent a regression . This indicates that multiple contact interactions are required to drive the cells to adhesion readiness and to prevent developmental slip-back.

Biochem Mol Biol Int, 1993 Mar, 29(4), 673 - 85
Behaviour of plasmid containing C4A2 repeats in S . cerevisiae and S . pombe; Shervington AA et al.; Plasmid, which combined the complete genome of BPV-I, yeast ARS, LEU yeast selectable marker gene, the NEO selectable marker gene and inverted (C4A2)n telomeric repeat gene sequences cloned originally from Tetrahymena thermophila, was constructed . It was introduced in either circular or linear form to Saccharomyces cerevisiae or Schizosaccharomyces pombe . Although both yeasts could replicate the plasmid extrachromosomally, irrespective of whether it was introduced as a circular or linear structure, the yeasts did differ in their ability to resolve a circular plasmid carrying the telomeric sequences into linear forms . S . cerevisiae was found to resolve the circular form of pCA/LEU/ARS to the linear structure, whereas circular pCA/LEU/ARS remained circular in S . pombe . On the other hand, pCA/LEU/ARS which had been previously linearised at the telomeric sequence was maintained as a linear structure in S . pombe and S . cerevisiae.

Biochemistry, 1993 Feb 23, 32(7), 1825 - 32
Binding and functional properties of two new extrinsic components, cytochrome c-550 and a 12-kDa protein, in cyanobacterial photosystem II; Shen JR et al.; Cytochrome c-550, a low-potential c-type cytochrome, and a 12-kDa protein were recently shown to be associated extrinsically and stoichiometrically with purified photosystem II (PSII) complex of the thermophilic cyanobacterium Synechococcus vulcanus {Shen, J.-R., Ikeuchi, M., & Inoue, Y . (1992) FEBS Lett . 301, 145-149} . The binding and functional properties of these two extrinsic components in PSII were studied by means of release-reconstitution and thermoluminescence techniques . The following results were obtained: (i) cyt c-550 rebound appreciably to cyanobacterial PSII in the absence of the 33- and 12-kDa extrinsic proteins, but the presence of these two proteins facilitated the rebinding, affording a full level of binding equal to that in native PSII . (ii) The 12-kDa protein did not rebind to PSII at all unless the 33-kDa protein or cyt c-550 was present . It rebound only partially in the presence of either of these two proteins, but it rebound maximally when reconstituted together with both of them . (iii) Reconstitution with cyt c-550 or the 12-kDa protein alone in the absence of the 33-kDa protein did not restore the O2-evolving activity of CaCl2-washed PSII . Reconstitution with cyt c-550 in combination with the 33-kDa protein appreciably enhanced the activity, but the activity restoration was much more marked and reached a level close to that of the original activity when all three extrinsic proteins were included.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 236 - 8
Nucleotide sequence of the genes encoding proline tRNA(UGG) and threonine tRNA(GGU) and consensus promoter model of Thermococcus celer; Klenk HP et al.; The nucleotide sequences of the genes encoding tRNA(Pro)(UGG) and tRNA(Thr)(GGU) from the extremely thermophilic archaeon (archaebacterium) Thermococcus celer have been determined . A consensus promoter model was deduced from the comparison of the upstream regions of several stable RNA genes with S1-mapped promoter regions of genes coding for ribosomal proteins and DNA-dependent RNA polymerase components.

Biochim Biophys Acta, 1993 Feb 13, 1156(2), 167 - 72
Thermostable beta-glucosidase and beta-xylosidase from Thermotoga sp . strain FjSS3-B.1; Ruttersmith LD et al.; A beta-D-glucosidase and a beta-D-xylosidase were purified to homogeneity from the thermophilic eubacterium Thermotoga sp . strain FjSS3-B.1 . Both enzymes were largely cell-associated and were probably associated with the 'toga' structures of this organism . Using SDS-PAGE they were found to have M(r) values of 75,000 and 92,000, respectively . The beta-glucosidase was active against cellobiose, sophorose and gentiobiose with Km values of 59 mM, 2.7 mM and 6 mM, respectively . The beta-xylosidase had a Km of 2 mM for xylobiose, showed strong activity against p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl alpha-L-arabinopyranoside, but was subject to strong substrate inhibition by p-nitrophenyl beta-D-xylopyranoside . Both enzymes were extremely thermostable, with half-lives of several hours at 98 degrees C . The thermostabilities of both enzymes were increased further by the addition of either trehalose or betaine.

Nucleic Acids Res, 1993 Feb 11, 21(3), 463 - 8
The helical repeat of DNA at high temperature; Duguet M; The increasing number of studies on thermophilic organisms addressed the question of DNA double helix parameters at high temperature . The present study shows that the helix rotation angle per base pair omega of an unconstrained DNA decreases linearly upon temperature increase, up to the premelting range . In the ionic conditions tested, this rule extends to temperatures up to 85 degrees C, which is a common growth temperature for many hyperthermophilic organisms . In addition, the torsional constant K of DNA decreases with temperature, indicating that the energy required to modify the DNA twist is lower at high temperature . These findings have several implications for people working on the structure and enzymology of DNA at high temperature.

Nucleic Acids Res, 1993 Feb 11, 21(3), 671 - 9
Characterization of ribonuclease P RNAs from thermophilic bacteria; Brown JW et al.; The catalytic RNA component of bacterial RNase P is responsible for the removal of 5' leader sequences from precursor tRNAs . As part of an on-going phylogenetic comparative characterization of bacterial RNase P, the genes encoding RNase P RNA from the thermophiles Thermotoga maritima, Thermotoga neapolitana, Thermus aquaticus, and a mesophilic relative of the latter, Deinococcus radiodurans, have been cloned and sequenced . RNAs transcribed from these genes in vitro are catalytically active in the absence of other components . Active holoenzymes have been reconstituted from the T.aquaticus and T.maritima RNAs and the protein component of RNase P from Escherichia coli . The RNase P RNAs of T.aquaticus and T.martima, synthesized in vitro, were characterized biochemically and shown to be inherently resistant to thermal disruption . Several features of these RNAs suggest mechanisms contributing to thermostability . The new sequences provide correlations that refine the secondary structure model of bacterial RNase P RNA.

FEBS Lett, 1993 Feb 8, 317(1-2), 96 - 100
Structure of the 16 S ribosomal RNA of the thermophilic cyanobacterium Chlorogloeopsis HTF ('Mastigocladus laminosus HTF') strain PCC7518, and phylogenetic analysis; Wilmotte A et al.; The thermophilic cyanobacterial strain, PCC7518, originally identified as 'Mastigocladus laminosus HTF' does not show branchings or heterocysts . The absence of branchings supports the later assignment to the genus Chlorogloeopsis . The absence of heterocysts may be the result of a mutation because heterocysts were observed in the original isolate . Alternatively, contamination may have happened . To solve this problem, the 16 S rRNA sequence was determined and used to infer a secondary structure model and build distance trees . The trees showed that strain PCC7518 belongs to the cluster of heterocystous species and has most probably lost the ability to produce heterocysts by mutation . It is only distantly related to Chlorogloeopsis fritschii PCC6718.

Biochim Biophys Acta, 1993 Feb 8, 1141(1), 45 - 51
Small subunits of Photosystem I reaction center complexes from Synechococcus elongatus . I . Is the psaF gene product required for oxidation of cytochrome c-553?
Hatanaka H, Sonoike K, Hirano M, Katoh S.
Photosystem I (PS I) reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus elongatus with nonionic detergents, digitonin or sucrose monolaurate, contained eight small subunit polypeptides . Two of the small polypeptides were identified by analysis of their N-terminal amino-acid sequences as the psaF and psaE gene products . Treatment with a cationic detergent, cetyltrimethylammonium bromide, resulted in depletion of five small subunits including the psaF gene product . Five PS I complexes isolated with an anionic detergent, sodium dodecylsulfate, contained zero to four small subunits but were all depleted of the psaF polypeptide . The function of the psaF gene product was examined by measuring reduction kinetics of flash-oxidized P-700 in the presence of different concentrations of cytochrome c-553 . Oxidized P-700 was rapidly reduced by the reduced cytochrome in all the PS I complexes that contained, at least, the psaC and psaD polypeptides and the second-order rate constants of electron transfer from cytochrome c-553 to P-700 were essentially the same between PS I complexes that contained the psaF polypeptide and those that lost this polypeptide . Thus, the psaF polypeptide is not required for the bimolecular reaction between P-700 and cytochrome c-553 . Mg2+ had a moderate stimulating effect on the rate of P-700 reduction whether PS I complexes were associated with the psaF gene product or not . The function of this subunit polypeptide is discussed.

J Mol Biol, 1993 Feb 5, 229(3), 782 - 4
Crystallization and preliminary X-ray analysis of an NAD(+)-dependent alcohol dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Pearl LH et al.; An NAD(+)-dependent alcohol dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus has been crystallized in the holo-enzyme and apo-enzyme forms . Crystals of the holo-enzyme grow from 2-methyl-2,4-pentanediol at pH 8.4 with the addition of NADH and at pH 7.0 with the addition of NADH and dimethyl sulphoxide . Crystals of the apo-enzyme grow at pH 6.3 from a mixture of polyethylene glycol 4000 and propan-2-ol . The holo-enzyme crystallizes in C2 with a dimer in the asymmetric unit, however the crystals are twinned and unsuitable for data collection . The apo-enzyme crystallizes in I4(1)22 (a = 126.82 A, b = 118.95 A) with a monomer in the asymmetric unit, and the single crystals diffract to 2.8 A.

J Biochem Biophys Methods, 1993 Feb, 26(1), 51 - 60
The use of phenyl-Sepharose for the affinity purification of proteinases; Prescott M et al.; Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC) . We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources . Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media . Purification factors in the range of 2.9-60 and enzyme activity yields in excess of 88% were obtained . In some cases homogeneous enzyme was obtained from culture supernatants in a single step . A number of other proteinases from mammalian sources were also retained . The specificity of the enzyme/support interaction was studied . Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site . Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp . strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts.

