Thermophiles

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1529 - 37
Proposal of Oscillochloridaceae fam . nov . on the basis of a phylogenetic analysis of the filamentous anoxygenic phototrophic bacteria, and emended description of Oscillochloris and Oscillochloris trichoides in comparison with further new isolates; Keppen OI et al.; The nucleotide sequences of the genes of 16S rRNAs were determined for the type strain Oscillochloris trichoides DG-6T and three new strains of Oscillochloris-like mesophilic filamentous green bacteria . Two major clusters have been found within the family Chloroflexaceae by phylogenetic-analysis: one cluster includes thermophilic species of Chloroflexus and the second includes mesophilic strains of Oscillochloris . The degree of relatedness of these clusters was below an intergeneric level, having only 82.5-86.5% of 16S rDNA sequence similarity . These phylogenetic data correlate well with the significant physiological, biochemical and chemotaxonomical differences between members of both groups . Therefore, the Oscillochloris and Chloroflexus clusters should be considered as two separate families . The description of the new family, Oscillochloridaceae fam . nov., and emended descriptions of the genus Oscillochloris and the species Oscillochloris trichoides are presented.

Enzyme Microb Technol, 2000 Sep 1, 27(6), 414 - 422
Optimisation of culture medium and conditions for alpha-l-Arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus; Gomes J et al.; The culture medium for Rhodothermus marinus was optimised on a shake-flask scale by using statistical factorial designs for enhanced production of a highly thermostable alpha-L-arabinofuranosidase (AFase) . The medium containing 3.6 g/l birch wood xylan and 8.2 g/l yeast extract yielded a maximum of 110 nkat/ml AFase activity together with 125 nkat/ml xylanase and 65 nkat/ml beta-xylosidase activity . In addition, low levels of beta-mannanase (30 nkat/ml), alpha-galactosidase (0.2 nkat/ml), beta-galactosidase (0.3 nkat/ml), endoglucanase (5 nkat/ml) and beta-glucosidase (30 nkat/ml) were detected in the culture filtrate . Among the various carbon sources tested, birchwood xylan was most effective for the formation of AFase and xylanase activities, followed by oat spelt and beechwood xylans, and xylan-rich lignocelluoses (e.g., starch-free sugar beet pulp and wheat bran) . Constitutive levels of enzyme activities were detected when the bacterium was grown on other polysaccharides and low-molecular-weight carbohydrates . A fermentation in a 5-l fermenter (3-l working volume) using the optimised medium yielded 60 nkat/ml AFase associated with 65 nkat/ml xylanase and 35 nkat/ml beta-xylosidase activities . The crude AFase displayed optimal activity between pH 5.5 and 7 and at 85 degrees C . It had half-lives of 8.3 h at 85 degrees C and 17 min at 90 degrees C . It showed high stability between pH 5 and 9 (24 h at 65 degrees C) . The combined use of AFase-rich xylanase and mannanase from R . marinus in the prebleaching of softwood kraft pulp gave a brightness increase of 1.8% ISO . To our knowledge, this is the first report on the production of a high AFase activity by an extreme thermophilic bacterium and this enzyme is the most thermostable AFase reported so far.

Biol Chem, 2000 May-Jun, 381(5-6), 367 - 75
Regulation of GTPases in the bacterial translation machinery; Sprinzl M et al.; Several GTPases participate in bacterial protein biosynthesis . Initiation factor 2 controls the formation of the ribosomal initiation complex and places initiator fMet-tRNAfMet in the ribosomal P-site . Elongation factors Tu and G are responsible for codon-specific binding of the aminoacyl-tRNA to the A-site, and peptidyl-tRNA to the P-site, respectively, during the elongation phase of protein biosynthesis . Release factor 3, a GTPase which is not ubiquitous, is involved in termination and release of the nascent polypeptide . Other translation factors, including initiation factors 1 and 3, elongation factor Ts, release factors 1 and 2, and ribosomal release factor do not belong to the family of GTP/GDP binding proteins . The guanosine nucleotide binding domains of the GTPases involved in translation are structurally related to the Galpha subunit of heterotrimeric G proteins and to the proteins of the Ras family . We have identified and sequenced all genes coding for translation factors in the extreme thermophile Thermus thermophilus . The proteins were overproduced in Escherichia coli, purified, biochemically characterised and used for crystallisation and structural analysis . Further biochemical investigations were aimed at gaining insight into the molecular mechanism underlying the regulation of the GTPase activity of the translation factors, and to elucidate the role of their ribosomal binding sites in this process.

J Mol Microbiol Biotechnol, 2000 Jul, 2(3), 331 - 8
Expression of the gene for the delta9 acyl-lipid desaturase in the thermophilic cyanobacterium; Kiseleva LL et al.; A single-copy gene resembling the gene for the delta9 acyl-lipid desaturase (desC) was cloned from the thermophilic cyanobacterium Synechococcus vulcanus . Expression of desC in Escherichia coli confirmed that it encodes the delta9 desaturase . The nucleotide sequence of the desC was characterized by high G+C content that is typical of the sequences of thermophilic bacteria . The deduced amino acid sequence exhibited low Cys content and high Arg/Lys ratio that are the attributes of thermostable enzymes . A low level of the desC mRNA was detected in the cells grown at 55 degrees C, the optimum growth temperature for S . vulcanus . About a 10-fold increase was observed in the levels of the transcript and the protein during the shift in temperature from 55 to 45 degrees C . At 35 degrees C the amount of the desC mRNA and of the enzyme accumulated in the cells, was 3 to 4 times smaller than at 45 degrees C . At both temperatures, however, lipids were desaturated at similar rates . These results suggest that in S . vulcanus the conversion of stearic acid into oleic acid may be controlled not only by the de novo synthesis of the delta9 desaturase but, possibly, by the activation of the pre-existing enzyme.

Anal Biochem, 2000 Aug 15, 284(1), 6 - 10
Two-dimensional agarose gel electrophoresis as a tool to isolate genus- and species-specific repetitive DNA sequences; Harms C et al.; Two-dimensional electrophoresis in agarose gels separates DNA-restriction fragments not only by molecular weight but also according to their AT-cluster content . The method produced genus-specific spot patterns of multicopy DNA fragments of grains as well as spot patterns of highly repetitive DNA fragments of ciliates, demonstrated for barley, spelt, and Tetrahymena . Further investigations in regard to their specificity by hybridization with three other grain species (wheat, oat, and rye) and three ciliate species (Tetrahymena thermophila, Tetrahymena pigmentosa, and Tetrahymena borealis) were performed . The DNA samples from spelt and Tetrahymena were demonstrated to be genus specific for Triticum and species specific for Tetrahymena pyriformis, respectively .

J Med Microbiol, 2000 Aug, 49(8), 713 - 8
Effect of probiotic bacteria on prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses in vitro; van der Mei HC et al.; The proliferation of yeasts in the mixed bacterial and fungal biofilms colonising silicone rubber voice prostheses in laryngectomised patients is the main cause of malfunctioning of the valve mechanism on the oesophageal side of the prostheses . Indwelling voice prostheses usually have to be replaced every 3-4 months . The consumption of probiotic bacteria is largely motivated by health claims related to the urogenital and lower digestive tract, but not to the upper digestive tract . The present study examined the influence of probiotic bacteria on the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses, as formed in a modified Robbins device . Exposure of oropharyngeal biofilms on voice prostheses to suspensions of Bifidobacterium infantis 420 or Enterococcus faecium 603 did not significantly reduce the number of yeasts in the biofilm . However, suspensions of Lactobacillus fermentum B54, L . rhamnosus 744 or L . lactis cremoris SK11 led to a reduction in the number of yeasts harvested from the voice prostheses . Suspensions of L . casei Shirota and Streptococcus thermophilus B significantly reduced the number of yeasts in the biofilm to 39% and 33%, respectively . The reduction brought about in yeast prevalence in the mixed biofilm was greatest by exposure to a suspension of L . lactis 53, with yeast prevalence only 4% of the control . In conclusion, the study demonstrated that the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses might be controlled by consumption of probiotic bacteria.

Rev Latinoam Microbiol, 1999 Jan-Mar, 41(1), 5 - 10
Behavior of enterotoxigenic strains of Staphylococcus aureus in milk fermented with a yogurt starter culture; Zuniga Estrada A et al.; The ability of a yogurt starter culture formed by Streptococcus salivarius subsp . thermophilus and Lactobacillus delbrueckii subsp bulgaricus to inhibit the growth of four enterotoxin type A and B producers Staphylococcus aureus strains (ATCC 6538, S6, FRI-100 and a strain isolated from milk) during fermentation of milk and subsequent storage was investigated . Sterile skim milk was inoculated with about 10(6) CFU/ml of S . aureus and with about 10(6) CFU of starter culture, and incubated at 42 degrees C during 8 h, followed by refrigeration at 4 degrees C . Samples were taken every 2 h during fermentation and every 2 days during storage . Viable count of lactic acid bacteria and S . aureus as well as pH, acidity, thermostable deoxyribonuclease (TNase) and staphylococcal enterotoxin A (SEA) production were evaluated . Behavior of four strains was similar; S . aureus survived the 8 h fermentation with LAB, and its population began to decrease from the first day of storage, being completely inhibited at 9-10 days . TNase and SEA production were positive in all samples taken along the study . It was demonstrated that enterotoxigenic strains of S . aureus were able to survive the fermentation of milk with a yogurt starter culture and they were inhibited after several days during storage of the fermented product, contrary to the general belief which considered it very difficult due to the low pH . Even though S . aureus was inhibited, TNase and SEA were demonstrable along the storage . Therefore, fermented milks may play an important role in the transmission of this organism.

Can J Microbiol, 2000 Jul, 46(7), 669 - 73
Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion; Rubinder K et al.; Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers . The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2) . Diploids derived from heterokaryons segregated to stable haploid recombinant strains . In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of alpha-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).

Eur J Biochem, 2000 Aug, 267(16), 5217 - 26
Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms; Marc F et al.; The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus . Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway) . In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M . jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T . neapolitana and B . stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor . M . jannaschii ArgJ is therefore a monofunctional enzyme, whereas T . neapolitana and B . stearothermophilus encoded ArgJ are bifunctional . Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism . Acetylated ArgJ intermediates were detected in semireactions using {14C}acetylCoA or {14C}N2-acetyl-L-glutamate as acetyl donors . In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis . Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E . coli . The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 205 - 10
Cysteine biosynthesis in the Archaea: Methanosarcina thermophila utilizes O-acetylserine sulfhydrylase; Borup B et al.; Two pathways for cysteine biosynthesis are known in nature; however, it is not known which, if either, the Archaea utilize . Enzyme activities in extracts of Methanosarcina thermophila grown with combinations of cysteine and sulfide as sulfur sources indicated that this archaeon utilizes the pathway found in the Bacteria domain . The genes encoding serine transacetylase and O-acetylserine sulfhydrylase (cysE and cysK) are adjacent on the chromosome of M . thermophila and possibly form an operon . When M . thermophila is grown with cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity is maximally expressed suggesting alternative roles for this enzyme apart from cysteine biosynthesis.

J Mol Biol, 2000 Aug 11, 301(2), 433 - 50
The crystal structure of adenylosuccinate lyase from Pyrobaculum aerophilum reveals an intracellular protein with three disulfide bonds; Toth EA et al.; Adenylosuccinate lyase catalyzes two separate reactions in the de novo purine biosynthetic pathway . Through its dual action in this pathway, adenylosuccinate lyase plays an integral part in cellular replication and metabolism . Mutations in the human enzyme can result in severe neurological disorders, including mental retardation with autistic features . The crystal structure of adenylosuccinate lyase from the hyperthermophilic archaebacterium Pyrobaculum aerophilum has been determined to 2.1 A resolution . Although both the fold of the monomer and the architecture of the tetrameric assembly are similar to adenylosuccinate lyase from the thermophilic eubacterium Thermotoga maritima, the archaebacterial lyase contains unique features . Surprisingly, the structure of adenylosuccinate lyase from P . aerophilum reveals that this intracellular protein contains three disulfide bonds that contribute significantly to its stability against thermal and chemical denaturation . The observation of multiple disulfide bonds in the recombinant form of the enzyme suggests the need for further investigations into whether the intracellular environment of P . aerophilum, and possibly other hyperthermophiles, may be compatible with protein disulfide bond formation . In addition, the protein is shorter in P . aerophilum than it is in other organisms . This abbreviation results from an internal excision of a cluster of helices that may be involved in protein-protein interactions in other organisms and may relate to the observed clinical effects of human mutations in that region .

Biochem Biophys Res Commun, 2000 Aug 11, 274(3), 727 - 31
A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor; D'Auria S et al.; Blood glucose is a clinically important analytes for diabetic health care . In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase . The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS) . The ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose . This decrease can be used to measure the glucose concentration . Our results suggest that enzymes which use glucose as their substrate can be used as reversible and nonconsuming glucose sensors in the absence of required cofactors . Moreover, the possibility of using inactive apoenzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes .

Biochemistry, 2000 Aug 8, 39(31), 9523 - 32
Experimental and theoretical analysis of the invasive signal amplification reaction; Lyamichev VI et al.; The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression . The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal . By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling . We have examined the performance of the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction . We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide . Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate . When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage . To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction . We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate . The experimental data are compared with a theoretical model of the invasive signal amplification reaction.

Biochemistry, 2000 Aug 8, 39(31), 9232 - 40
A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila; Tripp BC et al.; Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies . Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity . Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function . The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values . The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m) . These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat) . These results are consistent with Glu 84 functioning as a proton shuttle residue . The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m) . Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants . These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.

Biochemistry, 2000 Aug 8, 39(31), 9222 - 31
A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila; Iverson TM et al.; The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila . Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase . However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another . The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M . thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-) . Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme . Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated . In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations . This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases . A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site . On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.

Appl Environ Microbiol, 2000 Aug, 66(8), 3608 - 15
Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge; Imachi H et al.; The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis . For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C . After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum . Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M . thermoautotrophicum . In pure culture, the isolate was found to ferment pyruvate . 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species . To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules . Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens . They accounted for approximately 1.1% of the total cells in the sludge . These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.

Appl Environ Microbiol, 2000 Aug, 66(8), 3519 - 27
Correlation of activities of the enzymes alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase with exopolysaccharide biosynthesis by Streptococcus thermophilus LY03; Degeest B et al.; The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03 . This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses . Lactose and glucose were fermented by S . thermophilus LY03 only when they were used as sole energy and carbohydrate sources . Fructose was also fermented when it was applied in combination with lactose or glucose . Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances . A combination of lactose and glucose resulted in the largest amounts of EPS . Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced . Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S . thermophilus LY03.

Appl Microbiol Biotechnol, 2000 Jun, 53(6), 715 - 21
A highly thermostable endo-(1,4)-beta-mannanase from the marine bacterium Rhodothermus marinus; Politz O et al.; Rhodothermus marimus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan . Screening of expression libraries identified mannanase-positive clones . Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa . Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26 . Action pattern analysis categorised the R . marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity . When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa . The full-length protein of 113 kDa was only detected in crude extracts of R . marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa . Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 degrees C and pH 5.4, respectively . Purified, E . coil-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 degrees C and 90 degrees C, respectively . In contrast, R . marinus-derived protein retained 87% activity after 1 h at 90 degrees C . The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.

J Biol Chem, 2000 Dec 1, 275(48), 37824 - 8
Selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart; Hasegawa J et al.; Mesophilic cytochrome c(551) of Pseudomonas aeruginosa (PA c(551)) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), through only five amino acid substitutions . The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c(552) through careful structure comparison . Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c(551) could be partly attained through an enthalpic factor . The solution structure of the mutant showed that, as in HT c(552), there were tighter side chain packings in the mutated regions . Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy . Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.

J Biol Chem, 2000 Oct 20, 275(42), 32530 - 4
Loss of either of the two heme-binding cysteines from a class I c-type cytochrome has a surprisingly small effect on physicochemical properties; Tomlinson EJ et al.; Almost without exception, c-type cytochromes have heme covalently attached via two thioether linkages to the cysteine residues of a CXXCH motif . The reasons for the covalent attachment are not understood . Reported here is cytoplasmic expression in Escherichia coli of AXXCH and CXXAH variants of cytochrome c(552) from Hydrogenobacter thermophilus; remarkably, the single thioether bond proteins have, apart from an altered visible absorption spectrum, almost identical properties, including thermal stability and reduction potential, to the wild type CXXCH protein . In combination with previous work showing that an AXXAH variant of cytochrome c(552) is much less stable than the CXXCH form, it can be concluded that covalent attachment of heme via either of thioether bonds is sufficient to confer considerable stability and that these bonds contribute little to the setting of the reduction potential . The absence of AXXCH or CXXAH heme-binding motifs from bacterial cytochromes c may relate to the coexistence of the assembly pathway with that for formation of disulfide bonds in the bacterial periplasm.

J Biol Chem, 2000 Oct 27, 275(43), 33527 - 35
Quaternary structure of the lactose transport protein of Streptococcus thermophilus in the detergent-solubilized and membrane-reconstituted state; Friesen RH et al.; The quaternary structure of LacS, the lactose transporter of Streptococcus thermophilus, has been determined for the detergent-solubilized and the membrane-reconstituted state of the protein . The quaternary structure of the n-dodecyl-beta-d-maltoside-solubilized state was studied using a combination of sedimentation velocity and equilibrium centrifugation analysis . From these measurements it followed that the detergent-solubilized LacS undergoes reversible self-association with a monomer to dimer mode of association . The association constants were 5.4 +/- 3.6 and 4.4 +/- 1.0 ml mg(-1) as determined from the velocity and equilibrium sedimentation measurements, respectively . The experiments did not indicate significant changes in the shape of the protein-detergent complex or the amount of detergent bound in going from the monomeric to dimeric state of LacS . Importantly, a single Cys mutant of LacS is labeled by 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid in a substrate-dependent manner, indicating that the detergent-solubilized protein exhibits ligand binding activity . The quaternary structure of membrane-reconstituted LacS was determined by freeze-fracture electron microscopy analysis . Recent developments in the analysis of freeze-fracture images (Eskandari, S . P., Wright, E . M., Freman, M., Starace, D . M., and Zampighi, G . A . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 11235-11240) allowed us to directly correlate the cross-sectional area of the transmembrane segment to a dimeric state of the functionally membrane-reconstituted LacS protein . The cross-sectional area of the LacS protein was calibrated using the membrane-reconstituted transmembrane domain of the mannitol transporter enzyme II, an intramembrane particle for which the cross-sectional area was obtained from maps of two-dimensional crystals . The consequences of the determined quaternary structure for the transport function and regulation of LacS are discussed.

EMBO J, 2000 Aug 1, 19(15), 3857 - 69
Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8; Sugahara M et al.; The MutM {formamidopyrimidine DNA glycosylase (Fpg)} protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N-glycosylase activity) and cleaves both the 3'- and 5'-phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity) . The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 A resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn(2+) ion of the zinc finger . MutM is composed of two distinct and novel domains connected by a flexible hinge . There is a large, electrostatically positive cleft lined by highly conserved residues between the domains . On the basis of the three-dimensional structure and taking account of previous biochemical experiments, we propose a DNA-binding mode and reaction mechanism for MutM . The locations of the putative catalytic residues and the two DNA-binding motifs (the zinc finger and the helix-two-turns-helix motifs) suggest that the oxidized base is flipped out from double-stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes.

Mikrobiologiia, 2000 May-Jun, 69(3), 334 - 40
{Carbon metabolism in Sulfobacillus thermosulfidooxidans subsp . asporogenes, strain 41}; Tsaplina IA et al.; The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp . asporogenes, strain 41, grown either at an atmospheric content of CO2 in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO2 content increased to 5-10% . Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate cycle enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cellular extracts of strain 41 . During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5-10% resulted in the activation of growth and iron oxidation, a 20-30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.

Biophys J, 2000 Aug, 79(2), 756 - 66
Rotational mobility and orientational stability of a transport protein in lipid membranes; Spooner PJ et al.; A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide spin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin resonance . Bovine rhodopsin was also selectively spin-labeled and studied to aid the interpretation of the measurements . Observations of spin-labeled proteins in macroscopically aligned bilayers indicated that the spin label tends to orient so as to reflect the transmembrane orientation of the protein . Rotational correlation times of 1-2 micros for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers of dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the specific mixture of lipids used to reconstitute LacS (30 degrees C) . The rotational correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to decrease sharply and was determined to be 9 +/- 1 micros (mean +/- SD) for these samples . This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongated configuration within the membrane . Rather than changing its oligomeric state, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid.

Curr Microbiol, 2000 Sep, 41(3), 201 - 5
Susceptibility to heavy metals and cadmium accumulation in aerobic and anaerobic thermophilic microorganisms isolated from deep-sea hydrothermal vents; Llanos J et al.; Thirty thermophilic strains isolated from heavy metal-rich hydrothermal vent sites at Lau Basin were tested for their susceptibility to cadmium, zinc, cobalt, and nickel . The 14 aerobic spore formers belonging to the genus Bacillus, 6 anaerobic fermenters from the order Thermotogales, and 10 anaerobic sulfur reducers from the order Thermococcales could be clearly distinguished according to their metal susceptibilities . The Thermococcales were found to exhibit the highest resistance to cadmium and zinc, whereas Thermotogales were highly sensitive to these metals . In contrast, the Thermotogales displayed the highest resistance to cobalt ions . No clear distinction could be established between the metal susceptibilities of these strains and seven reference organisms used for comparative studies . Cadmium resistance, slightly inducible in some cadmium-resistant bacilli, was not plasmid mediated . The amount of cadmium immobilized by the Thermotogales was related to their level of resistance to this metal.

Curr Microbiol, 2000 Sep, 41(3), 177 - 81
Comparison of low-molecular-weight heat stress proteins encoded on plasmids in different strains of Streptococcus thermophilus; Solow BT et al.; Streptococcus thermophilus is used extensively for industrial fermentation of dairy products . Some strains of S . thermophilus are known to carry plasmids, and many of these plasmids are suspected of encoding low-molecular-weight heat stress proteins (Hsps) that may aid in survival under stressful conditions . In order to confirm the presence and examine the similarity of these low-molecular-weight Hsps, genes were identified and sequenced encoding Hsps on plasmids pER16 (4.5 kb), pER35 (10 kb), and pER36 (3.7 kb) from three different strains of S . thermophilus . The plasmid replication proteins were also sequenced to examine their relatedness . Amino acid sequence comparisons of the Hsps and of the replication proteins revealed a high degree of identity suggesting a common origin . Heat stress proteins enhance the viability of bacteria in extreme environments, and the presence of an Hsp encoded on a plasmid may enhance survival of S . thermophilus under harsh production conditions.

J Food Prot, 2000 Jul, 63(7), 916 - 20
Behavior of Listeria monocytogenes in pasteurized milk during fermentation with lactic acid bacteria; Pitt WM et al.; The behavior of Listeria monocytogenes in pasteurized milk during fermentation with starter and nonstarter lactic acid bacteria was investigated . Pasteurized milk was co-inoculated with approximately 10(4) CFU/ml of L . monocytogenes and 10(6) CFU/ml of Lactococcus lactis, Lactococcus cremoris, Lactobacillus plantarum, Lactobacillus bulgaricus, or Streptococcus thermophilus . Inoculated milks were incubated at 30 degrees C or 37 degrees C for 24 to 72 h . Listeria monocytogenes survived and also grew to some extent during incubation in the presence of all starter cultures; however, inhibition ranged from 83 to 100% based on maximum cell populations . During incubation with L . bulgaricus and L . plantarum, L . monocytogenes was completely inactivated after 20 h and 64 h of incubation at 37 degrees C and 30 degrees C, respectively . The pH of the fermenting milks declined steadily throughout the fermentation periods and was approximately 4.2 at the conclusion of the experimental period regardless both of the starter culture and pathogen combination or the temperature of incubation.

Can J Microbiol, 2000 Jun, 46(6), 559 - 64
Characterization of subterranean bacteria in the Hungarian Upper Permian Siltstone (Aleurolite) Formation; Farkas G et al.; The main purpose of this work was to study the microbiology of the Hungarian Upper Permian Siltstone (Aleurolite) Formation, to assess the safety of future underground repositories for nuclear waste . Sixty-seven air, groundwater, technical water, rock, and surface samples were collected aseptically from different depths . The number of aerobic and anaerobic isolates was 277 . The mesophilic minimum and maximum CFU counts of the air samples were 1.07-5.84 x 10(2).mL-1 (aerobic) and 0.22-1.04 x 10(2).mL-1 (anaerobic), respectively; those of the water samples were 0.39-1.25 x 10(5).mL-1 (aerobic) and 0.36-3.9 x 10(3).mL-1 (anaerobic); those of the technical water samples were 0.27-5.03 x 10(6).mL-1 (aerobic) and 4 x 10(5)-->10(6).mL-1 (anaerobic); and those of the aleurolite samples were 2.32 x 10(2)-2.47 x 10(5).g-1 (aerobic) and 0.45-9.5 x 10(2).g-1 (anaerobic) . In the groundwater, the thermophilic aerobic bacteria count was 0-2.4 x 10(2).mL-1 and the thermophilic anaerobic bacteria count was 0.43-4.6 x 10(4).mL-1 . The gases produced by the 16 gas-forming isolates were CO2 (aerobic isolates), and CO2 and H2 (anaerobic isolates) . About 20% of the aerobic isolates produced siderophores . The proportions of organic acid producers were lowest in aerobic and anaerobic isolates from the aleurolite, 13% and 14%, respectively . The highest proportions of acid producers in the aerobic and anaerobic isolates from the air samples were 63% and 54% . Altogether 160 of the aerobic isolates and 52 of the anaerobic isolates were spore formers . The radiosensitivity of the aerobic isolates was also determined; the D10 values of the sporeformers ranged between 0.8-2.44 kGy . Our results indicate that the sulfate-reducing bacteria and the production of complexing agents (siderophores) may contribute to the mobilization of radionuclides from underground repositories . As well, microbial gas production can influence the environmental conditions . The variability in bacterial radiotolerance indicates the biodiversity at this potential disposal site . These facts must be considered during the planning of a nuclear waste repository.

FEBS Lett, 2000 Jul 7, 476(3), 140 - 4
The heterotrimeric Thermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln); Becker HD et al.; Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) {Becker, H.D . and Kern, D . (1998) Proc . Natl . Acad . Sci . USA 95, 12832-12837} . Here we show that, like other bacteria, T . thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC) . We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme . The overproduced T . thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn) . Thus, the T . thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs . In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated.

J Bacteriol, 2000 Aug, 182(16), 4661 - 6
Sequencing, cloning, and high-level expression of the pfp gene, encoding a PP(i)-dependent phosphofructokinase from the extremely thermophilic eubacterium Dictyoglomus thermophilum; Ding YH et al.; The sequencing, cloning, and expression of the pfp gene from Dictyoglomus thermophilum, which consists of 1,041 bp and encodes a pyrophosphate-dependent phosphofructokinase, are described . A phylogenetic analysis indicates that the enzyme is closely related to the pyrophosphate-dependent enzyme from Thermoproteus tenax . The recombinant and native enzymes share a high degree of similarity for most properties examined.

J Biol Inorg Chem, 2000 Jun, 5(3), 354 - 63
Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study; Ioannidis N et al.; We compare the interaction of nitric oxide with the S states of the oxygen evolving complex (OEC) of photosystem II and the dinuclear Mn cluster of Thermus thermophilus catalase . Flash fluorescence studies indicate that the S3 state of the OEC in the presence of ca . 0.6 mM NO is reduced to the S1 with an apparent halftime of ca . 0.4 s at about 18 degrees C, compared with a biphasic decay, with approximate halftimes of 28 s for S3 to S2 and 140 s for S2 to S1 in the absence of NO . Under similar conditions the S2 state is reduced by NO to the S1 state with an approximate halftime of 2 s . These results extend a recent study indicating a slow reduction of the S1 state at -30 degrees C, via the S0 and S(-1) states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase . The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state . The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC . What is unique about the OEC is the rapid interaction of NO with the S3 state of the OEC, which is compatible with a metalloradical character of this state.

Nucleic Acids Res, 2000 Aug 1, 28(15), 2993 - 3001
Two Tetrahymena G-DNA-binding proteins, TGP1 and TGP3, share novel motifs and may play a role in micronuclear division; Lu Q et al.; G-DNA is a four-stranded DNA structure with diverse putative biological roles . We have previously purified and cloned a novel G-DNA-binding protein TGP1 from the ciliate Tetrahymena thermophila . Here we report the molecular cloning of TGP3, an additional G-DNA-binding protein from the same organism . The TGP3 cDNA encodes a 365 amino acid protein that is homologous to TGP1 (34% identity and 44% similarity) . The proteins share a sequence pattern that contains two novel repetitive and homologous motifs flanking an extensively hydrophilic and basic region . A nuclear fractionation experiment showed that TGP1 and TGP3 activities are localized predominantly in the nuclear fraction . To further investigate the biological roles of the proteins in vivo, we have generated separate macronuclear gene knockout (KO) strains (TGP1KO and TGP3KO) for each of the two genes . Southern blot analysis demonstrated that the macronuclear copies of each gene were completely disrupted . Mobility shift assays showed that the corresponding G-DNA-binding activity for each protein was abolished in the KO strains . Growth analysis showed that both KO strains grew at near wild-type rates, indicating that neither of the genes is essential for cell growth . Nevertheless, nuclear staining analysis revealed that both TGP1KO and TGP3KO cells have an increased occurrence (more than 2-fold) of extra micronuclei, implying faulty control of micronuclear division in the KO cells.

J Air Waste Manag Assoc, 2000 Jun, 50(6), 1037 - 44
Optimal loading rates and economic analyses for anaerobic digestion of poultry waste; Collins AR et al.; Four combinations of litter and carcasses from broiler chickens were examined utilizing a thermophilic, stirred-tank digester of demonstration size of approximately 10,000 gal . Under computed optimal loading rates, litter with paper bedding had the highest daily production of methane over an 8-day retention period . The greatest methane production per lb of volatile solids was achieved over 10 days with litter and paper bedding combined with carcasses . This research found that sufficient poultry litter is generated within 20 mi (32 km) of Moorefield, WV, to support a commercial-sized digester operation . However, anaerobic digestion of poultry waste cannot be financially supported by methane production alone . To be financially viable, anaerobic digestion requires a disposal fee for poultry waste and/or the sale of the digested solid effluent as an organic fertilizer to retail markets.

Lipids, 2000 Jun, 35(6), 587 - 93
Alterations in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA concentration in cultured chick aortic smooth muscle cells; Carazo A et al.; We observed and compared alterations in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase at the transcriptional level in unsynchronized, three-passage cultures of smooth-muscle cells from the aorta of chicks fed on a control diet (C-SMC) and those of chicks fed on a similar diet plus cholesterol (Ch-SMC) . Alterations in reductase mRNA concentrations in senescent cultures were much lower . We used a modification of the competitive (c) reverse transcription polymerase chain reaction method, using a Thermus thermophilus DNA polymerase (Tth pol) to quantify the very scarce species of HMG-CoA reductase mRNA in samples of cytoplasmic SMC mRNA . We cloned and sequenced a 199 bp cDNA fragment of chicken HMG-CoA reductase, which encoded a region of 66 amino acids belonging to the catalytic domain of the enzyme . HMG-CoA reductase mRNA concentrations from young C-SMC cultures rose 3.89-fold 4 h after the change of medium and returned to base levels between 8 to 12 h afterward . Concentrations in Ch-SMC cultures increased less (2.36-fold) 8 h after the change to fresh medium . Increases in reductase mRNA in senescent cultures of Ch-SMC and C-SMC measured under similar conditions were only 1.28- and 1.39-fold, respectively.

Biochemistry, 2000 Jul 18, 39(28), 8250 - 8
Mapping contacts between Escherichia coli alanyl tRNA synthetase and 2' hydroxyls using a complete tRNA molecule; Pleiss JA et al.; A dual-specific derivative of yeast tRNA(Phe) is described whose features facilitate structure-function studies of tRNAs . This tRNA has been made in three different bimolecular forms that allow modifications to be easily introduced into any position within the molecule . A set of deoxynucleotide substituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previously shown to interact with 2'-hydroxyls in the acceptor stem of the tRNA . Because the present experiments used a full-length tRNA, several contacts were identified that had not been previously found using microhelix substrates . Contacts at similar sites in the T-loop are seen in the cocrystal structure of tRNA(Ser) and Thermus thermophilus seryl-tRNA synthetase.

Mol Biol Evol, 2000 Jul, 17(7), 1050 - 60
Replicability and recurrence in the experimental evolution of a group I ribozyme; Hanczyc MM et al.; In order to explore the variety of possible responses available to a ribozyme population evolving a novel phenotype, five Tetrahymena thermophila group I intron ribozyme pools were evolved in parallel for cleavage of a DNA oligonucleotide . These ribozyme populations were propagated under identical conditions and characterized when they reached apparent phenotypic plateaus; the populations that reached the highest plateau showed a near 100-fold improvement in DNA cleavage activity . A detailed characterization of the evolved response in these populations reveals at least two distinct phenotypic trajectories emerging as a result of the imposed selection . Not only do these distinct solutions exhibit differential DNA cleavage activity, but they also exhibit a very different correlation with a related, but unselected, phenotype: RNA cleavage activity . In turn, each of these trajectories is underwritten by differing genotypic profiles . This study underscores the complex network of possible trajectories through sequence space available to an evolving population and uncovers the diversity of solutions that result when the process of experimental evolution is repeated multiple times in a simple, engineered system.

Lett Appl Microbiol, 2000 Jul, 31(1), 25 - 9
Production of cutinolytic esterase by filamentous bacteria; Fett WF et al.; Thirty-eight strains of filamentous bacteria, many of which are thermophilic or thermotolerant and commonly found in composts and mouldy fodders, were examined for their ability to produce cutinolytic esterase (cutinase) in culture media supplemented with cutin, suberin or cutin-containing agricultural by-products . Initially, the ability of culture supernatants to hydrolyse the artificial substrate p-nitrophenyl butyrate was determined by spectrophotometric assays . Only one bacterium, Thermoactinomyces vulgaris NRRL B-16117, exhibited cutinolytic esterase production . The enzyme was highly inducible, was repressed by the presence of glucose in the medium and hydrolysed both apple and tomato cutins . Inducers included apple cutin, apple pomace, tomato peel, potato suberin and commercial cork . Unlike similar fungal enzymes, the T . vulgaris cutinolytic esterase was not inducible by cutin hydrolysate . The cutinolytic esterase exhibited a half-life of over 60 min at 70 degrees C and a pH optimum of >/= 11.0 . This study indicates that thermophylic filamentous bacteria may be excellent commercial sources of heat-stable cutin-degrading enzymes that can be produced by fermentation of low cost feedstocks.

Br Med Bull, 2000, 56(1), 158 - 71
Control of bacterial spores; Brown KL; Bacterial spores are much more resistant than their vegetative counterparts . The most dangerous spore-former is Clostridium botulinum which produces a potent neurotoxin that can prove fatal . The most common food poisoning from a spore-former is caused by C . perfringens . Other food poisoning spore-formers include Bacillus cereus, B . subtilis and B . licheniformis . There are a number of non-pathogenic spore-formers including butyric and thermophilic anaerobes that cause significant economic losses to food producers . Some unusual spoilage complaints have been reported, for example, B . sporothermodurans in UHT milk, Alicyclobacillus acidoterrestris in apple and orange juice and Desulfotomaculum nigrificans in hot vending machines . Control of spore-formers requires an understanding of both the resistance and outgrowth characteristics of the spores.

Genetics, 2000 Jul, 155(3), 1119 - 25
Autonomously replicating macronuclear DNA pieces are the physical basis of genetic coassortment groups in Tetrahymena thermophila; Wong L et al.; The macronucleus of the ciliate Tetrahymena thermophila contains a fragmented somatic genome consisting of several hundred identifiable chromosome pieces . These pieces are generated by site-specific fragmentation of the germline chromosomes and most of them are represented at an average of 45 copies per macronucleus . In the course of successive divisions of an initially heterozygous macronucleus, the random distribution of alleles of loci carried on these copies eventually generates macronuclei that are pure for one allele or the other . This phenomenon is called phenotypic assortment . We have previously reported the existence of loci that assort together (coassort) and hypothesized that these loci reside on the same macronuclear piece . The work reported here provides new, rigorous genetic support for the hypothesis that macronuclear autonomously replicating chromosome pieces are the physical basis of coassortment groups . Thus, coassortment allows the mapping of the somatic genome by purely genetic means . The data also strongly suggest that the random distribution of alleles in the Tetrahymena macronucleus is due to the random distribution of the MAC chromosome pieces that carry them.

Biochem J, 2000 Jul 15, 349(Pt 2), 651 - 6
Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling; Shibuya H et al.; The thermostability of Streptomyces lividans xylanase B (SlxB-cat) was significantly increased by the replacement of its N-terminal region with the corresponding region from Thermomonospora fusca xylanase A (TfxA-cat) without observing a decrease in enzyme activity . In spite of the significant similarity between the amino acid sequences of the two xylanases, their thermostabilities are quite different . To facilitate an understanding of the contribution of structure to the thermostability observed, chimaeric enzymes were constructed by random gene shuffling and the thermostable chimaeric enzymes were selected for further study . A comparative study of the chimaeric and parental enzymes indicated that the N-terminus of TfxA-cat contributed to the observed thermostability . However, too many substitutions decreased both the thermostability and the activity of the enzyme . The mutants with the most desirable characteristics, Stx15 and Stx18, exhibited significant thermostabilities at 70 degrees C with optimum temperatures which were 20 degrees C higher than that of SlxB-cat and equal to that of TfxA-cat . The ability of these two chimaeric enzymes to produce reducing sugar from xylan was enhanced in comparison with the parental enzymes . These results suggest that these chimaeric enzymes inherit both their thermostability from TfxA-cat and their increased reactivity from SlxB-cat . Our study also demonstrates that random shuffling between a mesophilic enzyme and its thermophilic counterpart represents a facile approach for the improvement of the thermostability of a mesophilic enzyme.

Extremophiles, 2000 Jun, 4(3), 131 - 6
A novel microbial interaction: obligate commensalism between a new gram-negative thermophile and a thermophilic Bacillus strain; Rhee SK et al.; Obligately commensal interaction between a new gram-negative thermophile and a thermophilic Bacillus strain was investigated . From compost samples, a mixed culture showing tyrosine phenol-lyase activity was enriched at 60 degrees C . The mixed culture consisted of a thermophilic gram-negative strain, SC-1, and a gram-positive spore-forming strain, SK-1 . In mixed cultures, strain SC-1 started to grow only when strain SK-1 entered the stationary phase . Although strain SC-1 showed tyrosine phenol lyase activity, we could not isolate a colony with any nutrient medium . For the isolation and cultivation of strain SC-1, we added culture supernatant and cell extract of the mixed culture to the basal medium . The supernatant and cell extract of the mixed culture contained heat-stable and heat-labile factors, respectively, that are essential to the growth of strain SC-1 . During pure cultures of strain SK-1, the heat-stable growth factors were released during the growth phase and the heat-labile growth factors were produced intracellularly at the early stationary phase . Strain SC-1 was gram-negative and microaerophilic, and grows optimally at 60 degrees C . Based on these results, we propose a novel commensal interaction between a new gram-negative thermophile, strain SC-1, and Bacillus sp . strain SK-1.

Virology, 2000 Jul 5, 272(2), 409 - 16
SNDV, a novel virus of the extremely thermophilic and acidophilic archaeon Sulfolobus; Arnold HP et al.; We describe a novel virus, SNDV (Sulfolobus neozealandicus droplet-shaped virus), of the crenarchaeotal archaeon Sulfolobus, which was found in a carrier state in a Sulfolobus strain isolated from a field sample from New Zealand . SNDV particles are droplet-shaped and densely covered by thin tail fibers at their pointed ends . The virion consists of a core and a coat . The latter has the appearance of a beehive and has a surface that is either helically ribbed or a stack of hoops . The genome is cccDNA of 20 kb, which is modified by dam-like methylation . It is cleaved by only a few type II restriction enzymes e.g., DpnI but not MboI, demonstrating an N(6)-methylation of the adenine residue in GATC sequences . The DNA-modifying system differentiates between virus and host . We postulate a virus-encoded methylase that is active on hemimethylated DNA . The host range of SNDV is confined to few Sulfolobus strains from New Zealand . The virus persists in an unstable carrier state rather than as a prophage . Due to its uniqueness we propose to assign it to a novel virus family termed Guttaviridae .

Protein Expr Purif, 2000 Jul, 19(2), 289 - 97
Identification, purification, and characterization of a thermophilic imidase from pig liver; Su TM et al.; This study investigates thermophilic imidase activity of the liver . We demonstrate that imidase catalyzes the hydrolysis of imides at a temperature substantially higher than that of its native environment . Then, a thermophilic imidase is purified to homogeneity from pig liver, and its thermoproperties are studied . About 2500-fold of purification and 15% yield of imidase activity are obtained after ammonium sulfate precipitation, octyl, DEAE, chelation, and gel filtration chromatography . While avoiding heat treatment for the protein purification, this study also indicates that only one enzyme is responsible for the imidase activity . This homogenous enzyme prefers to catalyze hydrolysis of imides at above 60 degrees C rather than at the body temperature of a pig . Although stable at below 50 degrees C, imidase quickly loses its activity at above 65 degrees C . Thus, the temperature effect on imidase activity is limited mainly by its thermostability . Substrate specificity of imidase is also temperature dependent . Our results demonstrate that the hydrolysis of physiological substrates is the most temperature dependent and that of hydantoins is the least temperature dependent . When increasing the reaction temperature from 25 to 60 degrees C, specific activities increase 50- and 60-fold for dihydrouracil and dihydrothymine, respectively . The temperature effect on the K(m) and V(max) of imidase is substrate dependent .

Arch Environ Contam Toxicol, 2000 Aug, 39(2), 145 - 53
Membrane lipid composition of Bacillus stearothermophilus as affected by lipophilic environmental pollutants: an approach to membrane toxicity assessment; Donato MM et al.; The thermophilic eubacterium Bacillus stearothermophilus is used as a model to identify membrane perturbing effects of lipophilic compounds . A parallelism has been established between the toxicity of the organochlorine insecticide DDT and its metabolite, DDE, in bacterial growth and the effects on cell functions and physical perturbations induced at the membrane (Donato et al . 1997a, Arch Environ Contam Toxicol 33:109-116; Donato et al . 1997b, Appl Environ Microbiol 63:4948-495) . In the present work, the use of B . stearothermophilus as a model of screening for chemical toxicity has been implemented . Because the regulation of the lipid composition of the membrane is a common strategy in response to adverse growth conditions, we studied the effects of DDE on the lipid composition and the consequent alterations of membrane physical properties in comparison to the parental compound DDT . As expected, different adaptation responses were induced by the compounds, being DDT more effective as compared with DDE . Collected data are consistent with the stronger perturbations induced by DDT on growth and membrane functions . It is concluded that the membrane lipid composition of the bacterium is a very sensitive criterium to detect membrane-mediated toxic effects at low concentrations of lipophilic xenobiotics.

Nucleic Acids Res, 2000 Jun 1, 28(11), 2221 - 8
Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum; Sriskanda V et al.; We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum . The 561 amino acid MTH: ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K(m) 1.1 microM) . dATP can substitute for ATP, but CTP, GTP, UTP and NAD(+) cannot . MTH: ligase activity is thermophilic in vitro, with optimal nick-joining at 60 degrees C . Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction . Mutant K251A is unable to form the covalent ligase-adenylate intermediate (step 1) and hence cannot seal a 3'-OH/5'-PO(4) nick . Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3) . Mutant D253A is active in ligase-adenylate formation, but defective in activating the nick via formation of the DNA-adenylate intermediate (step 2) . D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick . A profound step 3 arrest, with accumulation of high levels of DNA-adenylate, could be elicited for the wild-type MTH: ligase by inclusion of calcium as the divalent cation cofactor . MTH: ligase sediments as a monomer in a glycerol gradient . Structure probing by limited proteolysis suggested that MTH: ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.

J Bacteriol, 2000 Jul, 182(14), 4104 - 7
Expression of the neutral protease gene from a thermophilic Bacillus sp . BT1 strain in Bacillus subtilis and its natural host: identification of a functional promoter; Vecerek B et al.; The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp . BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis . In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5' to the gene . In contrast, in heterologous B . subtilis this thermophilic npr promoter does not function, and expression of the npr gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X) . A functional promoter was identified 5' to ORF X that is required for efficient expression of the npr gene in Bacillus subtilis as verified by primer extension, reverse transcription-PCR, and 5' rapid amplification of cDNA ends experiments . These data suggest that transcriptional signals used in thermophilic Bacillus sp . BT1 strain are different from those used in B . subtilis.