Protein Sci, 1993 Feb, 2(2), 197 - 205
Sequence and expression of the gene for N10-formyltetrahydrofolate synthetase from Clostridium cylindrosporum; Rankin CA et al.; Sau3 A and Hind III restriction fragments of Clostridium cylindrosporum genomic DNA were used to isolate clones containing 80% of the N10-H4folate synthetase gene in a 5' fragment and the remaining 20% of the gene in the 3' fragment . These fragments were joined at a common SnaB I restriction site and expressed in Escherichia coli at a level equivalent to what is normally found in C . cylindrosporum . Sequence comparisons show a large degree of homology with genes from two other clostridial species, including a thermophile . Certain conserved sequences found in the three clostridial proteins and in the N10-H4folate synthetase portion of eukaryotic C1-H4folate synthases may represent consensus sequences for nucleotide and H4folate binding.

Eur J Biochem, 1993 Feb 1, 211(3), 549 - 54
Cloning, sequencing and expression of the gene encoding glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum; Bright JR et al.; The gene encoding glucose dehydrogenase has been identified by Southern analysis of doubly restricted genomic Thermoplasma acidophilum DNA, using two redundant 17-residue oligonucleotide probes reverse translated from protein N-terminal sequence data . A 1670-bp BamH1-EcoR1 restriction fragment was ligated into pUC19 and pUC18 (constructs pTaGDH1 and pTaGDH2, respectively) and cloned in Escherichia coli . The sequence of the whole fragment was determined, and a 1059-bp open reading frame identified as the gene encoding glucose dehydrogenase . Cell-free extracts from E . coli carrying construct pTaGDH1 displayed glucose dehydrogenase activity indistinguishable from controls, but extracts from cells carrying pTaGDH2 displayed a 600-fold increase in glucose dehydrogenase activity . For high-level expression and purification of native protein, the glucose dehydrogenase coding sequence was subcloned into pMEX8 . Glucose dehydrogenase purified from E . coli expressing the pMEX8 construct was indistinguishable by SDS/PAGE, N-terminal amino-acid sequence and kinetic analysis from the native enzyme purified from Tp . acidophilum . The derived 352-amino-acid sequence shows less than 20% identity with the glucose dehydrogenases of Bacillus subtilis and Bacillus megaterium but, by comparison with other eubacterial and eukaryotic dehydrogenase sequences, a portion of its sequence has been tentatively identified as a cofactor-binding region.

J Protein Chem, 1993 Feb, 12(1), 79 - 83
The complete amino acid sequence of ribosomal protein S8 from Thermus thermophilus; Reinbolt J et al.; Protein S8 from Thermus thermophilus consists of 138 amino acids of M(r) 15,840 . Its primary structure was established using peptide sequences from two different digests . Protein S8 from T . thermophilus shares a high percentage of identity with protein S8 from Thermus aquaticus . There are some consensus sequences between proteins S8 from eubacteria, archebacteria, chloroplasts, and cyanelles.

Mol Mar Biol Biotechnol, 1993 Feb, 2(1), 51 - 62
Identification and localization of bacterial endosymbionts in hydrothermal vent taxa with symbiont-specific polymerase chain reaction amplification and in situ hybridization techniques; Cary SC et al.; Invertebrates that contain endosymbiotic chemoautotrophic eubacteria are widely distributed in a variety of reducing marine habitats, including deepsea hydrothermal vents . The mechanisms of symbiont transmission in these invertebrates are not understood . To test the hypothesis that symbionts are transmitted via the eggs of hosts, we used group-specific hybridization probes complementary to 16S ribosomal RNAs (rRNAs) to look for symbionts in eggs and ovaries . 16S rRNA sequences were examined for domains unique to the symbionts of three vent animals: Calyptogena magnifica, Bathymodiolus thermophilus, and Riftia pachyptila . Three 16S rRNA-directed oligodeoxynucleotide hybridization probes (CG-1255R, RP-1243R, BT-1255R) specific for these endosymbionts were synthesized and evaluated by dot-blot hybridization . At higher stringencies, all three probes showed a high degree of specificity for their target endosymbionts rRNAs . The probes were also used as polymerase chain reaction (PCR) primers for detection of the symbiont 16S rRNA genes in genomic DNA isolated from host tissues known to contain symbionts . All three symbiont-specific probes were highly sensitive and specific as PCR primers; they successfully amplified 1 pg target DNA . However, all amplifications of extracted egg DNA from the vestimentiferan R . pachyptila with either universal eubacterial (Eub A/B) or the Riftia symbiont-specific (RP-1243R/Eub B) primer sets were unsuccessful . Nonradioactive in situ hybridizations were performed on ovarian tissue from the vestimentiferan Ridgea piscesae using RP-1243R, 3' end-labeled with digoxigenin-11-dUTP (Boehringer Mannheim) . The probe was subsequently detected with an alkaline phosphatase-conjugated immunoglobulin G antibody specific for the digoxigenin moeity . The probe bound only to the tissue of R . pisceasae coincident with the known location of symbiont cells and was not detected in any region of the ovary . These data indicate that transovarial symbiont transmission in the vestimentiferans does not take place and that symbiont acquisition is probably a post-spawning event.

Hindustan Antibiot Bull, 1993 Feb-May, 35(1-2), 33 - 42
Production of extracellular acidic lipase by Rhizopus arrhizus as a function of culture conditions; Kumar KK et al.; Thermophilic strain of Rhizopus arrhizus accumulates an acidic lipase in culture fluid when grown in a medium containing ground nut oil, milk powder and inorganic salts . Addition of 2.0% ground nut oil yielded the highest productivity of enzyme . Soyabean meal and arabinose were found to be the best nitrogen and carbon sources for enzyme production respectively . Addition of metal ions such as MnCl2, SnCl2 and CaCl2 increased the enzyme productivity by 4 fold . The enzyme productivity in the fermenter was much higher (310 U/ml) than in shake-flask (180 U/ml) . Crude lipase preparation showed pH and temperature activity optima at 3.5 and 45 degrees C respectively . The enzyme is thermostable and highly active in hydrolysing triglycerides and failed to hydrolyse-methyl esters of caprylate and palmitate.

J Biochem (Tokyo), 1993 Feb, 113(2), 245 - 50
Three-dimensional structure of F1-ATPase of thermophilic bacterium PS3 obtained by electron crystallography; Ishii N et al.; The three-dimensional molecular structure of the F1-ATPase from a thermophilic bacterium PS3 (TF1) at 3 nm resolution was reconstructed from a series of tilted, negatively stained electron microscopic images of two-dimensional crystals which were formed on a clean surface of mercury . It was shown that six ellipsoidal columns, each approximately 7 nm in length and approximately 3 nm in diameter, corresponding to the alpha and beta subunits, surrounded a central hollow cavity of approximately 2.8 nm in diameter . The cavity, however, did not penetrate the molecule, and a mass, most likely the gamma subunit, existed in the cavity at a depth of approximately 3.4 nm from the top.

Biochemistry, 1993 Jan 26, 32(3), 745 - 53
Structures of the modified folates in the thermophilic archaebacteria Pyrococcus furiosus; White RH; The structures of the modified folates present in Pyrococcus furiosus have been determined . This was accomplished largely by the characterization of the arylamines resulting from the air oxidative cleavage of the reduced modified folates present in these cells, using both chemical and enzymatic methods . Cell extracts separated on DEAE-Sephadex columns showed one major peak containing the arylamines derived from the modified folates . These arylamines were not retained on the DEAE-Sephadex columns, indicating that they contained no net negative charge . Purification of the azo dye derivatives of these arylamines on a Bio-Gel P-6 column showed the presence of three different compounds (compounds 1, 2, and 3) in an average amount of 4.1, 7.6, and 22 nmol/g dry weight of cells, respectively . Each of these compounds readily underwent mild acid hydrolysis (0.1 M HCl, 110 degrees C, 1 min) to produce the azo dye derivative of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane (pAPT) . The structure and stereochemistry (ribo) of the pAPT was the same as the pAPT present in methanopterin . In addition, compounds 1, 2, and 3 were each shown to contain 1 mol equiv of ribose and 1, 2, and 3 mol equiv of N-acetylglucosamine (gluNAc), respectively, and were designated as the azo dye derivatives of pAPT-ribose-gluNAc, pAPT-ribose-(gluNAc)2, and pAPT-ribose-(gluNAc)3 . Each of these compounds was readily cleaved to the azo dye derivative of pAPT-ribose by the enzymatic action of beta-N-acetylglucosaminidase, indicating that all the gluNAc residues were beta-linked.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1993 Jan 23, 1171(3), 295 - 8
Purification and characterisation of Bst LVI restriction endonuclease, a thermostable isoschizomer of ClaI from Bacillus stearothermophilus LV; Lobos C et al.; This work describes the purification and biochemical characterization of BstLVI restriction endonuclease, a thermostable isoschizomer of ClaI, from Bacillus stearothermophilus LV . The enzyme was purified by successive DEAE-cellulose, Affi-Gel Blue and Heparin-Sepharose CL-6B column chromatography . A molecular weight of 37,000 was determined for Bst LVI by gel filtration . As expected from thermophilic proteins, the enzyme showed a high stability towards heat and also to other known protein-denaturing agents.

J Mol Biol, 1993 Jan 20, 229(2), 561 - 3
Crystallization and preliminary X-ray analysis of the beta-galactosidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Pearl LH et al.; The beta-galactosidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus has been crystallized from polyethylene glycol 4000 in the presence of sodium acetate and acetate buffer at pH 4.6 . The protein crystallizes in P3(1)21 or P3(2)21 (a = 169.4, c = 98.29) and the crystals diffract beyond 2.5 A . The measured crystal density (approximately 1.28 g/cm3) is consistent with the presence of a tetramer (molecular mass 240 kDa) in the asymmetric unit . The specific volume of the crystals is 1.7 A3/Da, indicating a solvent content by volume of only 27%, which is amongst the lowest values observed for protein crystals, and indicates virtual close-packing of the tetramers.

Eur J Biochem, 1993 Jan 15, 211(1-2), 305 - 10
Ribonucleases from the extreme thermophilic archaebacterium S . solfataricus; Fusi P et al.; A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL-6B chromatography and Mono-S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus . They were referred to as p1, p2 and p3, according to their elution order from the Mono-S column . Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7-kDa DNA-binding proteins previously isolated in Sulfolobus strains {Choli, T., Wittman-Liebold, B . & Reinhardt, R . (1988) J . Biol . Chem . 263, 7087-7093} . The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE . Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure . Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7-7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate . The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity . This was also confirmed by incubating p2 with tRNA(fMet)Met (fMet, N-formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3'-phosphooligonucleotides.