J Bacteriol, 2000 Jul, 182(14), 3929 - 33
An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion; Xue H et al.; The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon Sulfolobus shibatae . Ssh10b constitutes about 4% of the cellular protein . Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of approximately approximately 12 bp, forming distinct shifts, until the DNA was coated with the protein . Binding of more Ssh10b resulted in the formation of smears of lower mobilities . The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not . Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperature-dependent fashion . While the ability of the protein to constrain supercoils was weak at 25 degrees C, it was enhanced substantially at 45 degrees C or higher temperatures (up to 80 degrees C) . Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.

Eur J Biochem, 2000 Jul, 267(13), 4290 - 9
Interaction of fMet-tRNAfMet and fMet-AMP with the C-terminal domain of Thermus thermophilus translation initiation factor 2; Szkaradkiewicz K et al.; Two polypeptides resistant against proteolytic digestion were identified in Thermus thermophilus translation initiation factor 2 (IF2): the central part of the protein (domains II/III), and the C-terminal domain (domain IV) . The interaction of intact IF2 and the isolated proteolytic fragments with fMet-tRNAfMet was subsequently characterized . The isolated C-terminal domain was as effective in binding of the 3' end of fMet-tRNAf Met as intact IF2 . N-Formylation of Met-tRNAfMet was required for its efficient binding to the C-terminal domain . This suggests that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is responsible for the recognition of fMet-tRNAfMet by IF2 during translation initiation . Moreover, it was demonstrated that fMet-AMP is a minimal ligand of IF2 . fMet-AMP inhibits fMet-tRNAfMet binding to IF2 as well as the activity of IF2 in the stimulation of ApUpG-dependent ribosomal binding of fMet-tRNAf Met . Specific interaction of fMet-AMP with IF2 was demonstrated by 1H-NMR spectroscopy . These findings indicate that fMet-AMP and the 3' terminal fMet-adenosine of fMet-tRNAfMet use the same binding site on the C-terminal domain of IF2 and imply that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is primarily responsible for the fMet-tRNAfMet binding and recognition by IF2.

Nature, 2000 Jun 8, 405(6787), 676 - 9
Filamentous microfossils in a 3,235-million-year-old volcanogenic massive sulphide deposit; Rasmussen B; The record of Archaean microfossils is sparse . Of the few bona fide fossil assemblages, most are from shallow-water settings, and they are typically associated with laminated, stromatolitic sedimentary rocks . Microfossils from deep-sea hydrothermal systems have not been reported in Precambrian rocks (> 544 million years old), although thermophilic microbes are ubiquitous in modern sea-floor hydrothermal settings, and apparently have the most ancient lineages . Here, I report the discovery of pyritic filaments, the probable fossil remains of thread-like microorganisms, in a 3,235-million-year-old deep-sea volcanogenic massive sulphide deposit from the Pilbara Craton of Australia . From their mode of occurrence, the micro-organisms were probably thermophilic chemotropic prokaryotes, which inhabited sub-sea-floor hydrothermal environments . They represent the first fossil evidence for microbial life in a Precambrian submarine thermal spring system, and extend the known range of submarine hydrothermal biota by more than 2,700 million years . Such environments may have hosted the first living systems on Earth, consistent with proposals for a thermophilic origin of life.

Enzyme Microb Technol, 2000 Jul 1, 27(1-2), 11 - 18
Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity; Burdette DS et al.; Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2 degrees ADH) was optimally active near 90 degrees C displaying thermostability half-lives of 1.2 days, 1.7 h, 19 min, 9.0 min, and 1.3 min at 80 degrees C, 90 degrees C, 92 degrees C, 95 degrees C, and 99 degrees C, respectively . Enzyme activity loss upon heating (90-100 degrees C) was accompanied by precipitation, but the soluble enzyme remaining after partial inactivation retained complete activity . Enzyme thermoinactivation was modeled by a pseudo-first order rate equation suggesting that the rate determining step was unimolecular with respect to protein and thermoinactivation preceded aggregation . The apparent 2 degrees ADH melting temperature (T(m)) occurred at approximately 115 degrees C, 20 degrees C higher than the temperature for maximal activity, suggesting that it is completely folded in its active temperature range . Thermodynamic calculations indicated that the active folded structure of the 2 degrees ADH is stabilized by a relatively small Gibbs energy (triangle upG(stab.)(double dagger) = 110 kJ mol(-1)) . 2 degrees ADH catalytic activities at 37 degrees C to 75 degrees C, were 2-fold enhanced by guanidine hydrochloride (GuHCl) concentrations between 120 mM and 190 mM . These results demonstrate the extreme resistance of this thermophilic 2 degrees ADH to thermal or chemical denaturation; and suggest increased temperature or GuHCl levels seem to enhance protein fixability and activity.

J Biol Chem, 2000 Sep 29, 275(39), 30019 - 28
Cloning, overexpression, and characterization of peroxiredoxin and NADH peroxiredoxin reductase from Thermus aquaticus; Logan C et al.; The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus . prx is located upstream from prxR, the two genes being separated by 13 bases . The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria . The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues . PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension . The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers . The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer . PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide . The activity of Prx is much less than has been observed with homologous proteins . Prx appears to be inactivated by cumene hydroperoxide . PrxR itself has low peroxidase activity.

J Biol Chem, 2000 Sep 15, 275(37), 28731 - 8
Hypermodification of tRNA in Thermophilic archaea . Cloning, overexpression, and characterization of tRNA-guanine transglycosylase from Methanococcus jannaschii; Bai Y et al.; tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides . Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA . From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned . The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized . The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ(0)) is inserted . The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ(0) as substrates, but not other 7-deazaguanine bases, and is able to modify tRNA from all three phylogenetic domains . The enzyme shows optimal activity at high temperature and acidic pH, consistent with the optimal growth conditions of M . jannaschii . The nature of the temperature dependence is consistent with a requirement for some degree of tRNA tertiary structure in order for recognition by the enzyme to occur.

Proteins, 2000 Aug 15, 40(3), 473 - 81
The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus; D'Auria S et al.; The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa . The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C . Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism . We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus . In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays . Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus . Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme . The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics . The data suggested an increase in the protein flexibility on increasing the temperature . Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures . Proteins 2000;40:473-481 .

J Biol Chem, 2000 Oct 6, 275(40), 31086 - 92
Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus solfataricus; Yano JK et al.; The structure of the first P450 identified in Archaea, CYP119 from Sulfolobus solfataricus, has been solved in two different crystal forms that differ by the ligand (imidazole or 4-phenylimidazole) coordinated to the heme iron . A comparison of the two structures reveals an unprecedented rearrangement of the active site to adapt to the different size and shape of ligands bound to the heme iron . These changes involve unraveling of the F helix C-terminal segment to extend a loop structure connecting the F and G helices, allowing the longer loop to dip down into the active site and interact with the smaller imidazole ligand . A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering of aromatic residues may provide the structural basis for the enhanced thermal stability of CYP119 . An additional feature of the 4-phenylimidazole-bound structure is a zinc ion tetrahedrally bound by symmetry-related His and Glu residues.

FEMS Microbiol Lett, 2000 Jun 15, 187(2), 151 - 4
Demonstration of the carbon-sulfur bond targeted desulfurization of benzothiophene by thermophilic Paenibacillus sp . strain A11-2 capable of desulfurizing dibenzothiophene; Konishi J et al.; Paenibacillus sp . strain A11-2, which had been primarily isolated as a bacterial strain capable of desulfurizing dibenzothiophene to produce 2-hydroxybiphenyl at high temperatures, was found to desulfurize benzothiophene more efficiently than dibenzothiophene . The desulfurized product was identified as o-hydroxystyrene by GC-MS and 1H-NMR analysis . Benzothiophene was assumed to be degraded in a way analogous to the 4S pathway, which has been well-known as a mode of dibenzothiophene degradation . These results suggest that benzothiophene desulfurization may share at least partially the reaction mechanism with dibenzothiophene desulfurization.

EMBO J, 2000 Jun 15, 19(12), 3119 - 31
Three telomerases with completely non-telomeric template replacements are catalytically active; Ware TL et al.; Telomerase is a reverse transcriptase minimally composed of a reverse transcriptase protein subunit and an internal RNA component that contains the templating region . Point mutations of template RNA bases can cause loss of enzymatic activity, reduced processivity and misincorporation in vitro . Here we report the first complete replacement of the nine base TETRAHYMENA: thermophila telomerase templating region in vivo with non-telomeric sequences . Rather than ablating telomerase activity, three such replaced telomerases (U9, AUN and AU4) were effective in polymerization in vitro . In vivo, the AU4 and AUN genes caused telomere shortening . We demonstrated the fidelity of the AUN and U9 telomerases in vitro and utilized AUN telomerase to demonstrate that 5' end primer recognition by telomerase is independent of template base pairing . However, the mutant AUN template telomerase catalyzed an abnormal DNA cleavage reaction . For these U-only and AU- substituted templates, we conclude that base-specific interactions between the telomerase template and protein (or distant parts of the RNA) are not absolutely required for the minimal core telomerase functions of nucleotide addition and base discrimination.

Science, 2000 Jun 16, 288(5473), 2048 - 51
A single-molecule study of RNA catalysis and folding; Zhuang X et al.; Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules . The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution . A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state . A rarely populated docked state, not measurable by ensemble methods, was observed . In the overall folding process, intermediate folding states and multiple folding pathways were observed . In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered . These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.

Appl Microbiol Biotechnol, 2000 May, 53(5), 591 - 5
Isolation of a delta7-cholesterol desaturase from Tetrahymena thermophila; Valcarce G et al.; Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to delta7-dehydrocholesterol (provitamin D3) . The activity was isolated in the microsomal fraction from Tetrahymena homogenates . Delta7-desaturase activity was stimulated fivefold by the addition of 6 mM ATP . Other cofactors assayed, including NAD, NADP, NADH or NADPH, had no significant effect . The activity was found in microsomes prepared from stationary-phase cultures of the ciliate, grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T . thermophila cells.

Appl Microbiol Biotechnol, 2000 May, 53(5), 517 - 24
Response of the thermophile Thermus sp . RQ-1 to hyperbaric air in batch and fed-batch cultivation; Belo I et al.; The effects of increased air pressure in a culture of the thermophilic microorganism Thermus sp . RQ-1 were investigated . Cell growth dependence on oxygen supply was investigated in a fermenter at atmospheric pressure . Total oxygen depletion from the medium for low values of kLa was observed during the exponential growth phase . It was possible with this strain to enhance the oxygen transfer rate by increasing the air pressure . Cell productivity was improved by pressurisation up to 0.56 MPa for batch cultivation; and an induction of the antioxidant enzymes, superoxide dismutase and catalase, was observed with the rise in pressure . Cell pre-cultivation under pressurised conditions conferred to the cells more resistance to an exposure to hydrogen peroxide and more sensitivity to paraquat (methyl viologen) . The usefulness of bioreactor pressurisation on the cultivation of Thermus sp . RQ-1 was demonstrated for fed-batch operation, with the attainment of higher cell densities . A two-fold increase in cell mass productivity was obtained by the use of hyperbaric air (0.5 MPa) . With the pressurisation of the head-space in the reactor, it was also possible to eliminate the loss of liquid by evaporation, which amounted to more than 10% at 70 degrees C and atmospheric pressure.

J Biol Chem, 2000 Sep 1, 275(35), 27062 - 8
A novel anti-tumor cytokine contains an RNA binding motif present in aminoacyl-tRNA synthetases; Kim Y et al.; Endothelial monocyte-activating polypeptide II (EMAP II) is a novel pro-apoptotic cytokine that shares sequence homology with the C-terminal regions of several tRNA synthetases . Pro-EMAP II, the precursor of EMAP II, is associated with the multi-tRNA synthetase complex and facilitates aminoacylation activity . The structure of human EMAP II, solved at 1.8 A resolution, revealed the oligomer-binding fold for binding different tRNAs and a domain that is structurally homologous to other chemokines . The similar structures to the RNA binding motif of EMAP II was previously observed in the anticodon binding domain of yeast Asp-tRNA synthetase (AspRSSC) and the B2 domain of Thermus thermophilus Phe-tRNA synthetase . The RNA binding pattern of EMAP II is likely to be nonspecific, in contrast to the AspRSSC . The peptide sequence that is responsible for cytokine activity is located, for the most part, in the beta1 strand . It is divided into two regions by a neighboring loop.

J Food Prot, 2000 Jun, 63(6), 758 - 62
Effect of carbon dioxide under high pressure on the survival of cheese starter cultures; van Hekken DL et al.; A new processing method that rapidly forms curds and whey from milk has the potential to improve cheesemaking procedures if cheese starter cultures can tolerate the processing conditions . The survival of Lactobacillus delbrueckii ssp . bulgaricus, Lactococcus lactis ssp . lactis, or Streptococcus thermophilus through this new process was evaluated . Inoculated milk containing 0, 1, or 3.25% fat or Lactobacillus MRS broth or tryptone yeast lactose broth (depending on microorganism used) was sparged with CO2 to a pressure of 5.52 MPa and held for 5 min at 38 degrees C . Broth contained 7.93 to 8.78 log CFU/ ml before processing and 7.84 to 8.66 log CFU/ml afterward . Before processing, milk inoculated with L bulgaricus, L . lactis, or S . thermophilus contained 6.81, 7.35, or 6.75 log CFU/ml, respectively . After processing, the curds contained 5.68, 7.32, or 6.50 log CFU/g, and the whey had 5.05, 6.43, or 6.14 log CFU/ml, respectively . After processing, the pHs of control samples were lower by 0.41 units in broth, 0.53 units in whey, and 0.89 units in curd . The pH of the processed inoculated samples decreased by 0.3 to 0.53 units in broth, 0.32 to 0.37 units in whey, and 0.93 to 0.98 units in the curd . Storing curds containing L . lactis at 30 degrees C or control curds and curds with L . bulgaricus or S . thermophilus at 37 degrees C for an additional 48 h resulted in pHs of 5.22, 5.41, 4.53, or 4.99, respectively . This study showed that milk inoculated with cheese starter cultures and treated with CO2 under high pressure to precipitate casein-produced curds that contained sufficient numbers of viable starter culture to produce lactic acid, thereby decreasing the pH.

Plant Cell Physiol, 2000 Apr, 41(4), 515 - 22
Effects of high-temperature treatments on a thermophilic cyanobacterium Synechococcus vulcanus; Inoue N et al.; Effects of high-temperature treatments on a thermophilic cyanobacterium, Synechococcus vulcanus, were studied, and the following results were obtained . (1) Oxygen evolution and the PSII photochemical reaction were the most sensitive sites and started to be inactivated at temperatures slightly higher than the cultivating temperature . (2) The decrease in the fluorescence Fv value reflected the inactivation of the charge separation reaction of PSII as well as that of the oxygen evolution reaction . (3) The dark fluorescence level, Fo, showed an increase at around 70 degrees C, which was partially reversed by further incubation at 50 degrees C . This increase reflected the inactivation of PSII reaction centers and probably dissociation of phycobilisomes from the PSII reaction center complexes . (4) At higher temperatures, phycobiliproteins disassembled and denatured in a pH-dependent manner, causing a large Fo decrease . (5) Cell membranes became leaky to low-molecular-weight substances at around 72 degrees C . (6) Inhibition of growth of the cells was recognized when the cells were pretreated at temperatures higher than 72 degrees C . Reversibility of the high-temperature effects and relationship between viability of the cells and the degradation of the cell membranes are discussed.

J Mol Biol, 2000 Jun 16, 299(4), 1051 - 60
An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase; Briand C et al.; The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T . thermophilus or Escherichia coli reveal a potential intermediate of the recognition process . The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes . While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains . The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state . Based on these data, a pathway for tRNA binding and recognition is proposed .

Biochemistry (Mosc), 2000 May, 65(5), 609 - 14
Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates; Rechkunova NI et al.; The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied . DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes . The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined . It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+ . The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+ . Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 963 - 70
Simultaneous saccharification and fermentation of cellulosic biomass to acetic acid; Borden JR et al.; A strain of Clostridium thermoaceticum (ATCC 49707) was evaluated for its homoacetate potential . This thermophilic anaerobe best produces acetate from glucose at pH 6.0 and 59 degrees C with a yield of 83% of theoretical . Enzyme hydrolysis of two substrates, a-cellulose and a pulp mill sludge, yielded 68% and 70% digestion, respectively . The optimum conditions for the simultaneous saccharification and fermentation (SSF) were substrate dependent: 55 degrees C, pH 6.0 for alpha-cellulose, and 55 degrees C, pH 5.5 for the pulp mill sludge . In the SSF with alpha-cellulose, the overall yield of acetate was strongly influenced by the enzyme loading . In a fed-batch operation of SSF with alpha-cellulose, an overall acetic acid yield of 60 wt% was obtained . Among the factors limiting the yields were incomplete digestion by the enzyme and the end-product inhibition . In the SSF of pulp mill sludge, inhibitors present in the sludge severely limited bacterial action . A large accumulation of glucose developed over the entire process, changing the intended SSF operation into a separate hydrolysis and fermentation operation . Despite a long lag phase of microbial growth, a terminal yield of 85% was obtained with this substrate.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 821 - 34
Use of corncob for endoxylanase production by thermophilic fungus Thermomyces lanuginosus IOC-4145; Damaso MC et al.; The production of cellulase-free endoxylanase by the thermophilic fungus Thermomyces lanuginosus was investigated in semisolid fermentation and liquid fermentation . Different process variables were investigated in semisolid fermentation, employing corncob as the carbon source . The best results were with the following conditions: grain size = 4.5 mm, solid:liquid ratio = 1:2, and inoculum size = 20% (v/v) . Corncob, xylan, and xylose were the best inducers for endoxylanase production . Additionally, organic nitrogen sources were necessary for the production of high endoxylanase activities . The crude enzyme had optimum activity at pH 6.0 and 75 degrees C, displaying a high thermostability . The apparent Km and Vmax were 1.77 mg of xylan/mL and 21.5 U/mg of protein, respectively.

J Appl Microbiol, 2000 Jun, 88(6), 975 - 82
Production and properties of hemicellulases by a Thermomyces lanuginosus strain; Singh S et al.; Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications . Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source . Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch . Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs . No cellulase activity was observed . The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5 . 5 and 9.5 were optimal for beta-xylanase activity . The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase . The pH optima of the other hemicellulases were between 5.0 and 6.5 . Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable . These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.

Eur J Biochem, 2000 Jun, 267(12), 3502 - 12
A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures; Georlette D et al.; The cloning, overexpression and characterization of a cold-adapted DNA ligase from the Antarctic sea water bacterium Pseudoalteromonas haloplanktis are described . Protein sequence analysis revealed that the cold-adapted Ph DNA ligase shows a significant level of sequence similarity to other NAD+-dependent DNA ligases and contains several previously described sequence motifs . Also, a decreased level of arginine and proline residues in Ph DNA ligase could be involved in the cold-adaptation strategy . Moreover, 3D modelling of the N-terminal domain of Ph DNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue . The recombinant Ph DNA ligase was overexpressed in Escherichia coli and purified to homogeneity . Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74 593 Da . Ph DNA ligase shows similar overall catalytic properties to other NAD+-dependent DNA ligases but is a cold-adapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpart E . coli DNA ligase . A kinetic comparison of three enzymes adapted to different temperatures (P . haloplanktis, E . coli and Thermus scotoductus DNA ligases) indicated that an increased kcat is the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreased Km for the nicked DNA substrate seems to allow T . scotoductus DNA ligase to work efficiently at high temperatures . Besides being useful for investigation of the adaptation of enzymes to extreme temperatures, P . haloplanktis DNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.

J Eukaryot Microbiol, 2000 May-Jun, 47(3), 185 - 90
Molecular mechanisms of microtubular organelle assembly in Tetrahymena; Gaertig J; Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell . Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm . These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Associated Proteins . For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia . It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of alpha- and two isotypes of 1-tubulin . However, Tetrahymena tubulins are modified secondarily by a host of posttranslational mechanisms . Each microtubule organelle type displays a unique set of secondary tubulin modifications . The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either alpha- or beta-tubulin . Both acetylation and polyglycylation of alpha-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way . However, the multiple polyglycylation sites on 1-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology . Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena.

Mol Microbiol, 2000 May, 36(4), 876 - 85
Growth phase-dependent expression and degradation of histones in the thermophilic archaeon Thermococcus zilligii; Dinger ME et al.; HTz is a member of the archaeal histone family . The archaeal histones have primary sequences and structural similarity to the eukaryal histone fold domain, and are thought to resemble the archetypal ancestor of the eukaryal nucleosome core histones . The effects of growth phase on the total soluble proteins from Thermococcus zilligii, isolated after various stages of growth from mid-logarithmic to late stationary phase, were examined by denaturing polyacrylamide gel electrophoresis . On entry into stationary phase, at least 11 proteins were detected that changed considerably in level . One of these proteins was identified by Western hybridization as HTz . The level of HTz decreased dramatically as cells entered stationary phase, and it could not be detected by late stationary phase . Unexpectedly, the Western hybridization detected a second protein, with an estimated molecular mass of approximately 14 kDa, which paralleled the decrease in level of HTz . Native purified HTz was shown to retain complete activity after prolonged incubation at the growth temperature of the organism, suggesting that the decrease in HTz was a specific cell-regulated process . Analysis of native purified HTz by electrospray ionization mass spectrometry revealed the molecular masses of HTz1 and HTz2 to be 7204 +/- 3 Da and 7016 +/- 3 Da respectively . The only non-covalent species that was detected corresponded to the molecular mass of an HTz1-HTz2 heterodimer . Northern analyses of T . zilligii total RNA with an htz1 gene probe indicated a rapid decrease in expression of htz1 with progression of the growth phase, and complete repression of htz1 transcript synthesis by late logarithmic phase . Three proteins that changed in level with growth phase were identified by N-terminal sequence analysis . The first was homologous to a hypothetical protein conserved in all Archaea sequenced to date, the second to the Sac10b family of archaeal DNA-binding proteins and the third to the C-terminal region of the leucine-responsive regulatory family of DNA-binding proteins (LRPs).

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1287 - 95
Thermanaerovibrio velox sp . nov., a new anaerobic, thermophilic, organotrophic bacterium that reduces elemental sulfur, and emended description of the genus Thermanaerovibrio; Zavarzina DG et al.; A moderately thermophilic, organotrophic bacterium with vibrioid cells was isolated from a sample of a cyanobacterial mat from caldera Uzon, Kamchatka, Russia, and designated strain Z-9701T . Cells of strain Z-9701T were curved, Gram-negative rods, 0.5-0.7 x 2.5-5.0 microm in size, with tapering ends and with fast, wavy movement by means of lateral flagella located on the concave side of the cell . Colonies were small, white, irregular or round, 0.2 mm in diameter, and with even edges . Strain Z-9701T was an obligate anaerobe with a temperature optimum at 60-65 degrees C and a pH optimum at 7.3 . It fermented glucose, fructose, mannose, N-acetyl-D-glucosamine, adonite, arginine, serine, peptone, yeast extract and Casamino acids . The fermentation products formed during growth on glucose were acetate, lactate, H2, CO2 and ethanol . Strain Z-9701T reduced elemental sulfur to H2S during organotrophic growth with glucose or peptides as energy and carbon sources . In the presence of S0, strain Z-9701T was capable of lithotrophic growth with molecular hydrogen as energy substrate and 0.1 g yeast extract l(-1) as carbon source . Sulfate, thiosulfate, nitrate, Fe(III) and sulfite were not reduced and did not stimulate growth . The G+C content of strain Z-9701T DNA was 54.6 mol% . The results of 16S rDNA sequence analyses revealed that strain Z-9701T belongs to the cluster within the Clostridium group formed by Thermanaerovibrio acidaminovorans, Dethiosulfovibrio peptidovorans, Anaerobaculum thermoterrenum and Aminobacterium colombiense, but the level of sequence similarity with the members of this cluster was not very high (87.6-92.2%) . Among these organisms, Thermanaerovibrio acidaminovorans is phenotypically close to strain Z-9701T . However, the two organisms showed a relatively low level of similarity of their 16S rRNA sequences (92.2%) and of DNA-DNA hybridization (15 +/- 1%) . Nevertheless, on the basis of the similar morphology and physiology of the new isolate and Thermanaerovibrio acidaminovorans, strain Z-9701T was placed in the genus Thermanaerovibrio and a new species, Thermanaerovibrio velox, proposed for it . The type strain is Z-9701T (= DSM 12556T).

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1239 - 46
Desulfacinum hydrothermale sp . nov., a thermophilic, sulfate-reducing bacterium from geothermally heated sediments near Milos Island (Greece); Sievert SM et al.; A thermophilic, sulfate-reducing bacterium, strain MT-96T, was isolated from an active, marine, shallow-water hydrothermal vent system . It used a large variety of substrates, ranging from simple organic compounds to long-chain fatty acids, as electron donors . Autotrophic growth was possible with H2 and CO2 in the presence of sulfate . Sulfate, thiosulfate and sulfite were used as electron acceptors . Sulfur and nitrate were not reduced . Fermentative growth was obtained with pyruvate, but not with fumarate or malate . Substrate oxidation was usually complete, leading to production of CO2, but at high substrate concentrations acetate accumulated . The oval-shaped cells were 0.8-1.0 microm in width and 1.5-2.5 microm in length . Cells were motile during the early-exponential-growth phase, but motility rapidly declined during later growth phases . Spores were not produced and cells stained Gram-negative . The temperature limits for growth were between 37 and 64 degrees C, with an optimum at 60 degrees C . Growth was observed at salinities ranging from 15 to 78 g NaCl l(-1), with optimum growth in the presence of 32-36 g NaCl l(-1) . This might reflect an adaptation to the elevated salinity of the hydrothermal fluid . The G+C content of the DNA was 59.5 mol% . Vitamins or other supplements were not required . Based on the 16S rRNA gene sequence, strain MT-96T belonged in the delta-subclass of the Proteobacteria . Strain MT-96T was found to be phenotypically and phylogenetically related to Desulfacinum infernum (< 95.3% sequence identity) and represents a new member of the genus Desulfacinum . The name Desulfacinum hydrothermale is proposed for this strain; the type strain is MT-96T (= DSM 13146).

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1155 - 62
Caloramator coolhaasii sp . nov., a glutamate-degrading, moderately thermophilic anaerobe; Plugge CM et al.; An obligately anaerobic, moderately thermophilic, glutamate-degrading bacterium (strain ZT) was isolated from an enrichment culture obtained from anaerobic thermophilic granular sludge . The cells were rod-shaped to filamentous and showed no motility or spore formation . The cell wall had a Gram-positive structure, which was revealed by electron microscopy . Optimum growth of the strain was observed under neutrophilic conditions at 50-55 degrees C . The doubling time of strain ZT grown in rich medium was approximately 1 h at optimal pH and temperature . Strain ZT was able to grow on a variety of organic compounds . Most carbon sources were converted to acetate, CO2, H2, and traces of propionate and lactate . Strain ZT oxidized glutamate to acetate, CO2, NH4+, traces of propionate and H2 . The doubling time on this substrate was 1-6 d . The strain fermented glutamate syntrophically in co-culture with Methanobacterium thermoautotrophicum Z-245T to the same products, but the co-culture had a fourfold higher growth rate . 16S rDNA sequence analysis revealed a relationship with Thermobrachium celere, Caloramator indicus and Caloramator proteoclasticus . The G+C content was 31.7 mol% . Based on its morphological, phylogenetic and physiological characteristics, it is proposed that strain ZT should be classified in the genus Caloramator as a new species, Caloramator coolhaasii.

J Biol Chem, 2000 Nov 3, 275(44), 34080 - 5
HPr(His approximately P)-mediated phosphorylation differently affects counterflow and proton motive force-driven uptake via the lactose transport protein of Streptococcus thermophilus; Gunnewijk MG et al.; The lactose transport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) . The IIA domain of LacS is phosphorylated on His-552 by the general energy coupling proteins of the PTS, which are Enzyme I and HPr . To study the effect of phosphorylation on transport, the LacS protein was purified and incorporated into liposomes with the IIA domain facing outwards . This allowed the phosphorylation of the membrane-reconstituted protein by purified HPr(His approximately P) of S . thermophilus . Phosphorylation of LacS increased the V(max) of counterflow transport, whereas the V(max) of the proton motive force (delta p)-driven lactose uptake was not affected . In line with a range of kinetic studies, we propose that phosphorylation affects the rate constants for the reorientation of the ternary complex (LacS with bound lactose plus proton), which is rate-determining for counterflow but not for delta p-driven transport.

J Biol Chem, 2000 Nov 3, 275(44), 34073 - 9
Phosphorylation state of HPr determines the level of expression and the extent of phosphorylation of the lactose transport protein of Streptococcus thermophilus; Gunnewijk MG et al.; The lactose transport protein (LacS) of Streptococcus thermophilus is composed of a translocator domain and a regulatory domain that is phosphorylated by HPr(His approximately P), the general energy coupling protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) . Lactose transport is affected by the phosphorylation state of HPr through changes in the activity of the LacS protein as well as expression of the lacS gene . To address whether or not CcpA-HPr(Ser-P)-mediated catabolite control is involved, the levels of LacS were determined under conditions in which the cellular phosphorylation state of HPr greatly differed . It appears that HPr(Ser-P) is mainly present in the exponential phase of growth, whereas HPr(His approximately P) dominates in the stationary phase . The transition from HPr(Ser-P) to HPr(His approximately P) parallels an increase in LacS level, a drop in lactose and an increase in galactose concentration in the growth medium . Because the K(m)(out) for lactose is higher than that for galactose, the lactose transport capacity decreases as lactose concentration decreases and galactose accumulates in the medium . Our data indicate that S . thermophilus compensates for the diminished transport capacity by synthesizing more LacS and phosphorylating the protein, which results in increased transport activity . The link between transport capacity and lacS expression levels and LacS phosphorylation are discussed.

Biochemistry, 2000 Jun 13, 39(23), 6791 - 8
Evidence for unfolding of the single-stranded GCCA 3'-End of a tRNA on its aminoacyl-tRNA synthetase from a stacked helical to a foldback conformation; Madore E et al.; The conformation of a tRNA in its initial contact with its cognate aminoacyl-tRNA synthetase was investigated with the Escherichia coli glutamyl-tRNA synthetase-tRNA(Glu) complex . Covalent complexes between the periodate-oxidized tRNA(Glu) and its synthetase were obtained . These complexes are specific since none were formed with any other oxidized E . coli tRNA . The three major residues cross-linked to the 3'-terminal adenosine of oxidized tRNA(Glu) are Lys115, Arg209, and Arg48 . Modeling of the tRNA(Glu)-glutamyl-tRNA synthetase based on the known crystal structures of Thermus thermophilus GluRS and of the E . coli tRNA(Gln)-glutaminyl-tRNA synthetase complex shows that these three residues are located in the pocket that binds the acceptor stem, and that Lys115, located in a 26 residue loop closed by coordination to a zinc atom in the tRNA acceptor stem-binding domain, is the first contact point of the 3'-terminal adenosine of tRNA(Glu) . In our model, we assume that the 3'-terminal GCCA single-stranded segment of tRNA(Glu) is helical and extends the stacking of the acceptor stem . This assumption is supported by the fact that the 3' CCA sequence of tRNA(Glu) is not readily circularized in the presence of T4 RNA ligase under conditions where several other tRNAs are circularized . The two other cross-linked sites are interpreted as the contact sites of the 3'-terminal ribose on the enzyme during the unfolding and movement of the 3'-terminal GCCA segment to position the acceptor ribose in the catalytic site for aminoacylation.

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6311 - 5
Solution structure of the RNA polymerase subunit RPB5 from Methanobacterium thermoautotrophicum; Yee A et al.; RPB5 is an essential subunit of eukaryotic and archaeal RNA polymerases . It is a proposed target for transcription activator proteins in eukaryotes, but the mechanism of interaction is not known . We have determined the solution structure of the RPB5 subunit from the thermophilic archeon, Methanobacterium thermoautotrophicum . MtRBP5 contains a four-stranded beta-sheet platform supporting two alpha-helices, one on each side of the beta-sheet, resulting in an overall mushroom shape that does not appear to have any structural homologues in the structural database . The position and conservation of charged surface residues suggests possible modes of interaction with other proteins, as well as a rationale for the thermal stability of this protein.

J Dairy Res, 2000 May, 67(2), 261 - 71
Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains; Valence F et al.; Intracellular peptidases of Lactobacillus helveticus may play a major role in the proteolysis of Swiss cheeses, provided that they are released through bacterial lysis . Experimental Swiss cheeses were manufactured on a small scale from thermized and microfiltered milk using as starters (in addition to Streptococcus thermophilus and Propionibacterium freudenreichii) one of two Lb . helveticus strains, ITGLH1 and ITGLH77, which undergo lysis to different extents in vitro . All the cheeses were biochemically identical after pressing . The viability of Lb . helveticus ITGLH1 and ITGLH77 decreased to a similar extent (96-98%) while in the cold room, but the concomitant release of intracellular lactate dehydrogenase in cheeses made with strain ITGLH1 was 5-7-fold that in cheeses made with ITGLH77 . Protein profiles and immunoblot detection of the dipeptidase PepD confirmed a greater degree of lysis of the ITGLH1 strain . Free active peptidases were detected in aqueous extracts of cheese for both strains, and proteolysis occurred principally in the warm room . Reversed-phase HPLC revealed a more extensive peptide hydrolysis for ITGLH1, which was confirmed by the greater release of free NH2 groups (+33%) and free amino acids (+75%) compared with ITGLH77 . As the intracellular peptidase activities of ITGLH1 and ITGLH77 have previously been shown to be similar, our results indicated that the extent of lysis of Lb . helveticus could have a direct impact on the degree of proteolysis in Swiss cheeses.

J Dairy Res, 2000 May, 67(2), 241 - 7
Fatty acid composition and freeze-thaw resistance in lactobacilli; Gomez Zavaglia A et al.; The fatty acid composition and freeze-thaw resistance of eight strains of thermophilic lactobacilli were studied . Seven of these contained the same polar and neutral lipids, the five major components making up 90% of the cellular fatty acid pool being 14:0, 16:0, 16:1, 18:1 and C19 cyclopropane (cyc19:0) . Strain comparison by means of cluster analysis based on the fatty acid ratios using the overlap coefficient revealed two well defined clusters . One was formed by three strains of species Lactobacillus delbrueckii subsp . lactis and Lb . delbrueckii subsp . delbrueckii, the other included five strains of the species Lb . delbrueckii subsp . bulgaricus, Lb . acidophilus and Lb . helveticus . Resistance of strains with a high content of unsaturated fatty acids (66-70%) decreased with increasing cyc19:0 concentrations . In contrast, in strains with a low concentration of unsaturated fatty acids (42-49%), increasing cyc19:0 levels were associated with increased freeze-thaw resistance.

Mol Cell Biochem, 2000 Mar, 206(1-2), 91 - 6
Purification, characterization and thermostability of lipase from a thermophilic Bacillus sp . J33; Nawani N et al.; A thermostable lipase produced by a thermophilic Bacillus sp . J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography . The enzyme is a monomeric protein having molecular weight of 45 kDa . It hydrolyzes triolein at all positions . The fatty acid specificity of lipase is broad with little preference for C12 and C4 . The Km and Vmax for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 microM min(-1) ml(-1) respectively . The immobilized enzyme was stable for 12 h at 60 degrees C . Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers . Lipase acquired a remarkable stability, since no deactivation occurred at 70 degrees C for 150 min in the presence of additives.

RNA, 2000 May, 6(5), 717 - 29
The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography; Lancaster L et al.; Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins . To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes . Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems . In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding . These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome . The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.

J Mol Biol, 2000 Jun 9, 299(3), 655 - 65
Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA; Schaper S et al.; The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system . The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene . The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family . Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction . Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E . coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding . The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes . Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP . The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins .

Biotechnol Prog, 2000 May-Jun, 16(3), 442 - 6
Affinity purification of fusion chaperonin Cpn60-(His)(6) from thermophilic bacterium Bacillus strain MS and its use in facilitating protein refolding and preventing heat denaturation; Teshima T et al.; The cpn60 gene from Bacillus strain MS, which is highly homologous to Bacillus stearothermophilus, was cloned . Cpn60 with a hexahistidine affinity tag (His)(6) fused to its C-terminus (cpn60-(His)(6)) was overproduced in Escherichia coli . Cpn60-(His)(6) was expressed in a soluble form in E . coli . and purified to homogeneity in a single step by nickel chelate affinity chromatography . Cpn60-(His)(6) formed a tetradecamer and had ATPase activity . Cpn60-(His)(6) mediated refolding of guanidine hydrochloride unfolded pig heart malic dehydrogenase (MDH) and Thermus flavus MDH at 25 and 70 degrees C, respectively, in an ATP-dependent manner . In addition, cpn60-(His)(6) prevented heat denaturation of pig heart MDH and T . flavus MDH at 30 and 80 degrees C, respectively, in an ATP-dependent manner . Therefore, cpn60-(His)(6) facilitates protein refolding and prevents heat denaturation of proteins across a wide temperature range.

J Cell Biol, 2000 May 29, 149(5), 1097 - 106
Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila; Xia L et al.; We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies . Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively . Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential . A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis . Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin . In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells . Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.

Appl Environ Microbiol, 2000 Jun, 66(6), 2330 - 5
Dynamic changes of intracellular pH in individual lactic acid bacterium cells in response to a rapid drop in extracellular pH; Siegumfeldt H et al.; We describe the dynamics of changes in the intracellular pH (pH(i)) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH(ex)) . Strains of Lactobacillus delbrueckii subsp . bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated . Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison . The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH(i) within a population . The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH(ex) from 7.0 to 5.0 . Under these conditions, the pH(i) of L . innocua remained neutral (between 7 and 8) . In contrast, the pH(i) values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH(ex) was decreased . No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases . Small differences between species were observed with regard to the initial pH(i) at pH(ex) 7.0, while different kinetics of pH(i) regulation were observed in different species and also in different strains of S . thermophilus.

Biosci Biotechnol Biochem, 2000 Apr, 64(4), 899 - 902
Efficient selection for thermostable protease in Thermus thermophilus; Takagi H et al.; An efficient procedure was established to select for thermostable proteases in an extreme thermophile, Thermus thermophilus . A non-protease-secreting mutant derived from T . thermophilus TH125 was used as host and the expression plasmid for aqualysin I from T . aquaticus YT-1 was constructed as a source of thermostable protease . T . thermophilus cells harboring the recombinant plasmid produced active aqualysin I into the medium and were able to grow on a minimal medium containing milk casein as the sole source of carbon and nitrogen.

Biochem Soc Trans, 1999 Dec, 27(6), 767 - 79
Colworth Medal Lecture . Enzymes in the quantum world; Scrutton NS; Studies on those enzymes and electron-transfer proteins involved in the catabolism of 'C1' substrates in methylotrophic bacteria have provided a wealth of information concerning the transfer of electrons and hydrogen by quantum tunnelling mechanisms . With regard to H-transfer, studies with MADH have provided the first example of ground-state tunnelling of hydrogen driven by the natural, thermally activated, low-frequency motions of the enzyme molecule . Subsequent studies with related enzymes (e.g . TMADH and bacterial sarcosine oxidase) and with thermophilic alcohol dehydrogenase suggest that vibrationally assisted tunnelling of hydrogen may be more widespread than originally assumed . Our studies of electron transfer in TMADH and ETF have established a role for large-scale protein dynamics in interprotein electron transfer, and have made a contribution to the ongoing debate concerning the mechanism of amine oxidation by enzymes . Moreover, our work has identified a hitherto unknown mechanism for the control of electron density in reduced flavin that influences the rate of electron transfer between redox centres within a protein molecule . Despite this progress, however, many questions still remain to be resolved . With the development of more sophisticated experimental techniques (and also continued financial support from the funding agencies!), the mechanistic uncertainties surrounding the quantum mechanical transfer of electrons and hydrogen in biological molecules should be transmogrified into the certainties one more readily acquaints with the classical world.

FEMS Microbiol Lett, 2000 Jun 1, 187(1), 69 - 76
Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain; Husson-Kao C et al.; The gene encoding Mur1, a Streptococcus thermophilus peptidoglycan hydrolase, was cloned by homology with acmA, the Lactococcus lactis major autolysin gene . Mur1 is a 24.7-kDa protein endowed with a putative signal peptide . Sequence analysis evidenced that Mur1 encompasses exactly the AcmA region containing the catalytic domain, but lacks the one containing amino acid repeats involved in cell wall binding . Mur1 appears to be expressed and cell-associated in S . thermophilus, as revealed by immunoblot analysis . These results suggest that the cell wall attachment mode of Mur1 differs from that of most peptidoglycan hydrolases described so far.

FEMS Microbiol Lett, 2000 Jun 1, 187(1), 47 - 52
Effects of a muramidase on a mixed bacterial community; Mercier C et al.; In bacterial communities one bacterium can influence the growth of other members of the population . These interactions may be based on nutritional factors or may occur via bacterial signaling molecules that are released in the medium . We present an example, showing that in addition to the above means of interactions, muramidases, enzymes that specifically cleave peptidoglycan chains, can also mediate interactions between bacteria . Using fluorescent in situ hybridization we demonstrate that Lactococcus lactis muramidase AcmA can hydrolyze the cell wall of Streptococcus thermophilus, without affecting viability . This intercellular activity of the lactococcal muramidase results in chain disruption of streptococci in vivo . Our data lead us to propose that chains can give growth advantages to streptococci in aerobic conditions.