Biochemistry, 1993 Jan 12, 32(1), 153 - 63
Thermal unfolding of a group I ribozyme: the low-temperature transition is primarily disruption of tertiary structure; Banerjee AR et al.; Little is known about the folding pathways of RNA . A particularly interesting RNA is L-21 Sca I, a linear form of the self-splicing intron from the precursor of the Tetrahymena thermophila large subunit (LSU) rRNA . Thermal unfolding of L-21 Sca I is studied by UV absorption and chemical mapping in 50 mM Na+ and 10 mM free Mg2+ at pH 7.5 . UV melting experiments identify two major transitions with maxima at 65 and 73 degrees C . Chemical mapping at the beginning and middle of the first transition suggests it primarily involves disruption of tertiary structure . Phylogenetic comparisons suggest a potential tertiary interaction between loops L2.1 and L9.1a . Chemical mapping and melting experiments on a truncated form of the intron lacking P9.1a, L-21 Nhe I, are consistent with this hypothesis . The results indicate that increasing temperature disrupts tertiary interactions before disrupting secondary structure . This suggests tertiary interactions are weaker than secondary interactions in this case . These results support an important assumption for RNA structure prediction: that secondary structure dominates the free energy of folding.

J Mol Biol, 1993 Jan 5, 229(1), 85 - 93
Cloning and overexpression of the triosephosphate isomerase genes from psychrophilic and thermophilic bacteria . Structural comparison of the predicted protein sequences; Rentier-Delrue F et al.; We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C . 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments . The TIM gene from psychrophilic bacteria Moraxella sp . TA137 was cloned and its nucleotide sequence determined . Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM . Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity . Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B . stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics . To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known . From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues.

J Biol Chem, 1993 Jan 5, 268(1), 601 - 7
Prokaryotic elongation factor Tu is phosphorylated in vivo; Lippmann C et al.; Covalent modification of proteins by phosphate transfer reactions constitutes a major mechanism of regulation in higher eukaryotes . Recently, phosphorylation of eukaryotic elongation factors has been described . Analysis of Escherichia coli proteins revealed several of them to be phosphorylated . Various lines of evidence lead us to conclude that one of these proteins is identical to elongation factor (EF) Tu, which can be phosphorylated in vivo at one of its threonine residues . Structural analysis showed that one fragment of the phosphorylated EF-Tu is highly resistant to tryptic digestion . Phosphorylation of eubacterial EF-Tu is not restricted to the E . coli factor but could also be demonstrated for Thermus thermophilus HB8 EF-Tu . Overexpression of tufA did not increase the number of EF-Tu molecules to be phosphorylated . This may indicate that a constant but limited amount of EF-Tu is modified, possibly for a specific function . Phosphorylation of EF-Tu could also be demonstrated in vitro . Upon analysis of subcellular fractions the highest kinase activity was found in the ribosomal fraction of E . coli . Protein sequencing of both the in vivo and in vitro phosphorylated protein revealed position 382 as the modified threonine residue.

J Biol Chem, 1993 Jan 5, 268(1), 325 - 9
Characterization of the metal centers of the corrinoid/iron-sulfur component of the CO dehydrogenase enzyme complex from Methanosarcina thermophila by EPR spectroscopy and spectroelectrochemistry; Jablonski PE et al.; The multienzyme carbon monoxide dehydrogenase complex from Methanosarcina thermophila contains at least two protein components: a CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and a cobalt-containing corrinoid/iron-sulfur component (Co/Fe-S) . The CO dehydrogenase complex has been shown to synthesize acetyl-CoA from CoA, CH3I, and CO as well as to cleave acetyl-CoA into its methyl, carbonyl, and CoA components as the first step in the catabolism of acetyl-CoA to methane and CO2 . Presumed to serve as an acceptor of the methyl group of acetyl-CoA en route to methane, the Co/Fe-S component contains iron, acid-labile sulfur, and a corrinoid cofactor (factor III) that is the site of methylation . Using EPR spectroscopy and spectroelectrochemistry, we characterized the cobalt and Fe-S centers of the Co/Fe-S component . The redox and EPR properties of the metal centers in the isolated Co/Fe-S component are similar to those of the Co/Fe-S component in the CO dehydrogenase enzyme complex, a result that indicates that any protein-protein interaction between components in the complex has little influence on the properties of the metal centers . The corrinoid is maintained in the base-off state with a formal equilibrium reduction potential (E'o) at pH 7.8 of -486 mV for the Co2+/1+ couple that facilitates reduction of the Co2+ state by approximately 12 kcal/mol relative to base-on cobamides . The Co/Fe-S component also contains a {4Fe-4S}2+/1+ cluster with an E'o at pH 7.8 of -502 mV, which is nearly isopotential with the Co2+/1+ couple of the cobamide.

J Struct Biol, 1993 Jan-Feb, 110(1), 84 - 9
Visualization of a ternary complex of the Escherichia coli Phe-tRNA(Phe) and Tu.GTP from Thermus thermophilus by scanning transmission electron microscopy; Blechschmidt B et al.; Scanning transmission electron microscopy (STEM) was used to visualize formation of a ternary complex between the T . thermophilus elongation factor (EF) Tu.GTP and the Escherichia coli Phe-tRNA(Phe) labeled with an undecagold (Au11) cluster at minor nucleotide 3-(3-amino-3-carboxypropyl) uridine at position 47 . The ternary complex was further characterized by the molecular mass and radius of gyration calculated from the mass distribution within the individual particles . Under conditions used for STEM imaging, the ternary complex is formed between Au11-labeled Phe-tRNA(Phe) and Tu.GTP in a yield up to 25% . The stoichiometry of EF-Tu.GTP to aminoacyl-tRNA (aa-tRNA) in the EF-Tu.GTP.aa-tRNA complex is 1:1, in agreement with the established view of the protein biosynthesis mechanism . The ternary complex is also formed, although to a lower extent, with GTP analogues (GMPPCP and GMPPNP, respectively), but not with Tu.GDP and nonaminoacylated tRNA(Phe) with Tu.GTP.

Arch Microbiol, 1993, 159(3), 213 - 9
N5,N10-methenyltetrahydromethanopterin cyclohydrolase from the extremely thermophilic sulfate reducing Archaeoglobus fulgidus: comparison of its properties with those of the cyclohydrolase from the extremely thermophilic Methanopyrus kandleri; Klein AR et al.; Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83 degrees C and 98 degrees C, respectively . Both Archaea contain an active N5,N10-methenyltetrahydromethanopterin cyclohydrolase . The enzyme from M . kandleri has recently been characterized . We describe here the purification and properties of the enzyme from A . fulgidus . The cyclohydrolase from A . fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M . kandleri . The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties . They differed, however, significantly with respect of the effect of K2HPO4 and of other salts on the activity and the stability . The cyclohydrolase from A . fulgidus required relatively high concentrations of K2HPO4 (1 M) for optimal thermostability at 90 degrees C but did not require salts for activity . Vice versa, the enzyme from M . kandleri was dependent on high K2HPO4 concentrations (1.5 M) for optimal activity but not for thermostability . Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts . The molecular basis for these differences are discussed.

J Eukaryot Microbiol, 1993 Jan-Feb, 40(1), 10 - 3
The Golgi apparatus of Tetrahymena thermophila; Kurz S et al.; Electron microscopic investigations reveal that the Golgi apparatus of Tetrahymena thermophila consists of numerous tiny dictyosomes, each consisting of one or two cisternae . The dictyosomes are localized predominantly in the cell cortex closely associated with the mitochondria, arranged in meridians alternating with the ciliary meridians . We estimated about 300-400 of these dictyosomes in the periphery of a cell, a value corresponding to the number of somatic cilia per cell . Cytochemical assays of thiamine pyrophosphatase and acid phosphatase, both marker enzymes of trans Golgi cisternae, resulted in deposits of lead or cerium phosphate in the outermost cisternae of the dictyosomes . In addition, cisternae located at the bases of the basal body/parasomal sac arrangements are stained . This indicates that these cisternae may belong to the Golgi apparatus of the cell.

Comp Biochem Physiol B, 1993 Jan, 104(1), 7 - 13
Evidence for alpha and Mu class glutathione S-transferases in a number of fungal species; Sheehan D et al.; 1 . The glutathione S-transferases of the following fungal species have been affinity purified on glutathione-Sepharose; Yarrowia lipolytica (NCYC825), Sporotrichum thermophile (ATCC16479), Talaromyces emersonii (ATCC26914) and Issatchenkia orientalis (CBS5147 and an isolate provided by Prof . Kumagai, Kyoto University) . 2 . SDS-PAGE analysis of affinity purified fungal extracts, followed by Western blots with polyclonal antisera to rat GSTs, was performed and indicated the presence of Alpha and Mu class glutathione S-transferases in the fungi immunologically and electrophoretically similar to the rat enzymes . 3 . Each extract gave a characteristic affinity-gradient elution pattern on glutathione-agarose.

FEMS Microbiol Lett, 1993 Jan 1, 106(1), 117 - 22
Effects of immobilization on growth, morphology, and DNA content of the ciliated protozoon Tetrahymena thermophila; Kiy T et al.; Morphological and physiological properties of Tetrahymena thermophila immobilized by encapsulation in calcium-alginate hollow spheres were found to be substantially different from those of suspended cells . Immobilized T . thermophila reached lengths of 70-100 microns, whereas the average cell of suspension cultures was about 40 microns long . Suspended cells appeared typically pear-shaped while immobilized cells developed a proboscis-like anterior end . Contrary to suspended T . thermophila, encapsulated cells were functionally deficient in phagocytosis although developing an oral apparatus . The diameter of the macronucleus of immobilized cells was about two times larger than the macronucleus of suspended cells and contained twice as much DNA, while the DNA content of the micronucleus remained unchanged . High cell density fermentations of suspended cells indicated that the alterations observed in immobilized cells were not due to close physical contacts between the cells.