J Biol Chem, 2000 Aug 11, 275(32), 24693 - 700
RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases; Ma WP et al.; DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity . In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand . By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates . Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity . There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates . Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold . Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity . The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously . The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 315 - 20
Thermobacillus xylanilyticus gen . nov., sp . nov., a new aerobic thermophilic xylan-degrading bacterium isolated from farm soil; Touzel JP et al.; An aerobic, thermophilic, xylanolytic, spore-forming bacterium, XETP (T = type strain; P = patent strain), has been isolated from farm soil situated underneath a manure heap in northern France . Strain XETP, which stained negative in the Gram test, occurs as short rods which sometimes form chains . Its spores are ellipsoidal, central to subterminal and occur in swollen sporangia . It grows at temperatures up to 63 degrees C and in the pH range 6.5-8.5 . When grown on glucose in optimal conditions, its doubling time was found to be 33 min . CO2 was observed to have a growth-stimulating effect at the start of the culture . In addition to glucose, the isolate utilizes xylose, arabinose, mannose, cellobiose, galactose, maltose, sucrose, xylan and starch . Growth is inhibited by 5% NaCl . The G+C content of strain XETP is 57.5 mol% . The 16S rDNA sequence analysis indicated that strain XETP falls into the radiation of the Bacillus-Lactobacillus-Streptococcus subdivision of the Gram-positive phylum . Its three closest phylogenetic relatives are 'Bacillus viscosus', Paenibacillus curdlanolyticus and Bacillus popilliae with identity values of 91.15, 90.94 and 90.92%, respectively . The major cellular fatty acids are 14-methyl pentadecanoic acid (16:0 iso), hexadecanoic acid (16:0) and 14-methyl hexadecanoic acid (17:0 anteiso) . On the basis of 16S rRNA sequence and chemotaxonomic characteristics, the isolate is different enough for it to be considered as a member of a new genus . It is therefore proposed that this isolate represents a new genus and species: Thermobacillus xylanilyticus . Strain XETP, the type strain of Thermobacillus xylanilyticus, has been deposited in the Collection Nationale de Cultures Microbiennes (CNCM I-1017) as a patent strain.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 209 - 17
Thermus igniterrae sp . nov . and Thermus antranikianii sp . nov., two new species from Iceland; Chung AP et al.; Several yellow-pigmented isolates, with optimum growth temperatures of about 65-70 degrees C, were recovered from hot springs in Iceland . Phylogenetic analysis of the 16S rDNA and DNA-DNA reassociation values showed that these organisms represented two new species of the genus Thermus . Strains RF-4T and HN1-8 had maximum temperatures for growth below 80 degrees C, while strains HN3-7T and HN2-7, unlike all other strains of the species of the genus Thermus except those belonging to Thermus thermophilus, grew at 80 degrees C . The new isolates from Iceland could not be distinguished easily from each other or from other strains of the species of the genus Thermus by biochemical characteristics; however, strains RF-4T and HN1-8 assimilated ribitol, a characteristic which was not detected in any of the other strains examined . Moreover, the species represented by strains RF-4T and HN1-8 and the species represented by strains HN3-7T and HN2-7 could be distinguished clearly from the other species of Thermus by their fatty acid composition . Strains RF-4T and HN1-8 have the highest combined levels of iso-15:0 and iso-17:0 and the lowest levels of iso-16:0 of any of the strains of the species of Thermus, while strains HN3-7T and HN2-7 are characterized by a very low iso-15:0/iso-17:0 ratio . On the basis of the phylogenetic analysis, DNA-DNA reassociation values, physiological and biochemical characteristics and fatty acid composition, the name Thermus igniterrae sp . nov . is proposed for the species represented by strains RF-4T and HN1-8 and the name Thermus antranikianii sp . nov . is proposed for the species represented by strains HN3-7T and HN2-7.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 119 - 25
Bifidobacterium thermacidophilum sp . nov., isolated from an anaerobic digester; Dong X et al.; A new phenotypic group of Bifidobacterium strains, isolated from an anaerobic digester for the treatment of waste water from a bean-curd farm, was described previously . In this study, the DNA-DNA relatedness between strain 36 (type strain, AS 1.2282T) of this new group and the type strains of other described Bifidobacterium species was analysed . The low level of DNA homology (0-58.9%) as well as comparison of the 16S rDNA sequences confirmed the distinct phylogenetic position of strain 36 . In addition, the new species differed from other Bifidobacterium species in its phenotypic characteristics, such as its growth at moderately thermophilic conditions (49.5 degrees C) and at relatively low pH (4.0), as well as its sugar-fermentation pattern . On the basis of phenotypic, genetic and phylogenetic studies, a new Bifidobacterium species, Bifidobacterium thermacidophilum sp . nov., was designated.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 43 - 53
Phylogenetic analysis of 18 thermophilic Methanobacterium isolates supports the proposals to create a new genus, Methanothermobacter gen . nov., and to reclassify several isolates in three species, Methanothermobacter thermautotrophicus comb . nov., Methanothermobacter wolfeii comb . nov., and Methanothermobacter marburgensis sp . nov; Wasserfallen A et al.; Using a combination of 16S rRNA analysis and antigenic fingerprinting consisting of new and published data, the phylogenetic position of 18 thermophilic isolates currently classified as Methanobacterium species was reinvestigated . The results were verified by independent methods, including, where applicable, plasmid and phage typing . Comparative analysis of 16S rRNA data for 30 strains belonging to the order Methanobacteriales strongly suggested that mesophilic and thermophilic Methanobacterium isolates are distantly related and should be assigned to separate genera . For the thermophilic strains the genus Methanothermobacter was initially proposed by Boone, Whitman and Rouviere . Furthermore, the results support a reclassification of 15 isolates in three species within the proposed genus: (i) Methanothermobacter thermautotrophicus comb . nov., containing eight isolates, six of which are able to utilize formate (type strain deltaHT); (ii) Methanothermobacter wolfeii comb . nov., containing four formate-utilizing isolates (type strain DSM 2970T); (iii) Methanothermobacter marburgensis sp . nov., containing three obligately autotrophic isolates (type strain MarburgT) . Of the nine isolates formerly referred to as Methanobacterium thermoformicicum, six were reclassified as Methanothermobacter thermautotrophicus and three as Methanothermobacter wolfeii.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 25 - 33
Desulfotomaculum alkaliphilum sp . nov., a new alkaliphilic, moderately thermophilic, sulfate-reducing bacterium; Pikuta E et al.; A new moderately thermophilic, alkaliphilic, sulfate-reducing, chemolithoheterotrophic bacterium, strain S1T, was isolated from a mixed cow/pig manure with neutral pH . The bacterium is an obligately anaerobic, non-motile, Gram-positive, spore-forming curved rod growing within a pH range of 8.0-9.15 (optimal growth at pH 8.6-8.7) and temperature range of 30-58 degrees C (optimal growth at 50-55 degrees C) . The optimum NaCl concentration for growth is 0.1% . Strain S1T is an obligately carbonate-dependent alkaliphile . The G+C content of the DNA is 40.9 mol% . A limited number of compounds are utilized as electron donors, including H2+acetate, formate, ethanol, lactate and pyruvate . Sulfate, sulfite and thiosulfate, but not sulfur or nitrate, can be used as electron acceptors . Strain S1T is able to utilize acetate or yeast extract as sources of carbon . Analysis of the 16S rDNA sequence allowed strain S1T (= DSM 12257T) to be classified as a representative of a new species of the genus Desulfotomaculum, Desulfotomaculum alkaliphilum sp . nov.

Eur J Biochem, 2000 Jun, 267(11), 3139 - 49
The 28.3 kDa FK506 binding protein from a thermophilic archaeum, Methanobacterium thermoautotrophicum, protects the denaturation of proteins in vitro; Ideno A et al.; Two families of FK506 binding protein (FKBP) type peptidyl-prolyl cis-trans isomerase (PPIase) have been found in Archaea . One is the 16-18 kDa short type FKBP family, and another is the 26-30 kDa long type FKBP family . The latter has a longer C-terminal region than the former . In this study, the 28.3 kDa long type FKBP gene from a thermophilic archaeum, Methanobacterium thermoautotrophicum, was expressed in Escherichia coli, and its gene product (MbFK) was characterized . The PPIase activity of MbFK was much lower than those of other FKBPs reported against oligopeptidyl substrates . MbFK protected green fluorescent protein (GFP) and rhodanese from thermal denaturation . Furthermore, MbFK suppressed the aggregation of chemically unfolded rhodanese and elevated the yield of its refolding although this activity was weaker than that of GroEL/ES . We made two deletion mutants, MbFK-N which lacked the C-terminal region, and MbFK-C which had only the C-terminal region . Far-UV CD spectra of these mutants showed that their secondary structures did not change from that of the wild-type . Whereas the PPIase activity of MbFK-N was low but detectable, that of MbFK-C was undetectable . The MbFK-C protected the thermal protein aggregation, and possessed a weak but significant aggregation suppressing activity against chemically unfolded protein . However, the MbFK-N did not suppress the aggregation of chemically unfolded rhodanese while it protected heat induced aggregation of rhodanese . These results may indicate that aggregation suppressing activity of MbFK-W against chemically unfolded protein are exerted mainly by its C-terminal domain while both domains contribute to thermal protein aggregation suppression.

J Biol Chem, 2000 May 26, 275(21), 16057 - 63
The structure and the characteristic DNA binding property of the C-terminal domain of the RNA polymerase alpha subunit from Thermus thermophilus; Wada T et al.; The C-terminal domain of the alpha subunit of the RNA polymerase (alphaCTD) from Escherichia coli (Ec) regulates transcription by interacting with many kinds of proteins and promoter upstream (UP) elements consisting of AT-rich sequences . However, it is unclear how this system is common in all eubacteria . We investigate the structure and properties of alphaCTD from an extremely thermophilic eubacterium, Thermus thermophilus (Tt) . The solution structure of Tt alphaCTD (85 amino acids) was determined by NMR, and the interaction between Tt alphaCTD and DNA with different sequences was investigated by means of chemical shift perturbation experiments . The tertiary structure of Tt alphaCTD is almost identical with that of Ec alphaCTD despite 32% sequence homology . However, Tt alphaCTD interacts with the upstream region sequence of the promoter in the Tt 16 S ribosomal protein operon rather than the Ec UP element DNA . The upstream region sequence of Tt is composed of 25 base pairs with 40% AT, unlike the Ec UP element with 80% AT . The DNA binding site in Tt alphaCTD is located on the surface composed of helix 4 and the loop preceding helix 4 . The electric charges on this surface are not remarkably localized like those of Ec alphaCTD.

J Dairy Sci, 2000 May, 83(5), 923 - 30
Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus; Ibrahim SA et al.; A classical chemical mutagenesis protocol was evaluated for increasing beta-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans . Two Bifidobacterium species (B . breve and B . longum) and one strain each of Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . To screen for beta-galactosidase (beta-gal) overproducing mutants, optimized EMS and MNNG mutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) . Colonies that exhibited a blue color were selected for quantitative beta-gal activities using the o-nitrophenyl-beta-galactoside (ONPG) assay . Seventy-five mutants were obtained out of more than 2 million colonies screened and showed increased beta-galactosidase activities compared with the wild-type strains . EMS gave a higher frequency of beta-gal overproducing mutants than MNNG for three of the four strains, S . thermophilus, B . breve, and B . longum, whereas the frequency of L . delbrueckii ssp . bulgaricus beta-gal mutants was similar with both mutagens . The highest beta-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L . delbrueckii ssp . bulgaricus; 104% for S . thermophilus; 70% for B . breve; and 222% for B . longum mutants . This food-grade classical approach has the ability to moderately increase beta-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.

J Agric Food Chem, 2000 May, 48(5), 1512 - 7
Origin of acetaldehyde during milk fermentation using (13)C-labeled precursors; Ott A et al.; Acetaldehyde formation by Lactobacillus delbrueckii subsp . bulgaricus and Streptococcus thermophilus during fermentation of cow's milk was investigated using (13)C-labeled glucose, L-threonine, and pyruvate with a recent static-and-trapped-headspace technique that does not require derivatization of acetaldehyde prior to gas chromatography-mass spectrometry . Over 90% and almost 100% of acetaldehyde originated from glucose during fermentation by L . delbrueckii subsp . bulgaricus and S . thermophilus, respectively, taking into account both singly and doubly labeled acetaldehyde . As both microorganisms showed threonine aldolase activity and formed labeled acetaldehyde from (13)C-labeled threonine during the fermentation of milk, this amino acid should also contribute to the acetaldehyde produced.

J Biol Inorg Chem, 2000 Apr, 5(2), 204 - 12
Protein control of the formation and decomposition of the CYP119 and CYP101 aryl-iron complexes; Tschirret-Guth RA et al.; CYP119, the first thermophilic P450 enzyme, reacts much more slowly than CYP101 (P450cam) with aryldiazenes to give sigma-bonded aryl-iron complexes . The CYP119 complexes are stable anaerobically at 80 degrees C but are readily oxidized by O2 to give the N-arylprotoporphyrin IX regioisomers . The aryl shift can also be initiated in the absence of O2 by K3Fe(CN)6 . In contrast, the corresponding CYP101 complexes are insensitive to O2 but decompose at temperatures above 50 degrees C owing to denaturation of the protein . The rate of the CYP119 aryl shift is decreased by electron-withdrawing substituents, with rho = -1.50 for both the O2- and K3Fe(CN)6-dependent reactions . A similar dependence (rho = -0.90) is observed for the K3Fe(CN)6-dependent CYP101 shift . The enthalpies and entropies of activation suggest that the CYP119 and CYP101 K3Fe(CN)6-mediated reactions are similar, but the CYP119 O2-dependent reaction proceeds via a different transition state . In all cases, the rate-determining step is oxidation of the aryl-iron complex . The temperature dependence of the O2- and K3Fe(CN)6-dependent CYP119 shifts provides evidence for temperature-dependent equilibration of two active site conformations . The oxygen sensitivity of the CYP119 aryl-iron complexes, and the temperature dependence of their rearrangement, reflect the unique active site properties of this thermophilic P450 enzyme.

J Biol Chem, 2000 Aug 4, 275(31), 23834 - 40
Close approximation of putative alpha -helices II, IV, VII, X, and XI in the translocation pathway of the lactose transport protein of Streptococcus thermophilus; Veenhoff LM et al.; The lactose transport protein (LacS) of Streptococcus thermophilus belongs to a family of transporters in which putative alpha-helices II and IV have been implicated in cation binding and the coupled transport of the substrate and the cation . Here, the analysis of site-directed mutants shows that a positive and negative charge at positions 64 and 71 in helix II are essential for transport, but not for lactose binding . The conservation of charge/side-chain properties is less critical for Glu-67 and Ile-70 in helix II, and Asp-133 and Lys-139 in helix IV, but these residues are important for the coupled transport of lactose together with a proton . The analysis of second-site suppressor mutants indicates an ion pair exists between helices II and IV, and thus a close approximation of these helices can be made . The second-site suppressor analysis also suggests ion pairing between helix II and the intracellular loops 6-7 and 10-11 . Because the C-terminal region of the transmembrane domain, especially helix XI and loop 10-11, is important for substrate binding in this family of proteins, we propose that sugar and proton binding and translocation are performed by the joint action of these regions in the protein . Indeed, substrate protection of maleimide labeling of single cysteine mutants confirms that alpha-helices II and IV are directly interacting or at least conformationally involved in sugar binding and/or translocation . On the basis of new and published data, we reason that the helices II, IV, VII, X, and XI and the intracellular loops 6-7 and 10-11 are in close proximity and form the binding sites and/or the translocation pathway in the transporters of the galactosides-pentosides-hexuronides family.

Proteins, 2000 Jul 1, 40(1), 58 - 70
Lower kinetic limit to protein thermal stability: a proposal regarding protein stability in vivo and its relation with misfolding diseases; Plaza del Pino IM et al.; In vitro thermal denaturation experiments suggest that, because of the possibility of irreversible alterations, thermodynamic stability (i.e., a positive value for the unfolding Gibbs energy) does not guarantee that a protein will remain in the native state during a given timescale . Furthermore, irreversible alterations are more likely to occur in vivo than in vitro because (a) some irreversible processes (e.g., aggregation, "undesirable" interactions with other macromolecular components, and proteolysis) are expected to be fast in the "crowded" cellular environment and (b) in many cases, the relevant timescale in vivo (probably related to the half-life for protein degradation) is expected to be longer than the timescale of the usual in vitro experiments (of the order of minutes) . We propose, therefore, that many proteins (in particular, thermophilic proteins and "complex" proteins systems) are designed (by evolution) to have significant kinetic stability when confronted with the destabilizing effect of irreversible alterations . We show that, as long as these alterations occur mainly from non-native states (a Lumry-Eyring scenario), the required kinetic stability may be achieved through the design of a sufficiently high activation barrier for unfolding, which we define as the Gibbs energy barrier that separates the native state from the non-native ensemble (unfolded, partially folded, and misfolded states) in the following generalized Lumry-Eyring model: Native State <--> Non-Native Ensemble --> Irreversibly Denatured Protein . Finally, using familial amyloid polyneuropathy (FAP) as an illustrative example, we discuss the relation between stability and amyloid fibril formation in terms of the above viewpoint, which leads us to the two following tentative suggestions: (a) the hot spot defined by the FAP-associated amyloidogenic mutations of transthyretin reflects the structure of the transition state for unfolding and (b) substances that decrease the in vitro rate of transthyretin unfolding could also be inhibitors of amyloid fibril formation .

EMBO J, 2000 May 15, 19(10), 2351 - 61
The 2 A crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue; Cusack S et al.; Leucyl-, isoleucyl- and valyl-tRNA synthetases are closely related large monomeric class I synthetases . Each contains a homologous insertion domain of approximately 200 residues, which is thought to permit them to hydrolyse ('edit') cognate tRNA that has been mischarged with a chemically similar but non-cognate amino acid . We describe the first crystal structure of a leucyl-tRNA synthetase, from the hyperthermophile Thermus thermophilus, at 2.0 A resolution . The overall architecture is similar to that of isoleucyl-tRNA synthetase, except that the putative editing domain is inserted at a different position in the primary structure . This feature is unique to prokaryote-like leucyl-tRNA synthetases, as is the presence of a novel additional flexibly inserted domain . Comparison of native enzyme and complexes with leucine and a leucyl- adenylate analogue shows that binding of the adenosine moiety of leucyl-adenylate causes significant conformational changes in the active site required for amino acid activation and tight binding of the adenylate . These changes are propagated to more distant regions of the enzyme, leading to a significantly more ordered structure ready for the subsequent aminoacylation and/or editing steps.

Biochemistry (Mosc), 2000 Apr, 65(4), 436 - 41
Phenylalanyl-tRNA synthetase interacts with DNA: studies on activity using deoxyribooligonucleotides; Ivanov KA et al.; Phenylalanyl-tRNA synthetase from Thermus thermophilus complexes with short (up to 30 nucleotide length) single-stranded DNA fragments more efficiently than with double-stranded fragments . The complexing between DNA and the protein significantly increases with deoxyribooligonucleotide longer than 20 nucleotides . Using affinity labeling, the binding site of DNA was located near the interface of the alpha- and beta-subunits . The binding sites of DNA and tRNAPhe do not overlap . Phenylalanyl-tRNA synthetase from E . coli also binds DNA.

Protein Eng, 2000 Apr, 13(4), 253 - 8
Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships; Nurachman Z et al.; Thermal stability of the Thermus thermophilus isopropylmalate dehydrogenase enzyme was substantially lost upon the deletion of three residues from the C-terminus . However, the stability was partly recovered by the addition of two, four and seven amino acid residues (called HD177, HD708 and HD711, respectively) to the C-terminal region of the truncated enzyme . Three structures of these mutant enzymes were determined by an X-ray diffraction method . All protein crystals belong to space group P2(1) and their structures were solved by a standard molecular replacement method where the original dimer structure of the A172L mutant was used as a search model . Thermal stability of these mutant enzymes is discussed based on the 3D structure with special attention to the width of the active-site groove and the minor groove, distortion of beta-sheet pillar structure and size of cavity in the domain-domain interface around the C-terminus . Our previous studies revealed that the thermal stability of isopropylmalate dehydrogenase increases when the active-site cleft is closed (the closed form) . In the present study it is shown that the active-site cleft can be regulated by open-close movement of the minor groove located at the opposite side to the active-site groove on the same subunit, through a paperclip-like motion.

Mikrobiologiia, 2000 Jan-Feb, 69(1), 113 - 9
{Physiological and phylogenetic diversity of thermophilic spore-forming hydrocarbon-oxidizing bacteria from oil fields}; Nazina TN et al.; The distribution and population density of aerobic hydrocarbon-oxidizing bacteria in the high-temperature oil fields of Western Siberia, Kazakhstan, and China were studied . Seven strains of aerobic thermophilic spore-forming bacteria were isolated from the oil fields and studied by microbiological and molecular biological methods . Based on the 16S rRNA gene sequences, phenotypic characteristics, and the results of DNA-DNA hybridization, the taxonomic affiliation of the isolates was tentatively established . The strains were assigned to the first and fifth subgroups of the genus Bacillus on the phylogenetic branch of the gram-positive bacteria . Strains B and 421 were classified as B . licheniformis . Strains X and U, located between B . stearothermophilus and B . thermocatenulatus on the phylogenetic tree, and strains K, Sam, and 34, related but not identical to B . thermodenitrificans and B . thermoleovorans, undoubtedly represent two new species . Phylogenetically and metabolically related representatives of thermophilic bacilli were found to occur in geographically distant oil fields.

J Biol Chem, 2000 Sep 8, 275(36), 27940 - 6
Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization; Nield J et al.; Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus . The analyses have yielded three-dimensional structures at 30-A resolution . Biochemical analysis of the C . reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure . Not only was the C . reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins . The particle isolated from S . elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex . The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography . By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C . reinhardtii and S . elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins . It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein . However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface . Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C . reinhardtii and S . elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.

Mol Cell Biol, 2000 Jun, 20(11), 4128 - 34
A novel chromodomain protein, pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila; Nikiforov MA et al.; Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes . These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends . Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated . Both processes occur concomitantly in the developing macronucleus . Two stage-specific protein factors involved in germ line DNA elimination have been described previously . Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus . Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide . Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally . DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

Extremophiles, 2000 Apr, 4(2), 83 - 90
Toward a molecular understanding of cold activity of enzymes from psychrophiles; Russell NJ; Despite the fact that a much greater proportion of the earth environment is cold rather than hot, much less is known about psychrophilic, cold-adapted microorganisms compared with thermophiles living at high temperatures . In particular, investigation of the molecular basis of cold-active enzymes from psychrophiles has only recently received concerted research attention, in measure as a result of the EC-funded project COLDZYME . This research effort has been stimulated by the realization that such cold-active enzymes offer novel opportunities for biotechnological exploitation . Only very recently has the first cold-active enzyme, alpha-amylase, been crystallized, and this success was followed rapidly by others . This effort has facilitated a direct approach to solving the three-dimensional structure of cold-active enzymes to complement the gene homology modeling that had been performed previously . Recently studies have highlighted how different adaptations are used by different enzymes to achieve conformational flexibility at low temperatures, and how such adaptations are not necessarily the opposite of those that confer thermostability to proteins in thermophilic counterparts . This review also highlights initial successes in engineering genetically improved thermal stability in cold-active enzymes to give improved catalysts for low-temperature biotechnology.

Extremophiles, 2000 Apr, 4(2), 77 - 82
Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp . CH34; Nies DH; In contrast to thermophilic or psychrophilic organisms, heavy metal-resistant bacteria do not supply enzymes that are active under harsh conditions, but are themselves tools for the evaluation and remediation of heavy metal-contaminated environments . Ralstonia sp . CH34 is a gram-negative bacterium with a remarkable set of resistance determinants, allowing this bacterium to live in extreme environments that are heavily contaminated with toxic metal ions . These heavy metal ions are mostly detoxified by inducible ion efflux systems that reduce the intracellular concentration of a given ion by active export . Because all metal resistance determinants in this bacterium are inducible, their regulatory systems can be used to develop biosensors that measure the biologically important concentrations of heavy metals in an environment . Resistance based on metal ion efflux detoxifies only the cytoplasm of the respective cell . Therefore, this resistance mechanism cannot be used directly to develop biotechnological procedures; however, metal ion efflux can protect a cell in a metal-contaminated environment . Thus, the cell can be enabled to mediate biochemical reactions such as precipitation of heavy metals with the carbon dioxide produced during growth or degradation of xenobiotics.

Extremophiles, 2000 Apr, 4(2), 71 - 6
Acidophiles in bioreactor mineral processing; Norris PR et al.; Mineral processing in bioreactors has become established in several countries during the past decade with industrial application of iron- and sulfur-oxidizing bacteria to release occluded gold from mineral sulfides . Cobalt extraction in bioreactors has also been commercialized, and development of high-temperature biooxidation of copper sulfides has reached pilot-plant scale . A variety of potentially useful mineral sulfide-oxidizing thermophiles have been recognized, but the most active strains have not been fully characterized.

Biosci Biotechnol Biochem, 2000 Mar, 64(3), 492 - 502
Purification and characterization of membrane-bound hydrogenase from Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium; Ishii M et al.; A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium . Solubilization and purification were done aerobically in the presence of Triton X-100 . Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order . Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction . The purified hydrogenase was shown to be a tetramer with alpha2beta2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit . The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 microM and with a turnover number around 2900 (min(-1)) . Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the {NiFe}-hydrogenases . Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported . Finally, a model is presented for energy and central metabolism of H . thermophilus strain TK-6.

Appl Microbiol Biotechnol, 2000 Apr, 53(4), 461 - 8
Simultaneous production of high activities of thermostable endoglucanase and beta-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus; Gomes I et al.; The culture-medium composition was optimised, on a shake-flask scale, for simultaneous production of high activities of endoglucanase and beta-glucosidase by Thermoascus aurantiacus using statistical factorial designs . The optimised medium containing 40.2 g l(-1) Solka Floc as the carbon source and 9 g l(-1) soymeal as the organic nitrogen source yielded 1130 nkat ml(-1) endoglucanase and 116 nkat ml(-1) beta-glucosidase activities after 264 h as shake cultures . In addition, good levels of beta-xylanase (3479 nkat ml(-1)) and low levels of filter-paper cellulase, beta-xylosidase, alpha-L-arabinofuranosidase, beta-mannanase, beta-mannosidase, alpha-galactosidase and beta-galactosidase were detected . Batch fermentation in a 5-1 laboratory fermentor using the optimised medium allowed the production of 940 nkat ml(-1) endoglucanase and 102 nkat ml(-1) beta-glucosidase in 192 h . Endoglucanase and beta-glucosidase showed optimum activity at pH 4.5 and pH 5, respectively, and they displayed optimum activity at 75 degrees C . Endoglucanase and beta-glucosidase showed good stability at pH values 4-8 and 4-7, respectively, after a prolonged incubation (48 h at 50 degrees C) . Endoglucanase had half-lives of 98 h at 70 degrees C and 4.1 h at 75 degrees C, while beta-glucosidase had half-lives of 23.5 h at 70 degrees C and 1.7 h at 75 degrees C . Alkali-treated bagasse, steam-treated wheat straw, Solka floc and Sigmacell 50 were 66, 48.5, 33.5 and 14.4% hydrolysed by a crude enzyme complex of T . aurantiacus in 50 h.

Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 703 - 11
{Comparative studies of airborne, cultivatable microorganisms at selected sites of waste managements, domestic animal husbandry and in the surrounding residential area}; Wust G et al.; During the last years, sampling of airborne microorganisms (including mesophilic bacteria, moulds, thermophilic bacteria and A . fumigatus) in waste disposal, recycling industry and control sampling locations in the urban and rural districts of Graz was conducted using Andersen 6-stage viable cascade impactors . In the present study additional sampling in livestock buildings has been conducted . Except mesophilic bacteria the emission in the area of waste disposal and recycling industry was significantly higher than in livestock buildings . In the surroundings of the livestock buildings the number of microorganisms was not increased, while the normal background level in the surroundings of the waste proceeding plants was reached from a distance of 150 to 300 meters.

Schriftenr Ver Wasser Boden Lufthyg, 1999, 104, 655 - 64
{Measuring the spread of airborne microorganisms in the area of composting sites}; Neef A et al.; Emission concentrations of culturable microorganisms were determined in the vicinity of three open or partly encapsulated composting facilities . Sampling was performed during so-called worst case situations which should promote aerial transport of emissions . Suitability of thermophilic organisms to detect an emitting influence of the plant was confirmed . Generally, concentrations decreased significantly with increasing distances from the plant at all three locations . At one plant 10(6) CFU m-3 thermophilic actinomycetes were found in a distance of 200 m . Partly increased concentrations could be determined even in distances above 500 m . Concentrations could vary within one hour to more than ten times.

Nat Struct Biol, 2000 May, 7(5), 380 - 3
Two exposed amino acid residues confer thermostability on a cold shock protein; Perl D et al.; Thermophilic organisms produce proteins of exceptional stability . To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis . Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol-1 at 70 degrees C) . 11.5 kJ mol-1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol-1 from hydrophobic interactions of Leu 66 at the carboxy terminus . The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively . The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.

Nat Struct Biol, 2000 May, 7(5), 371 - 4
Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation; Ralston CY et al.; For proteins, understanding tertiary interactions involved in local versus global unfolding has become increasingly important for understanding the nature of the native state ensemble, the mechanisms of unfolding, and the stability of both the native and intermediate states in folding . In this work we have addressed related questions with respect to RNA structure by combining chemical denaturation and hydroxyl radical footprinting methods . We have determined unfolding isotherms for each of 26 discrete sites of protection located throughout the Tetrahymena thermophila group I ribozyme . The cooperativity of folding, m-value, and the free energy, DeltaG degrees N-U, associated with formation of each tertiary contact was determined by analysis of the isotherms . The DeltaG degrees N-U values measured in this study vary from 1.7 +/- 0.2 to 7 . 6 +/- 1.2 kcal mol-1 . Thus, the stability of these discrete tertiary contacts vary by almost 104 . In addition, an intradomain contact and three interdomain contacts show high cooperativity (m-values of 1.1 +/- 0.2 to 1.7 +/- 0.3 kcal mol-1 M-1) indicating that these contacts exhibit global cooperatively in their folding behavior . This new approach to examining RNA stability provides an exciting comparison to our understanding of protein structure and folding mechanisms.

FEMS Microbiol Lett, 2000 May 15, 186(2), 235 - 8
Cloning, sequencing and expression in Escherichia coli of a thermophilic lipase from Bacillus thermoleovorans ID-1; Cho AR et al.; A thermophilic lipase of Bacillus thermoleovorans ID-1 was cloned and sequenced . The lipase gene codes 416 amino acid residues and contains the conserved pentapeptide Ala-X-Ser-X-Gly as other Bacillus lipase genes . The optimum temperature of the lipase is 75 degrees C, which is higher than other known Bacillus lipases . For expression in Escherichia coli, the lipase gene was subcloned in pET-22b(+) vector with a strong T7 promoter . Lipase activity was approximately 1.4-fold greater than under the native promoter.

Structure Fold Des, 2000 May 15, 8(5), 515 - 25
The crystal structure of the conserved GTPase of SRP54 from the archaeon Acidianus ambivalens and its comparison with related structures suggests a model for the SRP-SRP receptor complex; Montoya G et al.; BACKGROUND: Protein targeting to the endoplasmic reticulum in eukaryotes and to the cell membrane in prokaryotes is mediated by the signal recognition particle (SRP) and its receptor (SR) . Both contain conserved GTPase domains in the signal-peptide-binding proteins (SRP54 and Ffh) and the SR proteins (SRalpha and FtsY) . These GTPases are involved in the regulation of protein targeting . Most studies so far have focussed on the SRP machinery of mammals and bacteria, leaving the SRP system of archaea less well understood . RESULTS: We report the crystal structure of the conserved GTPase (NG-Ffh) from the thermophilic archaeon Acidianus ambivalens at 2.0 A resolution and of the Thr112-->Ala mutant, which is inactive in GTP hydrolysis . This is the first structure of an SRP component from an archaeon and allows for a detailed comparison with related structures from Escherichia coli and thermophilic bacteria . In particular, differences in the conserved consensus regions for nucleotide binding and the subdomain interfaces are observed, which provide information about the regulation of the GTPase . These interactions allow us to propose a common signalling mechanism for the SRP-SR system . CONCLUSIONS: The overall structure of SRP-GTPases is well conserved between bacteria and archaea, which indicates strong similarities in the regulation of the SRP-targeting pathway . Surprisingly, structure comparisons identified a homodimeric ATP-binding protein as the closest relative . A heterodimer model for the SRP-SR interaction is presented.

Structure Fold Des, 2000 May 15, 8(5), 493 - 504
Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey; Szilagyi A et al.; BACKGROUND: Proteins from thermophilic organisms usually show high intrinsic thermal stability but have structures that are very similar to their mesophilic homologues . From prevous studies it is difficult to draw general conclusions about the structural features underlying the increased thermal stability of thermophilic proteins . RESULTS: In order to reveal the general evolutionary strategy for changing the heat stability of proteins, a non-redundant data set was compiled comprising all high-quality structures of thermophilic proteins and their mesophilic homologues from the Protein Data Bank . The selection (quality) criteria were met by 64 mesophilic and 29 thermophilic protein subunits, representing 25 protein families . From the atomic coordinates, 13 structural parameters were calculated, compared and evaluated using statistical methods . This study is distinguished from earlier ones by the strict quality control of the structures used and the size of the data set . CONCLUSIONS: Different protein families adapt to higher temperatures by different sets of structural devices . Regarding the structural parameters, the only generally observed rule is an increase in the number of ion pairs with increasing growth temperature . Other parameters show just a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature . Proteins from extreme thermophiles are stabilized in different ways to moderately thermophilic ones . The preferences of these two groups are different with regards to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.

Structure Fold Des, 2000 Apr 15, 8(4), 363 - 71
Archaeal ribosomal protein L1: the structure provides new insights into RNA binding of the L1 protein family; Nevskaya N et al.; BACKGROUND: L1 is an important primary rRNA-binding protein, as well as a translational repressor that binds mRNA . It was shown that L1 proteins from some bacteria and archaea are functionally interchangeable within the ribosome and in the repression of translation . The crystal structure of bacterial L1 from Thermus thermophilus (TthL1) has previously been determined . RESULTS: We report here the first structure of a ribosomal protein from archaea, L1 from Methanococcus jannaschii (MjaL1) . The overall shape of the two-domain molecule differs dramatically from that of its bacterial counterpart (TthL1) because of the different relative orientations of the domains . Two strictly conserved regions of the amino acid sequence, each belonging to one of the domains and positioned close to each other in the interdomain cavity of TthL1, are separated by about 25 A in MjaL1 owing to a significant opening of the structure . These regions are structurally highly conserved and are proposed to be the specific RNA-binding sites . CONCLUSIONS: The unusually high RNA-binding affinity of MjaL1 might be explained by the exposure of its highly conserved regions . The open conformation of MjaL1 is strongly stabilized by nonconserved interdomain interactions and suggests that the closed conformations of L1 (as in TthL1) open upon RNA binding . Comparison of the two L1 protein structures reveals a high conformational variability of this ribosomal protein . Determination of the MjaL1 structure offers an additional variant for fitting the L1 protein into electron-density maps of the 50S ribosomal subunit.

J Biol Chem, 2000 May 12, 275(19), 14260 - 3
The role of the DELSEED motif of the beta subunit in rotation of F1-ATPase; Hara KY et al.; F(1)-ATPase is a rotary motor protein, and ATP hydrolysis generates torque at the interface between the gamma subunit, a rotor shaft, and the alpha(3)beta(3) substructure, a stator ring . The region of conserved acidic "DELSEED" motif of the beta subunit has a contact with gamma subunit and has been assumed to be involved in torque generation . Using the thermophilic alpha(3)beta(3)gamma complex in which the corresponding sequence is DELSDED, we replaced each residue and all five acidic residues in this sequence with alanine . In addition, each of two conserved residues at the counterpart contact position of gamma subunit was also replaced . Surprisingly, all of these mutants rotated with as much torque as the wild-type . We conclude that side chains of the DELSEED motif of the beta subunit do not have a direct role in torque generation.

J Biol Chem, 2000 May 12, 275(19), 14112 - 23
The active site of the thermophilic CYP119 from Sulfolobus solfataricus; Koo LS et al.; CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C . UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures . Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site . CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry . This catalytic activity is stable to preincubation at 80 degrees C for 90 min . Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state . Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away . CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants . Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues . Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.

Biomol Eng, 1999 Dec 31, 16(1-4), 67 - 72
Extremely high thermal stability of streptavidin and avidin upon biotin binding; Gonzalez M et al.; The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry . Biotin binding increases the midpoint of temperature Tm of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83 degrees C to 112 and 117 degrees C at full biotin saturation, respectively . This thermostability is the highest reported for proteins coming from either mesophilic or thermophilic organisms . In both proteins, biotin also increases the calorimetric enthalpy and the cooperativity of the unfolding . Thermal stability of STV was also evaluated in the presence of high concentrations of urea or guanidinium hydrochloride (GuHCl) . In 6 M GuHCl, STV remains as a tetramer and the Tm of the STV-biotin complex is centered at 108 degrees C, a few degrees below the value obtained in phosphate buffer . On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms . The main endotherm at a lower temperature has been ascribed to the dimeric liganded state with a Tm of 87 degrees C, and the higher temperature endotherm to the tetrameric liganded form with a Tm of 106 degrees C . As the thermostability of unliganded protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in structural ordering and compactness with the preservation of the tetramer integrity.

Biophys Chem, 2000 Apr 14, 84(2), 167 - 76
Pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius as probe to monitor the sodium concentrations in the blood; D'Auria S et al.; We describe the isolation and characterization of a pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius . This protein appears to be a tetramer composed of four 55-kDa subunits . The intrinsic tryptophan fluorescence of this protein is quenched by approximately 20% upon binding sodium, which occurs with a dissociation constant near 15 mM . Importantly, the intrinsic fluorescence of this pyruvate kinase does not appear to be affected by potassium, magnesium, and calcium at the concentrations found in whole blood . It appears that this pyruvate kinase can provide the basis for a selective protein sensor for sodium with minimal interference from other cations.

Biophys Chem, 2000 Apr 14, 84(2), 105 - 36
Calculation of the standard molal thermodynamic properties of aqueous biomolecules at elevated temperatures and pressures II . Unfolded proteins; Amend JP et al.; Equations of state for completely unfolded proteins have been generated from group additivity algorithms and the revised Helgeson-Kirkham-Flowers (HKF) equations of state to compute the standard molal thermodynamic properties of these molecules at elevated temperatures and pressures . The requisite equations of state parameters were computed from those of groups retrieved by regression of experimental calorimetric and densimetric data reported in the literature . This approach permits calculation of the standard molal thermodynamic properties as a function of temperature and pressure for any completely unfolded protein for which the amino acid sequence is known . Calculations of this kind have been carried out for 11 thermophilic proteins . The thermodynamic properties reported below can be combined with those for protein unfolding to compute the corresponding properties of completely folded (i.e . native) proteins.

Carbohydr Res, 2000 Apr 20, 325(3), 202 - 10
Identification and synthesis of a trisaccharide produced from lactose by transgalactosylation; Perrin V et al.; Enzymatic transgalactosylation of lactose by means of Streptococcus thermophilus, subspecies DN-001065, led to a mixture of D-galactose (approximately 4%), D-glucose (approximately 15%), lactose (approximately 51%), minor disaccharides (6%), trisaccharides (approximately 20%) and tetrasaccharides (3%) . The major trisaccharide (approximately 16%) was identified by NMR spectroscopy and chemical synthesis as being the known beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->4)-D-glucos e (3'-beta-D-galactopyranosyl-lactose) . It was purified from a mixture of peracetylated oligosaccharides by column chromatography followed by deacetylation . For the first time, 3'-beta-D-galactopyranosyl-lactose has been obtained on the 1 g scale, by resorting to simple techniques and equipment . NMR spectra have been unambiguously assigned.

Arch Microbiol, 2000 Feb, 173(2), 103 - 9
Purification and properties of the first-identified, archaeal, ATP-dependent 6-phosphofructokinase, an extremely thermophilic non-allosteric enzyme, from the hyperthermophile Desulfurococcus amylolyticus; Hansen T et al.; The ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Desulfurococcus amylolyticus was purified 1,500-fold to homogeneity . The enzyme had an apparent molecular mass of 140 kDa and was composed of a single type of subunit of 33 kDa suggesting a homotetrameric (alpha4) structure . The N-terminal amino acid sequence did not show significant similarity to ATP-PFKs isolated from eubacteria and eukarya . Kinetic constants of the enzyme were determined for both reaction directions at pH 6 and at 85 degrees C . Rate dependence on all substrates followed Michaelis-Menten kinetics . The apparent K(m)s for ATP and fructose 6-phosphate (forward reaction) were 0.28 and 1.17 mM, respectively; the apparent V(max) was about 41 U/mg . ATP could not be replaced by pyrophosphate (PPi) or ADP as phosphoryl donor, thus defining the enzyme as an ATP-dependent PFK . In addition to ATP (100%), the enzyme accepted GTP (97%), ITP (130%), UTP (84%), CTP (55%) and, less effectively, acetyl phosphate (13%) as phosphoryl donors . Enzyme activity was not allosterically regulated by classical effectors of ATP-PFKs such as ADP, AMP, and phosphoenolpyruvate or citrate . The enzyme also catalysed in vitro the reverse reaction with an apparent K(m) for fructose-1,6-bisphosphate and ADP of 16.7 and 0.5 mM, respectively, and an apparent V(max) of about 4.5 U/mg . Divalent cations were required for maximal activity; Mg2+, which was most effective, could be replaced partially by Ni2+, Mn2+ or Co2+ . The enzyme had a temperature optimum of 90 degrees C and showed a significant thermostability up to 100 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions . This is the first description of an ATP-dependent PFK from the domain of archaea, characterized as an extremely thermophilic, non-allosteric enzyme.

Syst Appl Microbiol, 1999 Dec, 22(4), 559 - 64
Thermodesulfobacterium hveragerdense sp . nov., and Thermodesulfovibrio islandicus sp . nov., two thermophilic sulfate reducing bacteria isolated from a Icelandic hot spring; Sonne-Hansen J et al.; Two thermophilic non-sporeforming sulfate-reducing bacteria (SRB) were isolated from microbial mats collected from an Icelandic hot spring . Strain JSP was a gram negative rod, with an average cell size of 2.8 x 0.5 microm . No flagella were found . Growth occurred between 55 and 74 degrees C with an optimum between 70 and 74 degrees C at pH 7.0 . The G+C content was 40 mol% . Strain R1Ha3 was a gram negative vibrio-shaped rod with an average cell size of 1.7 x 0.4 microm . Motility was observed mediated by one polar flagellum . The growth optimum at pH 7.0 was 65 degrees C, and growth occurred between 45 and 70 degrees C . The G+C content was 38 mol% . In the presence of sulfate, both strains used lactate, pyruvate and H2 as electron donors . In addition, strain R1Ha3 used formate . Pyruvate was the only substrate supporting fermentative growth of both strains . Growth occurred with sulfate as well as thiosulfate as electron acceptors . Furthermore, strain R1Ha3 reduced nitrate and strain JSP reduced sulfite . Neither of the strains were able to oxidize lactate completely to CO2 and neither of the strains contained desulfoviridin . 16S rDNA sequencing placed strain JSP in the genus Thermodesulfobacterium and strain R1Ha3 in the genus Thermodesulfovibrio . Based on the DNA-DNA hybridization studies and differences in morphology and physiology to their closest relatives the two new isolates were considered as new species . Strain JSP is named Thermodesulfobacterium hveragerdense and strain R1Ha3 Thermodesulfovibrio islandicus.

Enzyme Microb Technol, 2000 May 1, 26(8), 568 - 573
Immobilization of functionally unstable catechol-2,3-dioxygenase greatly improves operational stability; Fernandez-Lafuente R et al.; Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearothermophilus has been immobilized on highly activated glyoxyl agarose beads . The enzyme could be fully immobilized at 4 degrees C and pH 10.05 with a high retention of activity (around 80%) . Enzyme immobilized under these conditions showed little increase in thermostability compared with the soluble enzyme, but further incubation of immobilized enzyme at 25 degrees C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibiting a 100-fold increase in stability (c.f . the free enzyme) . The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentially independent of protein concentration whereas free enzyme was rapidly inactivated at low protein concentrations . An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catechol 2,3-dioxygenases at protein concentrations of 10 microg/ml . Immobilization increased the 'optimum temperature' for activity by 20 degrees C, retained activity at substrate concentrations where the soluble enzyme was fully inactivated and enhanced the resistance to inactivation during catalysis . These results suggest that the immobilization of the enzyme under controlled conditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and increased the rigidity of the subunit structures.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5156 - 60
Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding; Tomlinson EJ et al.; Cytochrome c(552) from Hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of Escherichia coli . The b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 M lower than for the wild-type protein . The reduction potential is 75 mV lower than that of the recombinant wild-type protein . The heme can be removed from the b type variant, thus generating an apo protein that has, according to circular dichroism spectroscopy, an alpha-helical content different from that of the holo b type protein . The latter is readily reformed in vitro by addition of heme to the apo protein . This reforming suggests that previously observed assembly of cytochrome c(552), which has the typical class I cytochrome c fold, in the E . coli cytoplasm is a consequence of spontaneous thioether bond formation after binding of heme to a prefolded polypeptide . These observations have implications for the general problem of c type cytochrome biogenesis.

Genome, 2000 Apr, 43(2), 306 - 16
Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequence analysis and evolutionary implications; Pavesi A et al.; Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction) . The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB . Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only . Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH) . A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A . officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots . Codon usage analysis showed a bias towards A/T ending codons . This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons . Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated . The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi . Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.

J Dairy Sci, 2000 Apr, 83(4), 666 - 73
Probiotic culture survival and implications in fermented frozen yogurt characteristics; Davidson RH et al.; Low-fat ice cream mix was fermented with probiotic-supplemented and traditional starter culture systems and evaluated for culture survival, composition, and sensory characteristics of frozen product . Fermentations were stopped when the titratable acidity reached 0.15% greater than the initial titratable acidity (end point 1) or when the pH reached 5.6 (end point 2) . Mix was frozen and stored for 11 wk at -20 degrees C . The traditional yogurt culture system contained the strains Streptococcus salivarius ssp . thermophilus and Lactobacillus delbrueckii ssp . bulgaricus . The probiotic-supplemented system contained the traditional cultures as well as Bifidobacterium longum and Lactobacillus acidophilus . We compared recovery of Bifodobacterium by three methods, a repair-detection system with roll-tubes and plates on modified bifid glucose medium and plates with maltose + galactose reinforced clostridial medium . Culture bacteria in both systems did not decrease in the yogurt during frozen storage . The roll-tube method with modified bifid glucose agar and repair detection system provided at least one-half log10 cfu/ml higher recovery of B . longum compared with recoveries using modified bifid glucose agar or maltose + galactose reinforced clostridial agar on petri plates . No change in concentrations of lactose or protein for products fermented with either culture system occurred during storage . Acid flavor was more intense when product was fermented to pH 5.6, but yogurt flavor was not intensified . The presence of probiotic bacteria in the supplemented system seemed to cause no differences in protein and lactose concentration and sensory characteristics.

J Dairy Sci, 2000 Apr, 83(4), 659 - 65
Pyroglutamic acid in cheese: presence, origin, and correlation with ripening time of Grana Padano cheese; Mucchetti G et al.; Pyroglutamic acid is present in many cheese varieties and particularly in high amounts (0.5 g/100 g of cheese) in extensively ripened Italian cheeses (Grana Padano and Parmigiano Reggiano) that are produced with thermophilic lactic acid bacteria as starters . The mechanism of pyroglutamic acid formation in cheese seems to be mostly enzymatic, as demonstrated by the presence of only L-pyroglutamic acid enantiomer . Thermophilic lactobacilli are involved in pyroglutamic acid production, as suggested by the low pyroglutamic acid content found in Bagos, a ripened Italian mountain cheese produced without addition of starter . Because milk pasteurization did not influence the pyroglutamic acid content in the ripened Grana Padano cheese, the formation of pyroglutamic acid mainly depends on the whey starter microflora rather than that of raw milk . Pyroglutamic acid concentration is linearly correlated (R2 = 0.94) with the age of Grana Padano cheese.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 209 - 13
Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions; Husson-Kao C et al.; The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products . The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses . Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic . These autolytic strains are characterized by a typical bell-shaped growth curve . Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition . The lysogenic character of this strain was evidenced . Taken together, our results indicate that the autolytic phenotype in S . thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.