Can J Microbiol, 1993 Jan, 39(1), 46 - 51
Use of bacteriophage for the selective isolation of thermophilic actinomycetes from composted eucalyptus bark; Kurtboke DI et al.; A method was developed to reduce the numbers of thermophilic bacteria on isolation plates, which in turn facilitated the detection and isolation of thermophilic actinomycetes . The method involves exposing the test material to bacteriophage suspensions prior to inoculation on isolation plates . This method was applied to composted eucalyptus bark samples, which were then inoculated on R8 and 1/2 TSA + 0.2% casein hydrolysate agar plates . The phage susceptibility of thermophilic bacteria provided a selective means of reducing their numbers on isolation plates and hence increased the numbers of Thermomonospora, Saccharopolyspora rectivirgula, and thermophilic Streptomyces spp . on these media in comparison with the numbers recorded from control plates.

FASEB J, 1993 Jan, 7(1), 196 - 200
5S rRNA modification in the hyperthermophilic archaea Sulfolobus solfataricus and Pyrodictium occultum; Bruenger E et al.; The 5S rRNAs from Sulfolobus solfataricus and Pyrodictium occultum were digested to nucleosides and analyzed using directly-combined HPLC/mass spectrometry . P . occultum 5S rRNA contains two modified nucleoside species, N4-acetylcytidine (ac4C) and N4-acetyl-2'-O-methylcytidine (ac4Cm) . Oligonucleotides were generated from P . occultum 5S rRNA by RNase T1 hydrolysis, and their molecular weights were determined using electrospray mass spectrometry and compared with those predicted from the P . occultum 5S RNA gene sequence . Deviation in mass between expected and observed molecular weights permitted ac4Cm to be located at position 35, in the nonanucleotide CAA-CACC{ac4Cm}G, and the ac4C in one or both of two (C,U)G trinucleotides . 2'-O-Methylcytidine is unambiguously characterized in S . solfataricus 5S rRNA, confirming earlier tentative assignments at the analogous sequence position (Stahl, D.A., Luehrsen, K.R., Woese, C.R., and Pace, N.R . (1981) Nucleic Acids Res., Vol . 9, pp . 6129-6137; Dams, E., Londei, P., Cammarano, P., Vandenberghe, A., and De Wachter, R . (1983) Nucleic Acids Res . Vol . 11, pp . 4667-4676) . Potential effects of the presence of ac4C and ac4Cm on thermal stabilization of 5S rRNA in thermophiles are discussed.

Mol Cell Biol, 1993 Jan, 13(1), 163 - 73
An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila; Wang T et al.; In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei . Biochemical data document the fact that HMG B is extremely labile in micronuclei . Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei . Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication . This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair . Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin . However, micronuclei are transcriptionally active during early stages of conjugation . Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented . Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration . We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

J Bacteriol, 1993 Jan, 175(1), 80 - 4
Membrane ATPase from the aceticlastic methanogen Methanothrix thermophila; Inatomi K et al.; A new isolate of the aceticlastic methanogen Methanothrix thermophila utilizes only acetate as the sole carbon and energy source for methanogenesis (Y . Kamagata and E . Mikami, Int . J . Syst . Bacteriol . 41:191-196, 1991) . ATPase activity in its membrane was found, and ATP hydrolysis activity in the pH range of 5.5 to 8.0 in the presence of Mg2+ was observed . It had maximum activity at around 70 degrees C and was specifically stimulated up to sixfold by 50 mM NaHSO3 . The proton ATPase inhibitor N,N'-dicyclohexylcarbodiimide inhibited the membrane ATPase activity, but azide, a potent inhibitor of F0F1 ATPase (H(+)-translocating ATPase of oxidative phosphorylation), did not . Since the enzyme was tightly bound to the membranes and could not be solubilized with dilute buffer containing EDTA, the nonionic detergent nonanoyl-N-methylglucamide (0.5%) was used to solubilize it from the membranes . The purified ATPase complex in the presence of the detergent was also sensitive to N,N'-dicyclohexylcarbodiimide, and other properties were almost the same as those in the membrane-associated form . The purified enzyme revealed at least five kinds of subunits on a sodium dodecyl sulfate-polyacrylamide gel, and their molecular masses were estimated to be 67, 52, 37, 28, and 22 kDa, respectively . The N-terminal amino acid sequences of the 67- and 52-kDa subunits had much higher similarity with those of the 64 (alpha)- and 50 (beta)-kDa subunits of the Methanosarcina barkeri ATPase and were also similar to those of the corresponding subunits of other archaeal ATPases . The alpha beta complex of the M . barkeri ATPase has ATP-hydrolyzing activity, suggesting that a catalytic part of the Methanothrix ATPase contains at least the 67- and 52-kDa subunits.

J Bacteriol, 1993 Jan, 175(1), 103 - 10
Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8; Borges KM et al.; A physical map of the chromosome of the extremely thermophilic eubacterium Thermus thermophilus HB8 has been constructed by using pulsed-field gel electrophoresis techniques . A total of 26 cleavage sites for the rarely cutting restriction endonucleases HpaI, MunI, and NdeI were located on the genome . On the basis of the sizes of the restriction fragments generated, the genome size was estimated to be 1.74 Mbp, which is significantly smaller than the chromosomes of Escherichia coli and other mesophiles . Partial digestion experiments revealed the order of the six HpaI bands on the chromosome . Hybridization of isolated restriction fragments to pulsed-field gel-separated restriction digestions confirmed the deduced order of the HpaI fragments and allowed ordering and alignment of the NdeI and MunI fragments . In addition, 16 genes or gene clusters cloned from several different Thermus strains were located on the T . thermophilus HB8 chromosomal map by hybridization of gene probes to pulsed-field gel-resolved restriction digestions.

Dev Biol, 1993 Jan, 155(1), 198 - 205
Costimulation-induced rounding in Tetrahymena thermophila: early cell shape transformation induced by sexual cell-to-cell collisions between complementary mating types; Fujishima M et al.; Mixing of starved cells of complementary mating types of Tetrahymena thermophila induces shortening of their longitudinal length within 10 min of mixing . This early morphogenetic transformation in preconjugant sexual interaction (costimulation period) was named "costimulation-induced rounding" (CIR) . CIR is the earliest morphological change that has ever been found in the costimulation period and differs from "synchronous rounding" in the vegetative cell cycle, because CIR cells are still able to form food vacuoles, while cells in synchronous rounding do not have this ability . When sexual cell-to-cell collisions between two mating types were hampered by unidirectional stirring for 20 min after mixing of the two mating types, both CIR and conjugation were delayed by 20 min . When secreted materials needed for the onset of costimulation were removed by washing the cells with 10 mM Tris-HCl, pH 7.4, before mixing the two mating types, both CIR and conjugation were delayed by about 30 min . CIR-like rounding was not induced by cell-free medium either from the opposite mating type or from mixed costimulated cells . These results indicated that CIR is induced when cells are activated to form conjugating pairs by cell-to-cell collisions between complementary mating types in the presence of secreted molecules.

Proteins, 1993 Jan, 15(1), 108 - 11
Crystallization and preliminary crystallographic analysis of ribonuclease H from Thermus thermophilus HB8; Okumura M et al.; Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength . The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with unit cell parameters a = b = 44.7 A, c = 314.7 A . They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 A resolution.

Plasmid, 1993 Jan, 29(1), 1 - 9
On two transposable elements from Bacillus stearothermophilus; Xu K et al.; Two transposable elements, IS5376 and IS5377, were identified in the thermophile Bacillus stearothermophilus CU21 based upon the following criteria: (1) both were found to appear on different plasmids introduced into the same host CU21; (2) signals of homology were found between the genomic DNA of CU21 and each of them; (3) different numbers of Southern hybridization bands were found for the genomic DNA of different strains of B . stearothermophilus; and (4) characteristic inverted repeats at both ends and direct repeats of the target DNA adjacent to them were found for both IS5376 and IS5377 . Two open reading frames (ORFs) were detected for IS5376 and one for IS5377 . The putative coding products of the ORFs are homologous to those of known ISs from mesophiles and are considered to be transposases . The results of analyses of nucleotide sequence and the deduced amino acid sequence suggest that IS5376 is a member of the IS21 family and that IS5377 is a member of the IS4 family.

Protein Eng, 1993 Jan, 6(1), 85 - 91
Structural study of mutants of Escherichia coli ribonuclease HI with enhanced thermostability; Ishikawa K et al.; Systematic replacement of the amino acid residues in Escherichia coli ribonuclease HI with those in the thermophilic counterpart has revealed that two mutations, His62-->Pro (H62P) and Lys95-->Gly (K95G), increased the thermostability of the protein . These single-site mutant proteins, together with the mutant proteins His62-->Ala (H62A), Lys95-->Asn (K95N) and Lys95-->Ala (K95A), were crystallized and their structures were determined at 1.8 A resolution . The crystal structures of these mutant proteins reveal that only the local structure around each mutation site is essential for the increase in thermostability . For each mutant protein, the stabilization mechanism is considered to be as follows: (i) H62P is stabilized because of a decrease in the entropy of the unfolded state, without a change in the native backbone structure; (ii) K95G is stabilized since the strain caused by the left-handed backbone structure in the typical 3:5 type loop is eliminated; and (iii) K95N is slightly stabilized by a hydrogen bond formed between the side-chain N delta-atom of the mutated aspargine residue and the main-chain carbonyl oxygen within the same residue.

Microbios, 1993, 75(302), 23 - 32
Tertiary and quaternary branched polyamines distributed in thermophilic Saccharococcus and Bacillus; Hamana K et al.; The distribution of long linear, tertiary and quaternary branched polyamines in twenty thermophilic Gram-positive strains belonging to the genera Saccharococcus, Bacillus or Amphibacillus were analysed by high-performance liquid chromatography and gas chromatography . The extremely thermophilic S . thermophilus contained a tertiary tetra-amine, N4-aminopropylspermidine, a linear penta-amine, thermopentamine, a tertiary penta-amine, N4-aminopropylspermine, and a quaternary penta-amine, N4-bis(amino-propyl)spermidine . N4-aminopropylspermine {NH2(CH2)3N((CH2)3NH2)(CH2)4NH(CH2)3NH2} first found in this study, was also detected in the extremely thermophilic 'B . caldolyticus' and 'B . caldotenax' and the moderately thermophilic B . stearothermophilus, B . smithii and B . thermocatenulatus.