J Biochem (Tokyo), 2000 May, 127(5), 931 - 7
FtsH recognizes proteins with unfolded structure and hydrolyzes the carboxyl side of hydrophobic residues; Asahara Y et al.; FtsH of Escherichia coli is an essential membrane-integrated ATP-dependent protease . We cloned a gene for an FtsH homolog (T . FtsH) from Thermus thermophilus HB8, expressed it in E . coli, and purified the expressed protein . ATPase activity of T.FtsH was activated by proteins with unfolded structure ( alpha-casein and pepsin), and T.FtsH digested these proteins in an ATP-, Zn(2+)-dependent manner . alpha-Lactalbumin was digested by T.FtsH when it was largely unfolded, but not in its native form . Analysis of the proteolytic products revealed that, in most cases, T.FtsH cleaved the C-terminal side of hydrophobic residues and produced a characteristic set of small peptides (<30 kDa) without releasing a large intermediate . Thus, T.FtsH recognizes the unfolded structure of the proteins and progressively digests them at the expense of ATP . A soluble domain of T.FtsH, which lacked the N-terminal two transmembrane helices, was also prepared but was found to retain neither ATPase nor protease activities . Thus, the membrane segment appeared to be indispensable for these activities of T.FtsH.

J Biol Chem, 2000 May 5, 275(18), 13955 - 61
V-Type H+-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus . Subunit structure and operon; Yokoyama K et al.; V-type ATPase (V(o)V(1)) capable of ATP-driven H(+) pumping and of H(+) gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus thermophilus . When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V(1) . We also isolated the V(o)V(1) operon, containing nine genes in the order of atpG-I-L-E-X-F-A-B-D, which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively . The last four genes were identified as those for V(1) subunits; atpA, B, D, and F encoded the A, B, gamma, and delta subunits, respectively . The first five genes, atpG-atpX, were identified as genes for the V(o) subunits . The product of atpL, the proteolipid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membrane-spanning domains rather than four . The hydrophobic 43-kDa product of atpI is the smallest member so far found of the eukaryotic 100-kDa subunit family . Its electrophoretic band overlapped with the band of the A subunit . Therefore, all the gene products were found in our purified V(o)V(1) . We isolated the A(3)B(3) subcomplex reconstituted from the isolated subunits and the A(3)B(3)gamma subcomplex from subunit-expressing Escherichia coli . Electron microscopic observation of these subcomplexes revealed that the gamma subunit of V(1) filled the central cavity of A(3)B(3) and might be central subunit, similar to the gamma subunit of F(1)-ATPase.

J Biol Chem, 2000 May 5, 275(18), 13708 - 12
Directed evolution of beta -glucosidase A from Paenibacillus polymyxa to thermal resistance; Gonzalez-Blasco G et al.; The beta-glucosidase encoded by the bglA gene from Paenibacillus polymyxa has a half-life time of 15 min at 35 degrees C and no detectable activity at 55 degrees C . We have isolated random mutations that enhance the thermoresistance of the enzyme . Following a directed evolution strategy, we have combined some of the isolated mutations to obtain a beta-glucosidase with a half-life of 12 min at 65 degrees C, in the range of resistance of thermophilic enzymes . No significant alteration of the kinetic parameters of the enzyme was observed . One of the mutants isolated in the screening for thermoresistant beta-glucosidase had the same resistance to denaturation as the wild type . This mutation caused the accumulation of enzyme in E . coli, probably due to its lower turnover . The structural changes responsible for the properties of the mutant enzymes have been analyzed . The putative causes increasing thermoresistance are as follows: the formation of an extra salt bridge, the replacement of an Asn residue exposed to the solvent, stabilization of the hydrophobic core, and stabilization of the quaternary structure of the protein.

J Biol Chem, 2000 May 5, 275(18), 13235 - 42
Interaction of UvrA and UvrB proteins with a fluorescent single-stranded DNA . Implication for slow conformational change upon interaction of UvrB with DNA; Yamagata A et al.; UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair . However, the molecular mechanism of damage recognition by these proteins is still not well understood . In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins . TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA . The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP . Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP . The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP . This time-dependent change could be separated into two phases . The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis . Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase . These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly . We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.

Biochim Biophys Acta, 2000 Jan 31, 1490(1-2), 115 - 20
Identification of archaeal genes encoding a novel stationary phase-response protein; Dinger ME et al.; A novel stationary phase-response protein has been identified in the acid-soluble protein extract of the thermophilic archaeon, Thermococcus zilligii . N-Terminal sequencing data were used to identify likely genes for homologues of the protein in the complete genome sequences of various archaeal species . The corresponding genes were identified and analysed . The genes encode a protein ranging from 83 to 92 amino acids in length, with a calculated pI ranging from 4.6 to 9.7 . The amino acid sequences of the genes were highly conserved, even between members belonging to the different archaeal kingdoms . The computed secondary structure of the protein indicates it consists of four large helical regions separated by short coiled regions . We propose this protein as a candidate regulator of gene expression in stationary phase.

Proc Int Conf Intell Syst Mol Biol . 1999;:262-71.
An exact method for finding short motifs in sequences, with application to the ribosome binding site problem; Tompa M; This is an investigation of methods for finding short motifs that only occur in a fraction of the input sequences . Unlike local search techniques that may not reach a global optimum, the method proposed here is guaranteed to produce the motifs with greatest z-scores . This method is illustrated for the Ribosome Binding Site Problem, which is to identify the short mRNA 5' untranslated sequence that is recognized by the ribosome during initiation of protein synthesis . Experiments were performed to solve this problem for each of fourteen sequenced prokaryotes, by applying the method to the full complement of genes from each . One of the interesting results of this experimentation is evidence that the recognized sequence of the thermophilic archaea A . fulgidus, M . jannaschii, M . thermoautotrophicum, and P . horikoshii may be somewhat different than the well known Shine-Dalgarno sequence.

J Biotechnol, 2000 Jan 7, 76(1), 83 - 92
Anaerobic thermophilic fermentation for acetic acid production from milk permeate; Talabardon M et al.; Fermentation of milk permeate to produce acetic acid under anaerobic thermophilic conditions (approximately 60 degrees C) was studied . Although none of the known thermophilic acetogenic bacteria can ferment lactose, it has been found that one strain can use galactose and two strains can use lactate . Moorella thermoautotrophica DSM 7417 and M . thermoacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of approximately 0.93 g g(-1) . Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found precisely to produce large amounts of lactate and acetate . However, it also produced significant amounts of ethanol, CO2 and H2 . The lactate yield was affected by cell growth . During the exponential phase, acetate, ethanol, CO2 and H2 were the main products of fermentation with an equimolar acetate/ethanol ratio, whereas during the stationary phase, only lactic acid was produced with a yield of 4 mol per mol lactose, thus reaching the maximal theoretical value . When this bacterium was co-cultured with M . thermoautotrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid concentration and an overall yield of acetate around 80% . Under such conditions, only 13% of the fermented lactose was converted to ethanol by C . thermolacticum.

J Bacteriol, 2000 May, 182(10), 2985 - 8
Sulfolobicins, specific proteinaceous toxins produced by strains of the extremely thermophilic archaeal genus Sulfolobus; Prangishvili D et al.; Several novel strains of "Sulfolobus islandicus" produced proteinaceous toxins, termed sulfolobicins, which killed cells of other strains of the same species, as well as of Sulfolobus solfataricus P1 and Sulfolobus shibatae B12, but not of the producer strains and of Sulfolobus acidocaldarius DSM639 . The sulfolobicin purified from the strain HEN2/2 had a molecular mass of about 20 kDa . It was found to be associated with the producer cells as well as with cell-derived S-layer-coated spherical membrane vesicles 90 to 180 nm in diameter and was not released from the cells in soluble form.

Can J Microbiol, 2000 Apr, 46(4), 295 - 303
Characterization of a major envelope protein from the rumen anaerobe Selenomonas ruminantium OB268; Kalmokoff ML et al.; Cell envelopes from the Gram-negative staining but phylogenetically Gram-positive rumen anaerobe Selenomonas ruminantium OB268 contained a major 42 kDa heat modifiable protein . A similarly sized protein was present in the envelopes of Selenomonas ruminantium D1 and Selenomonas infelix . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Triton X-100 extracted cell envelopes from S . ruminantium OB268 showed that they consisted primarily of the 42 kDa protein . Polyclonal antisera produced against these envelopes cross-reacted only with the 42 kDa major envelope proteins in both S . ruminantium D1 and S . infelix, indicating a conservation of antigenic structure among each of the major envelope proteins . The N-terminus of the 42 kDa S . ruminantium OB268 envelope protein shared significant homology with the S-layer (surface) protein from Thermus thermophilus, as well as additional envelope proteins containing the cell surface binding region known as a surface layer-like homologous (SLH) domain . Thin section analysis of Triton X-100 extracted envelopes demonstrated the presence of an outer bilayer over-laying the cell wall, and a regularly ordered array was visible following freeze-fracture etching through this bilayer . These findings suggest that the regularly ordered array may be composed of the 42 kDa major envelope protein . The 42 kDa protein has similarities with regularly ordered outer membrane proteins (rOMP) reported in certain Gram-negative and ancient eubacteria.

FEMS Microbiol Lett, 2000 May 1, 186(1), 67 - 71
Cloning and expression of an alpha-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterisation of the recombinant enzyme; Leveque E et al.; An alpha-amylase encoding gene from the extremely thermophilic Archaea Thermococcus hydrothermalis was cloned and expressed in Escherichia coli . The encoded alpha-amylase possesses molecular characteristics specific to the Archaea, especially from Pyrococcus species, with biochemical characteristics of the alpha-amylases from Thermococcus . The gene is 1374 bp long and encodes a protein of 457 amino acids composed of a 22 amino acid putative signal peptide and a 435 amino acid mature protein (calculated molecular mass 49236 Da) . The T . hydrothermalis recombinant alpha-amylase is optimally active at 75-85 degrees C and at pH 5.0-5.5.

J Biol Chem, 2000 Apr 28, 275(17), 12757 - 62
ATPase activity of a highly stable alpha(3)beta(3)gamma subcomplex of thermophilic F(1) can be regulated by the introduced regulatory region of gamma subunit of chloroplast F(1); Bald D et al.; A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic) . By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex . The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol . No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C . alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.

J Biol Chem, 2000 Apr 28, 275(17), 12388 - 92
Heat-inactivated proteins managed by DnaKJ-GrpE-ClpB chaperones are released as a chaperonin-recognizable non-native form; Watanabe YH et al.; Chaperones of Thermus thermophilus cooperate in reactivation of heat-inactivated proteins . The protein, inactivated at a high temperature in a TDnaKJ-GrpE set, recovered its activity during subsequent incubation with TClpB at moderate temperature (Motohashi, K., Watanabe, Y., Yohda, M., and Yoshida, M . (1999) Proc . Natl . Acad . Sci . U . S . A . 96, 7184-7189) . Here, we report that the addition of chaperonin (Tcpn) at moderate temperature improves the yield of the TDnaKJ-GrpE-ClpB-dependent reactivation . The trap-Tcpn, which binds substrate protein but does not release it, inhibits reactivation severely . Maximum recovery is gained at sub-stoichiometric amounts of each component of TDnaKJ, TGrpE, and TClpB relative to the substrate monomer . These observations indicate that, driven by ATP hydrolysis, TDnaKJ-GrpE-ClpB chaperones catalytically cooperate and release heat-inactivated protein as a non-native, chaperonin-recognizable folding intermediate.

J Biol Chem, 2000 Jun 23, 275(25), 19146 - 9
Uracil-DNA glycosylase in the extreme thermophile Archaeoglobus fulgidus; Sandigursky M et al.; Uracil-DNA glycosylase (UDG) is an essential enzyme for maintaining genomic integrity . Here we describe a UDG from the extreme thermophile Archaeoglobus fulgidus . The enzyme is a member of a new class of enzymes found in prokaryotes that is distinct from the UDG enzyme found in Escherichia coli, eukaryotes, and DNA-containing viruses . The A . fulgidus UDG is extremely thermostable, maintaining full activity after heating for 1.5 h at 95 degrees C . The protein is capable of removing uracil from double-stranded DNA containing either a U/A or U/G base pair as well as from single-stranded DNA . This enzyme is product-inhibited by both uracil and apurinic/apyrimidinic sites . The A . fulgidus UDG has a high degree of similarity at the primary amino acid sequence level to the enzyme found in Thermotoga maritima, a thermophilic eubacteria, and suggests a conserved mechanism of UDG-initiated base excision repair in archaea and thermophilic eubacteria.

Protein Eng, 2000 Mar, 13(3), 179 - 91
Factors enhancing protein thermostability; Kumar S et al.; Several sequence and structural factors have been proposed to contribute toward greater stability of thermophilic proteins . Here we present a statistical examination of structural and sequence parameters in representatives of 18 non-redundant families of thermophilic and mesophilic proteins . Our aim was to look for systematic differences among thermophilic and mesophilic proteins across the families . We observe that both thermophilic and mesophilic proteins have similar hydrophobicities, compactness, oligomeric states, polar and non-polar contribution to surface areas, main-chain and side-chain hydrogen bonds . Insertions/deletions and proline substitutions do not show consistent trends between the thermophilic and mesophilic members of the families . On the other hand, salt bridges and side chain-side chain hydrogen bonds increase in the majority of the thermophilic proteins . Additionally, comparisons of the sequences of the thermophile-mesophile homologous protein pairs indicate that Arg and Tyr are significantly more frequent, while Cys and Ser are less frequent in thermophilic proteins . Thermophiles both have a larger fraction of their residues in the alpha-helical conformation, and they avoid Pro in their alpha-helices to a greater extent than the mesophiles . These results indicate that thermostable proteins adapt dual strategies to withstand high temperatures . Our intention has been to explore factors contributing to the stability of proteins from thermophiles with respect to the melting temperatures (T(m)), the best descriptor of thermal stability . Unfortunately, T(m) values are available only for a few proteins in our high resolution dataset . Currently, this limits our ability to examine correlations in a meaningful way.

EMBO J, 2000 Apr 17, 19(8), 1766 - 76
Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from thermus thermophilus; Soulimane T et al.; Cytochrome c oxidase is a respiratory enzyme catalysing the energy-conserving reduction of molecular oxygen to water . The crystal structure of the ba(3)-cytochrome c oxidase from Thermus thermophilus has been determined to 2.4 A resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa . A structure-based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g . a new electron transfer pathway leading directly from Cu(A) to Cu(B) . Specific features of the ba(3)-oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O(2-), OH(-) or H(2)O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c . New aspects of the proton pumping mechanism could be identified.

Rapid Commun Mass Spectrom, 2000, 14(7), 585 - 9
Analysis of intact tetraether lipids in archaeal cell material and sediments by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry; Hopmans EC et al.; A method combining normal phase high performance liquid chromatography (HPLC) with positive ion atmospheric pressure chemical ionization mass spectrometry (APCI-MS) was developed for the analysis of intact glycerol dialkyl glycerol tetraethers (GDGTs) in archaeal cell material and sediments . All GDGTs previously reported to occur in the thermophilic archaeon Sulfolobus solfataricus could be identified based on their mass spectra and retention time . Positive ion mass spectra consisted of abundant protonated molecules and fragment ions corresponding to loss of water and the glycerol moiety . In addition, two novel GDGTs representing alternative combinations of biphytanyl moieties were observed . Using this method, the tetraethers present in the thermophilic archaeon Metallosphaera sedula and two sediment samples were characterized . This rapid method will greatly contribute to the establishment of the sedimentary record of these compounds and increase our understanding of archaea and their occurrence in widely different environments.

Gene, 2000 Apr 18, 247(1-2), 137 - 43
Cloning, sequencing, and characterization of the bifunctional xylosidase-arabinosidase from the anaerobic thermophile thermoanaerobacter ethanolicus; Mai V et al.; The gene for the bifunctional xylosidase-arabinosidase (xarB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus JW200 was cloned, sequenced, and expressed in Escherichia coli (Genebank Accession No . AF135015) . Analysis of the recombinant enzyme revealed activity against multiple substrates with the highest affinity towards p-nitrophenyl beta-D-xylopyranoside (pNPX) and highest activity against p-nitrophenyl alpha-L-arabinopyranoside (pNPAP), respectively . Thus, we classify this enzyme as a bifunctional xylosidase-arabinosidase . Even though both sequences are 96% identical on the amino acid level, excluding the amino-terminal end, a frame-shift mutation in the 5' region of the gene in T . brockii ATCC 33075 and a deletion in a downstream open reading frame in T . ethanolicus seem to have occurred through evolutionary divergence of these two species . This represents an interesting phenomenon of molecular evolution of bacterial species, as PCR analysis of the region around the deletion indicates that the deletion is not present in T . brockii ssp . finnii and T . brockii ssp . brockii type strain HTD4.

Cell Biol Int, 2000, 24(2), 121 - 3
Are cells rescued from 'low density death' by co-operation between phospholipases C AND D?
Rasmussen M, Rasmussen L.
Cells of the ciliate Tetrahymena thermophila die when transferred at low density to a lipid-free nutritionally complete medium . This death is prevented and they will start to proliferate if protein kinase C is activated and this activation is sustained . We propose that this takes place in two stages . Firstly, the phospholipase C pathway beginning with and specific for phosphatidylinositol leads to the formation of diacylglycerol and inositol tris -phosphate . Diacylglycerol activates protein kinase C, and inositol tris -phosphate via Ca(2+)phospholipase D (PLD) . Secondly, the protein kinase C response can now be sustained by diacylglycerol produced by phospholipase D, using phosphatidylcholine and phosphatidylserine as substrates . Should this switching from PI-specific phospholipase C (PLC) to phospholipase D fail, then the cell will die in the course of milliseconds during the minutes following inoculation .

Cell Biol Int, 1999, 23(12), 841 - 8
Rotokinesis, a novel phenomenon of cell locomotion-assisted cytokinesis in the ciliate Tetrahymena thermophila; Brown JM et al.; The mechanism responsible for final cell separation at the end of cytokinesis is currently unknown . Knockout strains of the ciliate, Tetrahymena thermophila lacking the kinesin-II homologous molecular motors, Kin1p and Kin2p are paralyzed due to their complete loss of cilia and undergo frequent cytokinesis failures . Observations of live dividing cells revealed that cleavage furrow ingression is normal in kinesin-II double knockout cells until the final stage of cell separation (Brown et al., 1999) . During closer inspection of dividing cells using video differential interference contrast microscopy, we found that wild-type cells undergo an extremely complex motile behavior near the end of cytokinesis . This process, which we have named rotokinesis, appears to facilitate the physical separation of daughter cells . Here we present recent work on Tetrahymena rotokinesis, and review studies in other organisms which suggest that the use of cell locomotion in the completion of cytokinesis is a general phenomenon of motile cell types .

J Food Prot, 2000 Apr, 63(4), 509 - 15
Thermal and chemical inactivation of indigenous Streptococcus thermophilus bacteriophages isolated from Argentinian dairy plants; Binetti AG et al.; Thermal and chemical resistance of five autochthonal bacteriophages of Streptococcus thermophilus, isolated from Cuartirolo cheese wheys and yogurt, was investigated . Times to obtain 99% inactivation of phages (T99) at 63 degrees C and 72 degrees C in three suspension media (enriched tryptic soy broth, reconstituted commercial nonfat skim milk, and tris magnesium gelatin buffer) were determined . The thermal resistance was dependent on the phages studied but not detectable counts (<10 PFU/ml) were only achieved by heating at 90 degrees C during 5 min . The data obtained for the three assayed media did not permit verifying significant differences among them . Sodium hypochlorite (100 ppm) provided a fast inactivation of bacteriophage particles (<10 PFU/ml after 5 min) . Ethanol, at concentrations of 75% and 100%, was also effective for phage destruction . Isopropanol was slightly less effective than ethanol at the same concentrations . Peracetic acid (0.15%) was also a very effective agent for phage inactivation . The results showed that these autochthonal bacteriophages were not completely inactivated neither by normal pasteurization treatments nor by some biocides commonly used in disinfection, except sodium hypochlorite and peracetic acid . The practical implications of these findings have pointed out the necessity of recognizing the importance of establishing adequate conditions to assure effective thermal and chemical treatments in dairy plants and laboratory environments.

J Food Prot, 2000 Apr, 63(4), 489 - 94
Rapid and specific enzyme immunoassay on hydrophobic grid membrane filter for detection and enumeration of thermophilic Campylobacter spp . from milk and chicken rinses; Wang H et al.; Six commercially available anti-Campylobacter antibodies were examined for their applicability in an enzyme immunoassay on hydrophobic grid membrane filters, both for the detection and enumeration of Campylobacter spp . When a panel of nine Campylobacter (seven Campylobacter jejuni and two Campylobacter coli) and eight non-Campylobacter strains were used in a dot-blot format enzyme immunoassay to test the specificity of these antibodies, only one polyclonal antibody (Biodesign) detected all Campylobacter strains . Escherichia coli O157:H7 produced weak nonspecific signals due to endogenous peroxidase activity . The specificity of this Biodesign antibody was further tested against 30 more Campylobacter strains and more than 600 non-Campylobacter strains on hydrophobic grid membrane filters grown on modified Campylobacter agar with charcoal and deoxycholate, a Campylobacter selective medium . All the Campylobacter strains were detected, whereas only two (Acinetobacter calcoaceticus, Salmonella Minnesota) of the approximately 130 non-Campylobacter strains, which grew on modified Campylobacter agar with charcoal and deoxycholate, gave false-positive signals . This simple, rapid, and specific enzyme immunoassay also detected Campylobacter spp . from inoculated milk and chicken rinses and naturally contaminated chicken rinses.

Acta Crystallogr D Biol Crystallogr, 2000 May, 56 ( Pt 5), 667 - 9
Crystallization and preliminary crystallographic analysis of Thermus thermophilus leucyl-tRNA synthetase and its complexes with leucine and a non-hydrolysable leucyl-adenylate analogue; Yaremchuk A et al.; Leucyl-tRNA synthetase from Thermus thermophilus (LeuRSTT) is the first LeuRS to be crystallized . Two crystal forms of the native enzyme have been obtained using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant . Crystals of the first form belong to space group I422 and have unit-cell parameters a = b = 312.4, c = 100.4 A . They diffract anisotropically to 3.5 A resolution in the c-axis direction and to only 6 A resolution in the perpendicular direction . Crystals of the second form, which can be obtained native or with leucine or a leucyl-adenylate analogue bound, belong to space group C222(1) and have unit-cell parameters a = 102 . 4, b = 154.1, c = 174.3 A . They diffract to 1.9 A resolution and contain one monomer in the asymmetric unit . Selenomethionated LeuRSTT has been produced and crystals of the second form suitable for MAD analysis have been grown.