Microbios, 1993, 74(301), 227 - 32
The effect of an electric field on the release of hexosaminidase in Tetrahymena; Bencsath M et al.; The activity of beta-D-hexosaminidase was detected by spectrofluorometry in the growth media of three species of Tetrahymena . The enzyme activity was about six times higher in T . pyriformis compared with the wild cells of T . thermophila, while the MS-1 mutant of T . thermophila manifested very low enzyme activity under the same experimental conditions . An electric field and electrical pulses produced significant though different increases in enzyme activity in media of wild and mutant T . thermophila . In contrast, T . pyriformis responded to the field pulses by decreasing the activity of the enzymes detected in the growth media.

Biosens Bioelectron, 1993, 8(2), 89 - 98
An integrated silicon thermophile as biosensor for the thermal monitoring of glucose, urea and penicillin; Bataillard P et al.; A new kind of calorimetric biosensor for the measurement of the heat (molar enthalpy change) of enzymatic reactions is presented . The device operates according to the Seebeck effect, the same principle on which thermocouples are based . The thermopile used in this work consists of an array of p-type silicon/aluminium strips integrated on a thin silicon membrane (5 microns) . Its sensitivity is about 1 V output voltage per watt of heating power, corresponding to a temperature resolution in the order of 10(-5) K and a heating power resolution of some tenths of a mu W in the flow system used . Furthermore, this performance is obtained without any control of external temperature because of the high common-mode thermal noise rejection ratio of the thermopile . The universal technique of calorimetry combined with the specificity of biochemical reactions makes this biosensor very versatile, with a broad range of possible applications . Glucose oxidase together with catalase for the determination of glucose, urease and penicillinase for the monitoring of urea and penicillin G, respectively, were immobilized directly onto the back side of the thermopile . The sensor was operated in conjunction with flow injection analysis which, in addition to its traditional advantages, allows preconditioning of the samples . Thus, artefacts due to mixing effects were suppressed and interference caused by differences in ionic strength between sample and carrier was strongly decreased . Detection limits between 1 and 2 mM were reported in the flow injection conditions described.

DNA Seq, 1993, 4(1), 1 - 9
Two putative insertion sequences flank a truncated glycogen branching enzyme gene in the thermophile Bacillus stearothermophilus CU21; Kiel JA et al.; We have isolated a region from the Bacillus stearothermophilus CU21 chromosome hybridizing strongly to a fragment of the B . caldolyticus glycogen operon . Sequence analysis of this region revealed the presence of a truncated glgB gene encoding the N-terminus of branching enzyme . A region highly similar to an internal fragment of B . caldolyticus glgC encoding ADP-glucose pyrophosphorylase was located approximately 1kb downstream from the incomplete glgB gene . The two truncated genes appeared to flank a sequence with characteristics of bacterial Insertion Sequences, which was designated RSBst-alpha . The presence of RSBst-alpha at this position indicates that integration of (an) IS-like element(s) may have been involved in deletion formation in the putative glycogen operon . Upstream of glgB an additional incomplete ORF was found with significant similarity to putative transposases from bacterial Insertion Sequences . This region was designated RSBst-beta . Both RSBst-alpha and RSBst-beta appeared to be present in multiple copies in the B . stearothermophilus CU21 chromosome.

Microbios, 1993, 76(308), 153 - 60
Differentiation of thermophilic species of Campylobacter, in particular C . coli and C . jejuni, with atypical characteristics, by analysis of protein-banding profiles on non-denaturing polyacrylamide gels; Kato Y et al.; The protein-banding patterns after electrophoresis of 23 strains of thermophilic Campylobacter, including 17 strains of C . coli and C . jejuni which had atypical characteristics with respect to the hydrolysis of hippurate and susceptibility to nalidixic acid, were characterized . Of the atypical strains of C . coli and C . jejuni 16 out of 17 gave protein-banding patterns which were essentially identical to those of typical strains, and distinct patterns were obtained from strains of two other species . The banding patterns of soluble proteins on non-denaturing polyacrylamide gels after electrophoresis appear to be useful for the differentiation of atypical strains of C . coli and C . jejuni . Strain 11791 which was originally identified as C . coli was identified as C . lari from its protein-banding profile.

Annu Rev Microbiol, 1993, 47, 791 - 819
The cellulosome: the exocellular organelle of Clostridium; Felix CR et al.; The cellulolytic enzyme complex of the anaerobic thermophile Clostridium thermocellum is reviewed . This complex, called the cellulosome, is cell associated and has a mass of from 2 x 10(6) to 6.5 x 10(6) Daltons . It consists of from 14 to 26 different polypeptides . Cellulosomes form larger complexes, polycellulosomes, with masses from 50 x 10(6) to 80 x 10(6) Daltons . The cellulosome efficiently hydrolyzes crystalline cellulose whereas individual polypeptides alone or in mixtures do not . Many of the polypeptides are catalytically active and can be characterized as endoglucanases, xylanases, and cellodextrinases . Several of the polypeptides have been sequenced including the largest subunit, CipA, that is a glycoprotein with a mass of 210 kDa . CipA has a cellulose-binding domain and nine internal repeated sequences postulated to bind eight catalytic subunits and a special peptide (ORF3p) . The ORF3p anchors the CipA to the cell surface . CipA can be characterized as a scaffold holding the catalytic subunits that line up with the cellulose fiber . This arrangement allows a multiple cutting of the cellulose glucan chain . A similar system has been observed for other cellulosome-like complexes, notably Clostridium cellulovorans.

Cell Motil Cytoskeleton, 1993, 25(3), 243 - 53
Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single alpha- and beta-tubulin; Gaertig J et al.; We have cloned and sequenced the two beta-tubulin genes of the ciliated protozoan Tetrahymena thermophila . The two genes encode identical 443 amino acid peptides which are 99.7% identical to the beta-tubulin proteins of T . pyriformis and 95% identical to human beta 1 tubulin . T . thermophila contains only one alpha-tubulin gene (Callahan et al., 1984: Cell 36:441-445) . Thus, all of the extremely diverse microtubule structures in this unicellular organism can be formed from a single alpha- and a single beta-tubulin peptide . We have also carried out a phylogenetic analysis of 84 complete beta-tubulin peptide sequences . This analysis supports two hypotheses regarding beta-tubulin evolution and function: 1) Multifunctional beta-tubulins are under greater evolutionary constraint than beta-tubulins present in specialized cells or in cells with very few microtubule related functions, which can evolve rapidly; and 2) Cells which form axonemes maintain a homogeneous population of tubulins.

Arch Microbiol, 1993, 160(3), 186 - 92
Two N5,N10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme; Klein AR et al.; It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor . We describe here the presence in this Archaeon of a second N5,N10-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F420-dependent . This enzyme was purified and characterized . The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7 +/- 0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa . The enzyme activity increased to an optimum with increasing salt concentrations . Optimal salt concentrations were e.g . 2 M (NH4)2SO4, 2 M Na2HPO4, 1.5 M K2HPO4, and 2 M NaCl . In the absence of salts the enzyme exhibited almost no activity . The salts affected mainly the Vmax rather than the Km of the enzyme . The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group . In the presence of 1 M (NH4)2SO4 the Vmax was 4000 U/mg (kcat = 2400 s-1) and the Km for N5,N10-methylenetetrahydromethanopterin and for coenzyme F420 were 80 microM and 20 microM, respectively . The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K2HPO4 at 90 degrees C for one hour . The N-terminal amino acid sequence was found to be similar to that of the F420-dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.

Biochimie, 1993, 75(12), 1159 - 66
Ternary complex between elongation factor Tu.GTP and Phe-tRNA(Phe); Forster C et al.; The effect of aminoacylation and ternary complex formation with elongation factor Tu.GTP on the tertiary structure of yeast tRNA(Phe) was examined by 1H-NMR spectroscopy . Esterification of phenylalanine to tRNA(Phe) does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA . Complex formation of Phe-tRNA(Phe) with elongation factor Tu.GTP results in a broadening of all imino proton resonances of the tRNA . The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA(Phe) upon binding to elongation factor Tu.GTP . All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA(Phe)-elongation factor Tu-GTP ternary complex . Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA(Phe) preserves its typical L-shaped tertiary structure in the complex . At high (> 10(-4) M) ligand concentrations a complex between tRNA(Phe) and elongation factor Tu-GDP can be detected on the NMR time-scale . Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure . Using the known tertiary structures of yeast tRNA(Phe) and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.

Biochimie, 1993, 75(12), 1091 - 8
Phenylalanyl-tRNA synthetase from Thermus thermophilus has four antiparallel folds of which only two are catalytically functional; Mosyak L et al.; Phenylalanyl-tRNA synthetase from Thermus thermophilus has an alpha 2 beta 2 type quaternary structure and is one of the most complicated members of the synthetase family . Identification of PheRSTT as a member of class II aaRSs was based only on sequence alignment of the small alpha-subunit with other synthetases . The three-dimensional crystal structure of the catalytic and 'catalytic-like' domains at 2.9 A resolution in PheRSTT is described . The alpha-subunit contains an antiparallel fold which includes signature motifs 1, 2 and 3, characteristic of class II synthetases . One of the three structural domains of the beta-subunit (alpha'-domain) is formed by a seven-stranded antiparallel beta-sheet surrounded by alpha-helices similar to catalytic domains in SerRS, AspRS and the alpha-subunit of PheRSTT . The alpha beta heterodimer (alpha and alpha') exhibits essentially the same topology in the intersubunit region as in the known alpha 2 structures of class II aaRS's . The multimerization area of whole PheRSTT molecule comprises a quasi-tetrahedral four-helix bundle.

Nutr Cancer, 1993, 20(3), 261 - 70
Antigenotoxic properties of lactic acid bacteria in the S . typhimurium mutagenicity assay; Pool-Zobel BL et al.; A high percentage of human tumors is reported to be related to dietary habits . One way to improve the nutritional impact is to increase the intake of protective factors, such as inhibitors of DNA damage and other types of anticarcinogens . Specific strains of lactic acid bacteria used to ferment milk are promising candidates that may be antimutagenic and anticarcinogenic . We have studied the antimutagenicity of 10 isolated strains of beneficial lactic acid bacteria . Four types of fermented milk products were also studied for their protective properties . The effect of these bacteria on the yield of revertants induced by nitrosated beef extract was investigated in the Salmonella typhimurium mutagenicity assay . Eight of 10 isolated Lactobacillus strains reduced the yield of his+ revertants almost back to the levels of the untreated controls . Different fermented fresh yogurts containing viable bacteria (probably Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus thermophilus or Lactobacillus acidophilus and Bifidobacteria) showed protective effects as well . The degree of suppressing revertants was independent of the yogurt's fat content . In contrast, yogurt products that had been heat treated were not inhibitory . The other fresh fermented milk products (e.g., buttermilk, kefir, and "Dickmilch") were not antimutagenic in this study . The results imply that some bacteria used in milk processing have an antimutagenic potential and that this property is specific for the bacterial strain.

J Bacteriol, 1993 Jan, 175(2), 474 - 8
Nitrogen fixation by the thermophilic green sulfur bacterium Chlorobium tepidum; Wahlund TM et al.; The thermophilic green sulfur bacterium Chlorobium tepidum grew with N2, NH4+, or glutamine as the sole nitrogen source under phototrophic (anaerobic-light) conditions . Growth on N2 required increased buffering capacity to stabilize uncharacterized pH changes that occurred during diazotrophic growth . Increased sulfide levels were stimulatory for growth on N2 . Levels of nitrogenase activity (acetylene reduction) in N2-grown C . tepidum cells were very high, among the highest ever reported for anoxygenic phototrophic bacteria . Maximal acetylene reduction rates in C . tepidum cells were observed at 48 to 50 degrees C, which is about 15 degrees C higher than the optimum temperature for nitrogenase activity in mesophilic chlorobia, and nitrogenase activity in C . tepidum responded to addition of ammonia by a "switch-off/switch-on" mechanism like that in phototrophic purple bacteria . C . tepidum cells assimilated ammonia mainly via the glutamine synthetase-glutamate synthase pathway, elevated levels of both of these enzymes being present in cells grown on N2 . These results show that N2 fixation can occur in green sulfur bacteria up to at least 60 degrees C and that regulatory mechanisms important in control of nitrogenase activity in mesophilic anoxygenic phototrophs also appear to regulate thermally active forms of the enzyme.

Antonie Van Leeuwenhoek, 1993-94, 64(3-4), 357 - 86
A polyphasic taxonomic study of thermophilic bacilli from a wide geographical area; White D et al.; Two hundred and thirty-four thermophilic Bacillus strains isolated from geographically widespread locations were examined by phenotypic characterisation followed by numerical analysis . The strains were distributed between eighteen cluster-groups which were subsequently evaluated in DNA base composition and DNA sequence homology studies . The inclusion of type and reference strains unambiguously identified strains related to B . licheniformis, B . pallidus, B . smithii, B . stearothermophilus, B . thermocloacae and B . thermoglucosidasius . Other reference strains included in distinctive groups were 'B . caldotenax', together with 'B . caldovelox' and 'B . caldolyticus', B . kaustophilus and 'B . thermodenitrificans' . An emended description of B . kaustophilus is provided . It is proposed that 'B . caldotenax' and 'B . thermodenitrificans' should be accepted as validly described species . Members of other clusters that appeared to have distinctive characteristics, including beta-glucanase production and the ability to degrade tyrosine, may provide the nuclei of further novel species.

Antonie Van Leeuwenhoek, 1993-94, 64(3-4), 341 - 55
A biphasic approach to the determination of the phenotypic and genotypic diversity of some anaerobic, cellulolytic, thermophilic, rod-shaped bacteria; Rainey FA et al.; A biphasic approach, involving a numerical phenetic and a phylogenetic study, was used to determine diversity among some anaerobic, cellulolytic, thermophilic, rod-shaped bacteria . Ninety two characters were determined for 51 strains in the numerical taxonomy study, and partial 16S rDNA sequences from 16 isolates were compared . Both the phenetic and phylogenetic data indicate diversity within this group of organisms, and reveal the lack of similarity between sporogenous and asporogenous isolates . The results of the phylogenetic study demonstrate the lack of relationship of the majority of the strains studied to previously studied thermophilic bacteria . In general, good correlation exists between the two data sets, but discrepancies arise when strains with a high level of similarity are examined . The need for caution in the interpretation of data obtained from such a biphasic approach is discussed.

Antonie Van Leeuwenhoek, 1993-94, 64(3-4), 325 - 40
Biosystematics and diversity amongst novel carboxydotrophic actinomycetes; O'Donnell AG et al.; Fifty-four carboxydotrophic actinomycetes isolated from soils and composts were compared through 119 unit characters with representative mesophilic and thermophilic streptomycetes . The data were examined using the Jaccard, pattern and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm . Acceptable cophenetic correlation and test error values allowed confidence to be placed in the resultant numerical taxonomies . The carboxydotrophic actinomycetes, which were distinct from cluster-groups corresponding to the mesophilic and thermophilic streptomycetes, formed two major cluster-groups the members of which were examined for the presence of diagnostic chemical markers . All but two of the carboxydotrophic actinomycetes had a profile of chemical properties consistent with their assignment to the genus Streptomyces . Quantitative fatty acid data were examined using the SIMCA package and the two statistically significant groups obtained corresponded with the cluster-groups circumscribed in the numerical phenetic analysis . Members of the two groups were also distinguished on the basis of their phospholipid composition . The two strains that contained meso-as opposed to LL-diaminopimelic acid in their peptidoglycan also showed a distinct chemotaxonomic profile . It was concluded that the carboxydotrophic actinomycetes form a novel and taxonomically diverse group.

Biosci Biotechnol Biochem, 1993 Jan, 57(1), 93 - 7
Thermolabile alanine racemase from a psychotroph, Pseudomonas fluorescens: purification and properties; Yokoigawa K et al.; A psychotrophic bacterium that produces a thermolabile alanine racemase was isolated from raw milk, and identified as Pseudomonas fluorescens TM5-2 . The enzyme was purified to homogeneity from the cell extract, and characterized to be compared with enzymes from mesophiles (Bacillus subtilis and Salmonella typhimurium) and a thermophile (Bacillus stearothermophilus) . The enzyme has a molecular weight of about 76,000 and consists of two subunits identical in molecular weight (38,000) . The enzyme contains two mol of pyridoxal 5'-phosphate per mol as a coenzyme . The amino acid composition was different from those of other alanine racemases in content of valine . The amino acid sequence of the amino terminal region (from 1Met to 25Gly) had 21-33% homology with those of other alanine racemases . Kinetic parameters of the enzyme were similar to those of other alanine racemases . The enzyme is extremely labile over 30 degrees C, and shows the high catalytic activity even at 0 degrees C; it is thermolabile and psychotrophic.

Nucleic Acids Res, 1992 Dec 25, 20(24), 6525 - 33
Replication-dependent and independent regulation of HMG expression during the cell cycle and conjugation in Tetrahymena; Wang T et al.; Two abundant high-mobility-group (HMG)-like proteins, HMG B and HMG C, exist in the ciliated protozoan, Tetrahymena thermophila . Of these, HMG C is specific to transcriptionally active macronuclei, while HMG B is found in macronuclei and in transcriptionally inactive micronuclei {1} . Using Northern and in situ analyses, we show that the genes encoding HMG B and HMG C are not expressed uniformly throughout the vegetative cycle or during the sexual process, conjugation . Elevated expression of both genes is observed during macronuclear S phase of the vegetative cycle and during endoreplication of developing new macronuclei in later stages of conjugation . Interruption of any of these macronuclear DNA replications by aphidicolin leads to a rapid drop in the message levels of HMG B and HMG C . These results resemble what is typically observed for replication-dependent nucleosomal histones and differ from the apparent lack of cell cycle regulation observed for HMG genes in vertebrates . A specific-induction of HMG B mRNA is also observed early in conjugation and during this interval, inhibition of micronuclear DNA synthesis by aphidicolin does not affect the message level of HMG B . Thus, during conjugation, expression of HMG B shows both replication-dependent and independent regulation . Results similar to these with HMG B are obtained with histone H4II gene, a gene which is also expressed during micro- and macronuclear S phases during the vegetative cycle . These results demonstrate surprising complexity in the expression of HMG genes in Tetrahymena and lend support to the hypothesis that cell cycle regulation plays an important role in directing HMG-like proteins to the appropriate nucleus {2} . Interestingly, expression of neither HMG gene is perfectly synchronized with that of histone H4II gene during the developmental program suggesting that important differences exist between vegetatively growing (cell cycle control) and conjugating (developmental control) cells.

Nucleic Acids Res, 1992 Dec 25, 20(24), 6501 - 7
Modular organization of related Archaeal plasmids encoding different restriction-modification systems in Methanobacterium thermoformicicum; Nolling J et al.; Nucleotide sequence comparison of the related 13513-bp plasmid pFV1 and the 11014-bp plasmid pFZ1 from the thermophilic archaeon Methanobacterium thermoformicicum THF and Z-245, respectively, revealed a homologous, approximately 8.2 kb backbone structure that is interrupted by plasmid-specific elements . Various highly conserved palindromic structures and an ORF that could code for a NTP-binding protein were identified within the backbone structure and may be involved in plasmid maintenance and replication . Each plasmid contains at comparable locations a module which specifies components of different restriction-modification (R/M) systems . The R/M module of pFV1 contained, in addition to the genes of the GGCC-recognizing R/M system MthTI, an ORF which may be involved in repair of G-T mismatches generated by deamination of m5C at high temperatures.

Vet Rec, 1992 Dec 19-26, 131(25-26), 574 - 6
Introduction and spread of thermophilic campylobacters in broiler flocks; Evans SJ; Campylobacteriosis is the most commonly reported infectious cause of human gastroenteritis in developed countries and broiler chickens are considered to be the major food-borne source of the infection . The control of the infection in man depends upon its control in broiler flocks but the epidemiology in poultry is poorly understood . Up to 50 per cent of broiler flocks may be infected and most of the birds in an infected flock carry the organisms until slaughter . Vertical transmission through the egg appears unlikely but there are many other potential sources of the infection for the chicks; direct contact with infected animals or birds has been proposed and there is also evidence for indirect transmission through drinking water or insect vectors . It is suggested that the cross-sectional studies discussed in this review should be followed by well designed case-control studies to test the aetiological hypotheses put forward.

Eur J Biochem, 1992 Dec 15, 210(3), 971 - 81
Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri; Breitung J et al.; N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield . The monomeric enzyme had an apparent molecular mass of 35 kDa . The N-terminal amino acid sequence of the polypeptide was determined . The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity . The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4 . The salts KCl, NaCl and NH4Cl did not activate the enzyme . The dependence of activity on salt concentration showed a sigmoidal curve . For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required . A detailed kinetic analysis revealed that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT . At the optimal salt concentration and at 65 degrees C, the Vmax was 2700 U/mg (1 U = 1 mumol/min), the Km for CHO-MFR was 50 microM and the Km for H4MPT was 100 microM . At 90 degrees C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65 degrees C . Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization . The efficiency of salts in protecting formyltransferase from heat inactivation at 90 degrees C decreased in the order K2HPO4 = (NH4)2SO4 >> KCl = NH4Cl = NaCl >> Na2SO4 > Na2HPO4 . The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type . The properties of the enzyme from M . kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.

FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 523 - 8
Cellulose degradation by Clostridium thermocellum: from manure to molecular biology; Beguin P et al.; Clostridium thermocellum, a Gram-positive, thermophilic anaerobe produces a highly active cellulase system . This system, termed the cellulosome, is a complex composed of at least 14-18 different types of components organized around a large, cellulose-binding protein . Combining recombinant DNA technology and protein biochemistry has proved to be a successful approach in unravelling some important features of the system.

Biochemistry, 1992 Dec 15, 31(49), 12493 - 9
Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius; In't Veld G et al.; The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated . Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L . lactis . Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S . acidocaldarius lipid content . Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S . acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S . acidocaldarius lipids . Membrane fluidity decreased with increasing S . acidocaldarius lipid content . These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.

Biochim Biophys Acta, 1992 Dec 7, 1140(2), 157 - 62
Added subunit beta of CF1 as well as gamma/delta/epsilon restore photophosphorylation in partially CF1-depleted thylakoids; Engelbrecht S et al.; We investigated the ability of subunits beta, gamma, delta, and epsilon of CF1, the F1-ATPase of chloroplasts, to interact with exposed CF0 in EDTA-treated, partially CF1-depleted thylakoid membranes . We measured the ability of subunits beta, gamma, delta, and epsilon to stimulate the rate of photophosphorylation under continuous light and, for subunit beta, also the ability to diminish the proton leakage through exposed CF0 by deceleration of the decay of electrochromic absorption transients under flashing light . The greatest effect was caused by subunit beta, followed by gamma/delta/epsilon . Pairwise combinations of gamma, delta, and epsilon or each of these subunits alone were only marginally effective . Subunit gamma from the thermophilic bacterium PS 3 in combination with chloroplast delta and epsilon was as effective as chloroplast gamma . The finding that the small CF1 subunits in concert and the beta subunit by itself specifically interacted with the exposed proton channel CF0, qualifies the previous concept of subunit delta acting particularly as a plug to the open CF0 channel . The interactions between the channel and the catalytic portion of the enzyme seem to involve most of the small, and at least beta of the large subunits.

Biochim Biophys Acta, 1992 Dec 7, 1140(2), 175 - 83
Resonance Raman studies of Rieske-type proteins; Kuila D et al.; Resonance Raman (RR) spectra are reported for the {2Fe-2S} Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength . Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K . By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO . The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1 . One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions . We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins . In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster . The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of {2Fe-2S} ferrodoxins . The profiles are bimodal with maxima near 490 nm and > approx . 550 nm . By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile . Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin . This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues . Taken as a whole, the data are consistent with an St2FeSb2Fe{N(His)}t2 structure for the Rieske-type cluster.

J Mol Biol, 1992 Dec 5, 228(3), 850 - 61
Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles; Hummel R et al.; The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance . However, its intracellular localization and biological function have remained elusive . It has generally been assumed that the F-antigen is confined phylogenetically to vertebrates . Now we have cloned and characterized a gene from the ciliated protozoan Tetrahymena thermophila encoding a protein which clearly is homologous with the rat F-antigen . The coding region of the Tetrahymena F-antigen (TF-ag) gene specifies a 46,051 M(r) protein and is interrupted by three introns . In accordance with the predicted molecular mass of the TF-ag protein, antibodies raised against a cro-lacZ'-TF-ag fusion protein specifically recognized a 45,000 M(r) protein in Western blots of total T . thermophila protein . Immunoelectron microscopy demonstrated that the TF-ag is associated with membranes of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking . Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded . The abundance of the TF-ag protein, however, declined only moderately during prolonged periods of starvation demonstrating that extensive release of the TF-ag did not take place . In combination these results suggest that the TF-ag protein is a recycled constituent of the intracellular membrane network in T . thermophila.

J Mol Biol, 1992 Dec 5, 228(3), 743 - 58
Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena; Christian EL et al.; We have developed a quantitative substitution interference technique to examine the role of Pro-Rp oxygens in the phosphodiester backbone of RNA, using phosphorothioates as a structural probe . This approach is generally applicable to any reaction involving RNA in which the precursor and reaction products can be separated . We have applied the technique to identity structural requirements in the group I intron from Tetrahymena thermophila for catalysis of hydrolysis at the 3' splice site; 44 phosphate oxygens are important in 3' splice site hydrolysis . These include four or five oxygens previously observed to be important in exon ligation . Although phosphate oxygens having a functional significance can be found throughout the intron, the strongest phosphorothioate effects are closely associated with positions in the highly conserved intron core, which are likely to be involved in tertiary interactions, substrate recognition and catalysis.

FEMS Microbiol Lett, 1992 Dec 1, 78(2-3), 261 - 4
Reversible expression of flagella in Campylobacter spp; Diker KS et al.; The in vitro phase variation of flagella and the transition rates between flagellate and aflagellate phenotypes in Campylobacter species including C . jejuni, C . coli, C . lari (thermophilic campylobacters), C . fetus subsp . fetus, C . fetus subsp . venerealis and C . hyointestinalis were investigated . The change from the flagellate to aflagellate phenotype was detected in all of the 12 Campylobacter strains studied . When measured in a motility medium, flagellate to aflagellate transition in thermophilic campylobacters, C . fetus and C . hyointestinalis strains occurred at a rate of 1.8 x 10(-3) to 7.5 x 10(-3), 3.0 x 10(-4) to 7.8 x 10(-4) and 1.8 x 10(-5) to 7.7 x 10(-6) per cell per generation, respectively . Transition from aflagellate to flagellate phenotype occurred at a rate of 5.8 x 10(-6) to 9.3 x 10(-6) per cell per generation in thermophilic campylobacters and 1.0 x 10(-6) to 1.5 x 10(-6) in C . fetus strains . No reversion from aflagellate to flagellate phenotype could be detected in C . hyointestinalis strains . It was concluded that the ability to reversibly express flagella was inherent in the wild-type strains and the transition rates for both directions were consistent for each strain.

Appl Environ Microbiol, 1992 Dec, 58(12), 3864 - 7
The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme; Gibbs MD et al.; The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues . The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase . Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.

J Bioenerg Biomembr, 1992 Dec, 24(6), 601 - 9
Energy transduction and transport processes in thermophilic bacteria; Konings WN et al.; Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure . The stability of the integral membrane proteins of thermophiles make them particularly amenable to study . Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates . Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons . Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition . Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics . Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport . Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations . The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.

Genetics, 1992 Dec, 132(4), 1017 - 31
Uniparental cytogamy: a novel method for bringing micronuclear mutations of Tetrahymena into homozygous macronuclear expression with precocious sexual maturity; Cole ES et al.; A new method of inducing self-fertilization, uniparental cytogamy, yields homozygous germinal and somatic genotypes in the ciliate Tetrahymena thermophila . Progeny are highly fertile and show a marked tendency for precocious sexual maturity . This method is highly effective in protocols designed to generate and express nonlethal dominant or recessive mutations.

Eur J Biochem, 1992 Dec 1, 210(2), 621 - 7
Chromatin structure and conserved sequence elements in genes encoding ribosomal proteins in Tetrahymena thermophila; Norgaard P et al.; The chromatin structure of the macronuclear genes encoding ribosomal proteins S25 and L1 in the ciliated protozoan Tetrahymena thermophila was analyzed . Using the indirect end-labelling technique, DNase-I-hypersensitive regions were located in the promoter regions as well as in the 3' regions of the genes . The DNase-I-hypersensitive regions were present in chromatin of exponentially growing cells, where the rate of ribosomal-protein gene transcription is high, and in chromatin from starved cells, where transcription of ribosomal-protein genes is severely depressed . Micrococcalnuclease-digestion experiments revealed that the promoter regions of the S25 gene and the L1 gene are devoid of nucleosomes in exponentially growing cells . In starved cells, no nucleosomal organisation of the promoter region of the L1 gene could be detected, whereas nucleosomal structures were discernible in the promoter region of the S25 gene . A conspicuous polypurine sequence motif, AARGGGAAA, is present within or adjacent to the DNase-I-hypersensitive regions in the promoter of the S25 and the L1 gene, and interestingly, the same motif is found also in the promoter regions of the genes encoding ribosomal proteins L21 and L37.

Gene, 1992 Dec 1, 122(1), 53 - 62
Cloning and sequencing of the genes encoding glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase (gap operon) from mesophilic Bacillus megaterium: comparison with corresponding sequences from thermophilic Bacillus stearothermophilus; Schlapfer BS et al.; The structural genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and the N-terminal part of triosephosphate isomerase (TIM) from mesophilic Bacillus megaterium DSM319 have been cloned as a gene cluster (gap operon) by complementation of an Escherichia coli gap amber mutant . Subsequently, the entire tpi gene, encoding TIM, was isolated by colony hybridization using a homologous probe . Nucleotide (nt) sequence analysis revealed an unidentified open reading frame (urf1) of 1029 bp located 50 nt upstream from the start codon of the gap gene . Gene expression from subclones containing different coding regions was studied by enzyme assay and SDS-PAGE . Both GAPDH and TIM are synthesized in transformed E . coli cells, whereas PGK is not . There is no unequivocal evidence for urf1 expression . Two putative promoter sites are present: one 100 nt upstream from urf1 and one 200 nt upstream from the pgk gene . An inverted repeat following the second promoter site is postulated to be involved in the transcriptional regulation of the operon . Each coding region shows a G+C content of 40% attained by the adaptation of the G+C content of the third base in the codon to compensate the G+C content of the first and second bases . The deduced amino acid (aa) sequences of B . megaterium GAPDH, PGK and TIM were compared with those from the thermophilic Bacillus stearothermophilus by antisymmetrical matrices . The detected characteristic thermophilic-mesophilic exchange pattern concerning aa substitutions between hydrophobic-polar and charged-charged residues corresponds to data obtained for thermophilic and mesophilic lactate dehydrogenases (LDH) . The determination of the thermostability of these enzymes revealed two regions of stability for B . megaterium TIM at high enzyme concentrations . Heat treatment seems to be responsible for the conversion of two differently active conformations or the induction of a new quaternary structure.

J Bacteriol, 1992 Dec, 174(23), 7859 - 63
Identification of the gene encoding transcription factor NusG of Thermus thermophilus; Heinrich T et al.; The nusG gene of Thermus thermophilus HB8 was cloned and sequenced . It is located 388 bp downstream from tufB, which is followed by the genes for ribosomal proteins L11 and L1 . No equivalent to secE preceding nusG, as in Escherichia coli, could be detected . The nusG gene product was overproduced in E . coli . A rabbit antiserum raised against the purified recombinant NusG reacted exclusively with one protein band of T . thermophilus crude extracts in Western blot (immunoblot) analyses, and no cross-reaction of the antiserum with E . coli NusG was observed . Recombinant NusG and the reacting T . thermophilus wild-type protein had identical sizes on sodium dodecyl sulfate-polyacrylamide gels . T . thermophilus and E . coli NusG have 45% identical and 22.5% similar amino acids, and similarities between the two proteins are most pronounced in carboxy-terminal regions . The T . thermophilus nusG gene could not rescue a nusG-deficient E . coli mutant strain.

J Biochem (Tokyo), 1992 Dec, 112(6), 792 - 5
Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii; Aono S et al.; A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii . This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin . The oxidized ferredoxin showed the characteristic EPR spectrum for {3Fe-4S}1+ (1.2 spin/mol Fd) . This signal disappeared upon reduction with dithionite and new signals due to {3Fe-4S}0 and {4Fe-4S}1+ (0.7 spin/mol Fd) appeared . The quantitation of EPR signals and the iron content reveal that B . schlegelii ferredoxin contains one {3Fe-4S}1+/0 and one {4Fe-4S}2+/1+ cluster . The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.

Bioorg Khim, 1992 Dec, 18(12), 1473 - 7
{BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2}; Kovalevskaia NP et al.; A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies . The enzyme is an isoschizomer of SduI from Streptococcus durans . BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments . Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C . The phage T4 glucosylated DNA is not cleaved by the enzyme.

J Biochem (Tokyo), 1992 Dec, 112(6), 811 - 5
Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures; Tanaka T et al.; Aspartate aminotransferase (AspAT) {EC 2.6.1.1} of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M . thermoformicicum strain SF-4 . AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M . thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate . The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit . Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor . Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes . Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form . Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4 . They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.

Cesk Epidemiol Mikrobiol Imunol, 1992 Dec, 41(6), 366 - 71
{Natural foci of tick-borne encephalitis in the Slovak Republic and its relation to the natural ecosystem}; Minar J; To evaluate the incidence of natural foci of tick-borne encephalitis in the Slovak Republic data of the hygiene service on the morbidity from tick-borne encephalitis during the period from 1961-1988 were used as well as data from the literature and results of the author's field studies on the incidence of the common tick . The main foci of tick-borne encephalitis are in the West Slovakian region, in the Zahorske lowland, in the Vah valley up to the distrikt of Povazska Bystrica, in the area of the Low Carpathian mountains, Tribec, Vtacnik, the Nitra and Hron hills, and Kovacov hills, in the Central Slovakian region in the Krupin hills and in the East Slovakian region in the Slovak karst and Slanske hills . The incidence of common ticks and foci of tick-borne encephalitis is linked to the original oak grove communities . In Slovakia the latter comprise hornbean and oak forests, oak groves and thermophile oak forests . Areas of original communities of beech woods and spruces which grow in higher altitudes do not provide favourable conditions for the development of the common tick . Rare foci of tick-borne encephalitis of a mountainous type found in Slovakia survive probably due to the extremely favourable microclimatic conditions in these areas . Also the hygrophilous communities of alders, moorlands and dry steppes, original as well as cultivated ones, are not suitable for the common tick . This is why ticks are not found in central, northern and northeastern Slovakia, in the Rye island and lowland along the Tisa river.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Eng, 1992 Dec, 5(8), 769 - 74
Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase; Kallwass HK et al.; Lactate dehydrogenases are of considerable interest as stereospecific catalysts in the chemical preparation of enantiomerically pure alpha-hydroxyacid synthons . For such applications in synthetic organic chemistry it would be desirable to have enzymes which tolerate elevated temperatures for prolonged reaction times, to increase productivity and to extend their applicability to poor substrates . Here, two examples are reported of significant thermostabilizations, induced by site-directed mutagenesis, of an already thermostable protein, the L-lactate dehydrogenase (EC 1.1.1.27, 35 kDa per monomer subunit) from Bacillus stearothermophilus . Thermal inactivation of this enzyme is accompanied by irreversible unfolding of the native protein structure . The replacement of Arg171 by Tyr stabilizes the enzyme against thermal inactivation and unfolding . This stabilizing effect appears to be based on improved interactions between the subunits in the core of the active dimeric or tetrameric forms of the enzyme . The thermal stability of L-lactate dehydrogenase variants with an active site Arg residue, either in the 171 (wild-type) or in the 102 position, is further increased by sulfate ions . The two stabilizing effects are additive, as found for the Arg171Tyr/Gln102Arg double mutant, for which the stability of the protein in 100 mM sulfate solution reaches that of L-lactate dehydrogenases from extreme thermophiles . All mutant proteins retain significant catalytic activity, both in the presence and absence of stabilizing salts, and are viable catalysts in preparative scale reactions.

Appl Environ Microbiol, 1992 Dec, 58(12), 3964 - 9
Uncultivated cyanobacteria, Chloroflexus-like inhabitants, and spirochete-like inhabitants of a hot spring microbial mat; Weller R et al.; Analysis of 16S rRNA sequences retrieved as cDNA (16S rcDNA) from the Octopus Spring cyanobacterial mat has permitted phylogenetic characterization of some uncultivated community members, expanding our knowledge or diversity within this microbial community . Two new cyanobacterial 16S rRNA sequences were discovered, raising to four the number of cyanobacterial sequence types known to occur in the mat . None of the sequences found is that of the cultivated thermophilic cyanobacterium Synechococcus lividus . A new 16S rRNA sequence characteristic of green nonsulfur bacteria and their relatives was discovered, raising to two the number of such sequences known to exist in the mat . Both are unique among the 16S rRNA sequences of cultivated members of this group, including an Octopus Spring isolate of Chloroflexus aurantiacus and Heliothrix oregonensis, whose sequences we report herein . Two spirochete-like 16S rRNA sequences were discovered . One can be placed in the leptospira subdivision of the spirochete group, but the other has such a loose affiliation with the spirochete group that it might actually belong to an as yet unrecognized subdivision or even to a new eubacterial line of descent.

Ann N Y Acad Sci, 1992 Nov 30, 671, 366 - 76
The alpha 3 beta 3 and alpha 1 beta 1 complexes of ATP synthase; Kagawa Y et al.; Two catalytic structures of H(+)-motive ATP synthase (Fig . 1), the alpha 3 beta 3 oligomer (M(r) = 319,581) and alpha 1 beta 1 promoter (M(r) = 106,527) (Fig . 2), were isolated using high pressure liquid chromatography (Fig . 3) and polyacrylamide gel electrophoresis (Figs . 4 and 5) . These were reconstituted from the alpha and beta subunits of thermophilic F1 (TF1), and the alpha 3 beta 3 oligomer was also crystallized . Common to both F1 and the alpha 3 beta 3 oligomer were the nucleotide specificity, the two Km values, the presence of protomer-oligomer activities, and the one-hit--one-kill phenomenon . A synchrotron experiment on the ATP hydrolysis cycle revealed the dynamic shrinkage and expansion of F1(44) that correspond, respectively, to the ATP-induced association and ADP-induced dissociation of the alpha 3 beta 3 oligomer . The oligomer, like mitochondrial F1 and TF1, exhibited two kinds of ATPase activity: one was cooperative and was inhibited by only one inhibitor per hexamer, and the other was inhibited by three inhibitors per hexamer.

Nucleic Acids Res, 1992 Nov 25, 20(22), 5963 - 70
Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus; Schlegl J et al.; We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E . coli and Thermus thermophilus, an extreme thermophilic eubacterium . Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes . Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies . Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T . thermophilus than by the corresponding E . coli activities . Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria . Our data further indicate that (pre-)tRNA interacts stronger with E . coli than T . thermophilus M1 RNA . Thus, low cleavage efficiencies of truncated model substrates by T . thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate . In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.

Science, 1992 Nov 20, 258(5086), 1355 - 8
Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods; Bevilacqua PC et al.; Fluorescence-detected stopped-flow and equilibrium methods have been used to study the mechanism for binding of pyrene (pyr)-labeled RNA oligomer substrates to the ribozyme (catalytic RNA) from Tetrahymena thermophila . The fluorescence of these substrates increases up to 25-fold on binding to the ribozyme . Stopped-flow experiments provide evidence that pyr experiences at least three different microenvironments during the binding process . A minimal mechanism is presented in which substrate initially base pairs to ribozyme and subsequently forms tertiary contacts in an RNA folding step . All four microscopic rate constants are measured for ribozyme binding of pyrCCUCU.

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