AIHAJ, 2000 Jan-Feb, 61(1), 56 - 63
Influence of building maintenance, environmental factors, and seasons on airborne contaminants of swine confinement buildings; Duchaine C et al.; Eight swine confinement buildings, selected to cover the widest possible range of cleanliness, were visited twice during winter and once during summer to verify the range, seasonal variations, and correlations between biological and chemical contaminants . Physical aspects were graded for dirtiness (1 = clean, 10 = dirty), ventilation, air temperature, number of animals, building, and room size . Air samples were taken to measure relative humidity, CO2, ammonia, total dust, and microbiological counts and/or identification (bacteria and molds); endotoxin levels also were measured . During winter, average measurements and ranges were: CO2 = 0.304% (0.254 to 0.349%); ammonia = 19.6 ppm (1.9 to 25.9 ppm); dust = 3.54 mg/m3 (2.15 to 5.60 mg/m3) . There were 883 cfu/m3 (547 to 2862 cfu/m3) of molds, 4.25 x 10(5) cfu/m3 (1.67 x 10(5) to 9.30 x 10(5) cfu/m3) of total bacteria, 29 cfu/m3 (3 to 94 cfu/m3) of thermophilic actinomycetes) . A significant decrease in bacterial levels (p = 0.04), dust (p = 0.0008), ammonia (p = 0.005), and CO2 (p < 0.0001) was observed during summer sampling when compared with winter levels . Mold counts were positively correlated (p = 0.03) with dirtiness scores, while bacterial counts were negatively correlated with this parameter (p < 0.002), whereas bacteria and endotoxins were correlated with the number of animals (p < 0.05) . Ambient gases (CO2 and ammonia) correlated with each other (p = 0.006) . Bacteria were the most important contaminant in swine confinement buildings, and endotoxin levels found were also very high (mean = 4.9 x 10(3) EU/m3) . We conclude that a wide range of air contamination exists in swine confinement buildings of different maintenance . There is a decrease in some of these contaminants during summer . Observed dirtiness of the swine confinement buildings has a poor predictive value concerning air quality.

Enzyme Microb Technol, 2000 Apr 1, 26(7), 502 - 508
Thermal stability of beta-xylanases produced by different Thermomyces lanuginosus strains; Singh S et al.; The thermostability of beta-xylanases produced by nine thermophilic Thermomyces lanuginosus strains in a coarse corn cob medium was assessed . The xylanase produced by T . lanuginosus strain SSBP retained 100% of its activity after 6 h at temperatures up to 65 degrees C . In comparison seven ATCC strains and the DSM 5826 strain of T . lanuginosus only retained 100% xylanase activity at temperatures up to 60 degrees C . Culture filtrates of T . lanuginosus strain SSBP grown on coarse corn cobs, oatspelts xylan, birchwood xylan, wheatbran, locust beangum, and sugar cane bagasse, retained 100% xylanase activity at temperatures up to 60 degrees C . The xylanase produced on corn cobs was the most thermostable and showed an increase of approximately 6% from 70 degrees C to 80 degrees C . The T(1/2) of all strains at 70 degrees C at pH 6.5 varied greatly from 63 min for strain ATCC 28083 to 340 min for strain SSBP . The xylanase of strain SSBP was much less thermostable at pH 5.0 and pH 12.0 with T(1/2) values of 11.5 min and 15 min, respectively at 70 degrees C . At 50 degrees C, the enzyme of T . lanuginosus strain SSBP produced on coarse corn cobs was stable within the pH range of 5.5-10.0 . Furthermore, the enzyme retained total activity at 60 degrees C for over 14 days and at 65 degrees C for over 48 h . The xylanase of T . lanuginosus strain SSBP possesses thermo- and pH stability properties that may be attractive to industrial application.

Electrophoresis, 2000 Mar, 21(5), 949 - 55
Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis; Perrin C et al.; Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products . The two-dimensional electrophoresis (2-DE) protein profile was obtained from three independent analyses of 2-DE gels of soluble proteins of the strain PB18 . About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively . Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots . Eleven were located in the PB18 2-DE profile after silver staining . These preliminary results contribute to the setting up of a two-dimensional image (or reference map) of the proteins from S . thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress . Bidimensional autoradiographs of two strains (PB18 and ST105) of S . thermophilus grown in exponential phase at 42 degrees C with {35S}methionine were compared with an image analysis system . Among the eleven located proteins in the 2-DE silver-stained profile, nine were found in PB18 and eight in ST105 autoradiographs . One protein was specific to PB18 . The eight proteins could play the role of internal 2-D PAGE markers of p/ and Mr for S . thermophilus.

Antonie Van Leeuwenhoek, 2000 Feb, 77(2), 117 - 33
Molecular ecology of hydrothermal vent microbial communities; Jeanthon C; The study of the structure and diversity of hydrothermal vent microbial communities has long been restricted to the morphological description of microorganisms and the use of enrichment culture-based techniques . Until recently the identification of the culturable fraction required the isolation of pure cultures followed by testing for multiple physiological and biochemical traits . However, peculiar inhabitants of the hydrothermal ecosystem such as the invertebrate endosymbionts and the dense microbial mat filaments have eluded laboratory cultivation . Substantial progress has been achieved in recent years in techniques for the identification of microorganisms in natural environments . Application of molecular approaches has revealed the existence of unique and previously unrecognized microorganisms . These have provided fresh insight into the ecology, diversity and evolution of mesophilic and thermophilic microbial communities from the deep-sea hydrothermal ecosystem . This review reports the main discoveries made through the introduction of these powerful techniques in the study of deep-sea hydrothermal vent microbiology.

Gene, 2000 Apr 4, 246(1-2), 295 - 301
Molecular cloning of profilin from Tetrahymena thermophila; Wilkes DE et al.; The actin-binding protein profilin was isolated from Tetrahymena thermophila by affinity chromatography, and the peptide sequence was determined for part of the protein . The cDNA sequence was obtained by using the peptide sequence, reverse transcription-PCR and 5' and 3' RACE . The cDNA coded for a profilin of 16680Da, which made it among the largest known profilins, and it had a predicted isoelectric point of 8.27 . The deduced amino acid sequence was divergent from other profilins, having more than 26% identity only with profilin from Tetrahymena pyriformis . The sequence contained insertions that are also present in profilins from Tetrahymena pyriformis and Trypanosoma brucei . There appeared to be only a single profilin gene and one transcript from this gene.

J Bacteriol, 2000 May, 182(9), 2520 - 9
The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons; Paul L et al.; Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM) . Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time . A single copy of mttB, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1 . However, two other nearly identical copies of mtbB1, designated mtbB2 and mtbB3, were also found in the genome . A 6.8-kb transcript was detected with probes to mttB and mtbB1, as well as to mtbC and mttC, encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to mttP, encoding a putative membrane protein which might function as a methylamine permease . These results indicate that these genes, found on the chromosome in the order mtbC, mttB, mttC, mttP, and mtbB1, form a single transcriptional unit . A transcriptional start site was detected 303 or 304 bp upstream of the translational start of mtbC . The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon . Each of the three DMA methyltransferase gene copies from Methanosarcina barkeri contained an amber codon at the same position, followed by a downstream UAA or UGA codon . The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of mtbB1, mtbB2, or mtbB3 if termination occurred at the UAA or UGA codon rather than the in-frame amber codon . The mttB gene from Methanosarcina thermophila contained a UAG codon at the same position as the M . barkeri mttB gene . The UAG codon is also present in mttB transcripts . Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis from methylamine contain in-frame amber codons that are suppressed during expression of the characterized methyltransferases.

Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 364 - 8
Sequence of the two operons encoding the four core subunits of the cytochrome b(6)f complex from the thermophilic Cyanobacterium synechococcus elongatus; Schneider D et al.; The genes encoding cytochrome f (petA), cytochrome b(6) (petB), the Rieske FeS-protein (petC), and subunit IV (petD) of the cytochrome b(6)f complex from the thermophilic cyanobacterium Synechococcus elongatus were cloned and sequenced . Similar to other cyanobacteria, the structural genes are arranged in two short, single-copy operons, petC/petA and petB/petD, respectively . In addition, five open reading frames with homology to known orfs from the cyanobacterium Synechocystis PCC 6803 were identified in the immediate vicinity of these two operons.

Mol Microbiol, 2000 Apr, 36(1), 114 - 22
Sequence and expression of a halobacterial beta-galactosidase gene; Holmes ML et al.; Studies of gene expression in haloarchaea have been greatly hindered by the lack of a convenient reporter gene . In a previous study, a beta-galactosidase from Haloferax alicantei was purified and several peptide sequences determined . The peptide sequences have now been used to clone the entire beta-galactosidase gene (designated bgaH) along with some flanking chromosomal DNA . The deduced amino acid sequence of BgaH was 665 amino acids (74 kDa) and showed greatest amino acid similarity to members of glycosyl hydrolase family 42 {classification of Henrissat, B., and Bairoch, A . (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities . Biochem J 293: 781-788} . Within this family, BgaH was most similar (42-43% aa identity) to enzymes from extremely thermophilic bacteria such as Thermotoga and Thermus . Family 42 enzymes are only distantly related to the Sulfolobus LacS and Escherichia coli LacZ enzymes (families one and two respectively) . Three open reading frames (ORFs) upstream of bgaH were readily identified by database searches as glucose-fructose oxidoreductase, 2-dehydro-3-deoxyphosphogluconate aldolase and 2-keto-3-deoxygluconate kinase, enzymes that are also involved in carbohydrate metabolism . Downstream of bgaH there was an ORF which contained a putative fibronectin III motif . The bgaH gene was engineered into a halobacterial plasmid vector and introduced into Haloferax volcanii, a widely used strain that lacks detectable beta-galactosidase activity . Transformants were shown to express the enzyme; colonies turned blue when sprayed with Xgal and enzyme activity could be easily quantitated using a standard ONPG assay . In an accompanying publication, Patenge et al . (2000) have demonstrated the utility of bgaH as a promoter reporter in Halobacterium salinarum.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 771 - 9
Syntrophothermus lipocalidus gen . nov., sp . nov., a novel thermophilic, syntrophic, fatty-acid-oxidizing anaerobe which utilizes isobutyrate; Sekiguchi Y et al.; A new anaerobic, thermophilic, syntrophic, fatty-acid-oxidizing bacterium designated strain TGB-C1T was isolated from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor . The cells were slightly curved rods and were weakly motile . Spore formation was not observed . The optimal temperature for growth was around 55 degrees C and growth occurred in the range 45 to 60 degrees C . The pH range for growth was 5.8-7.5, and the optimum pH was 6.5-7.0 . Crotonate was the only substrate that allowed the strain to grow in pure culture . However, in co-culture with the thermophilic, hydrogenotrophic Methanobacterium thermoautotrophicum strain delta H, the isolate could syntrophically oxidize saturated fatty acids with 4-10 carbon atoms, including isobutyrate . During the degradation of isobutyrate by the co-culture, isobutyrate was isomerized to butyrate, which was then oxidized . The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate or Fe(III) as electron acceptor . The DNA base composition was 51.0 mol% . 16S rDNA sequence analysis revealed that the strain belongs to the family Syntrophomonadaceae, but it was only distantly related to other known species of beta-oxidizing syntrophs . Hence, the name Syntrophothermus lipocalidus is proposed for TGB-C1T as a new species of a new genus.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 735 - 42
Tepidimonas ignava gen . nov., sp . nov., a new chemolithoheterotrophic and slightly thermophilic member of the beta-Proteobacteria; Moreira C et al.; A bacterial isolate with an optimum growth temperature of about 55 degrees C was recovered on a medium composed of one part Kligler's iron agar and four parts of Thermus Agar from the host spring at Sao Pedro do Sul in central Portugal . Phylogenetic analyses using the 16S rRNA gene sequence of strain SPS-1037T indicated that the new organism represented a new genus and species of beta-Proteobacteria . The major fatty acids of strain SPS-1037T are C16:0 and C17:0 . Ubiquinone 8 is the major respiratory quinone, and the major polar lipids are phosphatidylethanolamine and phosphatidylglycerol . The new isolate is aerobic and chemolithoheterotrophic . Thiosulfate and tetrathionate were oxidized to sulfate . The growth yield of the organism was improved by the addition of thiosulfate to media containing organic carbon sources, but the organism did not grow autotrophically under the conditions examined . Heterotrophic growth of strain SPS-1037T occurs on amino acids and organic acids, but this organism does not assimilate carbohydrates . On the basis of the phylogenetic analyses, and physiological and biochemical characteristics, it is proposed that strain SPS-1037T represents a new genus and a new species for which the name Tepidimonas ignava is proposed.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 517 - 23
Saccharococcus caldoxylosilyticus sp . nov., an obligately thermophilic, xylose-utilizing, endospore-forming bacterium; Ahmad S et al.; Several closely related, xylanolytic, thermophilic bacilli were isolated from local soils on xylose-containing minimal medium . On the basis of morphology and biochemical characteristics, one of the isolates, designated strain S1812T (T = type strain), was studied further . Strain S1812T was a xylanolytic, sporulating, Gram-positive, rod-shaped bacterium . Its Gram-positive nature was confirmed by electron microscopic examination of thin sections of the cells . The isolate was a thermophilic (optimum temperature for growth, 65 degrees C), facultative anaerobe that grew on a wide range of carbon sources including glucose, lactose, starch and xylose . It expressed high levels of both xylose isomerase and xylulokinase on xylose and also on glucose . The DNA G + C content was 44 mol% . rRNA gene sequence analysis placed strain S1812T in Bacillus cluster 5; it was more closely related to Saccharococcus thermophilus than to thermophilic Bacillus species . DNA-DNA hybridization also indicated its close relationship to S . thermophilus . Based on the evidence presented, it is proposed that strain S1812T be designated Saccharococcus caldoxylosilyticus sp . nov . Strain S1812T is the type strain (= ATCC 700356T = DSM 97-987T).

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 505 - 9
Streptomyces thermocoprophilus sp . nov., a cellulase-free endo-xylanase-producing streptomycete; Kim B et al.; The taxonomic position of a thermophilic actinomycete strain isolated from poultry faeces was examined using a polyphasic approach . The isolate, designated B19T, was assigned to the genus Streptomyces on the basis of chemotaxonomic and morphological criteria . An almost complete 16S rRNA gene (rDNA) sequence obtained for the test strain was compared with those of representative streptomycetes, notably thermophilic streptomycetes . 16S rDNA sequence data not only supported the assignment of the strain to the genus Streptomyces but also showed that the isolate formed a distinct phyletic line within the evolutionary branch composed of Streptomyces thermodiastaticus and related species . The strain was distinguished from related validly described Streptomyces species by a number of phenotypic properties . It is, therefore, proposed that strain B19T be classified in the genus Streptomyces as Streptomyces thermocoprophilus sp . nov.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 451 - 7
Alicyclobacillus hesperidum sp . nov . and a related genomic species from solfataric soils of São Miguel in the Azores; Albuquerque L et al.; Several acidophilic, slightly thermophilic or thermophilic Gram-positive isolates were recovered from solfataric soil at Furnas on the Island of Sao Miguel in the Azores . Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms represented two novel species of the genus Alicyclobacillus . Strains FR-11T and FR-1b had an optimum growth temperature of about 50 degrees C, whereas strains FR-3 and FR-6T had an optimum growth temperature of about 60 degrees C . Biochemical, physiological and chemotaxonomic characteristics did not distinguish isolates FR-3 and FR-6T from the type strain of Alicyclobacillus acidocaldarius; however, strains FR-11T and FR-1b could be easily distinguished from the type strain of Alicyclobacillus acidoterrestris by the carbon source assimilation pattern and the fatty acid composition . On the basis of the phylogenetic analysis, physiological and biochemical characteristics, and fatty acid composition the name Alicyclobacillus hesperidum is proposed for the species represented by strains FR-11T and FR-1b; a formal name for the new genomic species represented by strains FR-3 and FR-6T is not proposed at this time.

Genetics, 2000 Mar, 154(3), 1155 - 67
Tetrahymena macronuclear genome mapping: colinearity Of macronuclear coassortment groups and the micronuclear map on chromosome 1l; Wickert S et al.; The genetics of the ciliate Tetrahymena thermophila are richer than for most other eukaryotic cells, because Tetrahymena possesses two genomes: a germline (micronuclear) genome that follows a Mendelian model of genetic transmission and a somatic (macronuclear) genome, derived from the micronuclear genome by fragmentation, which follows a different genetic transmission model called phenotypic assortment . While genetic markers in the micronucleus fall into classical linkage groups under meiotic recombination and segregation, the same markers in the macronucleus fall into coassortment groups (CAGs) under phenotypic assortment by the random distribution of MAC chromosome pieces . We set out to determine whether genomic mapping in the macronucleus by genetic means is feasible . To investigate the relationship between the micronuclear map and coassortment groups, we systematically placed into CAGs all of the markers lying on chromosome 1L that are also found in the macronucleus . Sixteen CAGs were identified, 7 of which contain at least two loci . We have concluded that CAGs represent a fundamental genetic feature of the MAC . The MIC and MAC maps on 1L are colinear; that is, CAGs consist exclusively of markers that map to a continuous segment in a given region of the micronuclear map, with no intervening markers from other CAGs . These findings provide a solid foundation for exploiting the MAC chromosome pieces to build a physical map of the Tetrahymena genome.

Genetics, 2000 Mar, 154(3), 1141 - 53
Tetrahymena micronuclear genome mapping . a high-resolution meiotic map of chromosome 1l; Wickert S et al.; The ciliate Tetrahymena thermophila is a useful model organism that combines diverse experimental advantages with powerful capabilities for genetic manipulation . The genetics of Tetrahymena are especially rich among eukaryotic cells, because it possesses two distinct but related nuclear genomes within one cytoplasm, contained separately in the micronucleus (MIC) and the macronucleus (MAC) . In an effort to advance fulfillment of Tetrahymena's potential as a genetic system, we are mapping both genomes and investigating the correspondence between them . With the latter goal especially in mind, we report here a high-resolution meiotic linkage map of the left arm of chromosome 1, one of Tetrahymena's five chromosomes . The map consists of 40 markers, with an average spacing of 2.3 cM in the Haldane function and a total length of 88.6 cM . This study represents the first mapping of any large region of the Tetrahymena genome that has been done at this level of detail . Results of a parallel mapping effort in the macronucleus, and the correspondence between the two genomes, can be found in this issue as a companion to this article.

Eur J Med Res . 2000 Mar 27;5(3):127.
Histologically proven extrinsic allergic alveolitis with severe obstructive pulmonary emphysema
Bickhardt J, Kunze P, Rolle A, Matthiessen A.
Anamnesis: 61-year old man with progressive shortness of breath on exercise . Cough and expectoration during the last 6 years . - Exposure: Driver of cereals, massive exposure to mouldy and pest contaminated grains . Gave up his profession in 1979 due to dyspnea with short (2-3 h) latency after exposure . Since 1980 intermittent exposure during occasional jobs; renewed symptomatology . Aspergillus fumigatus detected on agar plates inoculated with material from wet areas in bathroom and kitchen . - Clinical symptoms: Barely audible vesicular breathing, barrel-shaped thorax, inspiratory-intercostal retraction . - Bodyplethysmography: Obstructive pulmonary emphysema with FEV1 0.8 l, TLC 7.8 l, RV/TLC relation 67% . - Precipitin-detection: Significantly increased IgG against Fusarium . Other moulds including Aspergillus: negative; thermophilic actinomycetes: negative; pigeon and chicken: negative; Ouchterlony with native material from patients flat: negative . - CT including HR-CT: Bilateral-substantial emphysema, no bullae, no ground glass-opacity, no signs for interstitial lung diseases, no mediastinal enlargement of lymph nodes . - Alpha-1-Antitrypsin: 1.67 to 2.3 g/l (normal range), phenotype M1 . - Histology: In resected material from right-side lung-volume-resection detection of pulmonary emphysema as well as lymphocyte infiltration and numerous epitheloid cell granulomas with Langhans'giant cells without caseation assessed as residues of an exogenous allergic alveolitis . - Conclusion: In a patient with lung volume reduction surgery due to severe emphysema histologically a persistent exogenous allergic alveolitis was detected, which might have caused the emphysema.

Biotechnol Prog, 2000 Mar-Apr, 16(2), 296 - 8
Protein purification by ultrafiltration using a beta-galactosidase fusion tag; Sakhamuru K et al.; The use of beta-galactosidase (465 kDa) as a fusion tag for ultrafiltration-based protein purification has been investigated . The target protein studied was thermophilic glucose dehydrogenase (157 kDa, GDH) from Thermoplasma acidophilum . An expression vector was constructed comprising the lacZ gene fused to a factor Xa cleavage sequence that was attached to the 5' end of the GDH gene . This gene fusion was expressed in Escherichia coli JM109 to yield a soluble protein that exhibited activities for both enzymes . Cleavage of this fusion protein (622 kDa) by factor Xa gave two smaller proteins that showed individual beta-galactosidase and GDH activity . A two-stage diafiltration process for protein purification was used in an ultrafiltration stirred cell . In the first stage, a 500 kDa membrane was used to retain the fusion protein and transmit smaller E . coli host proteins . Approximately 80% of the GDH activity was retained in this step . Following cleavage, the second stage utilized a 300 kDa membrane to fractionate the beta-galactosidase and GDH . No beta-galactosidase was detected in the permeate solutions, and 97% of the GDH activity was recovered in the permeate.

Protein Sci, 2000 Mar, 9(3), 466 - 75
An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study; Georis J et al.; In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp . S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes . Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity . A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability . Indeed, the optimum activity temperature (70 vs . 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C . The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity . Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1 . Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively . This interaction should increase the stability of the N-terminal part of Xyl1 . Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2) . Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.

J Eukaryot Microbiol, 2000 Mar-Apr, 47(2), 139 - 47
Macronuclear development in conjugants of Tetrahymena thermophila, which were artificially separated at meiotic prophase; Kiersnowska M et al.; Conjugant pairs of Tetrahymena thermophila were mechanically separated by vigorous pipetting at the early stages of meiotic prophase . The complete sequence of conjugational nuclear events including the appearance of pronuclei, development of the new macronuclei (postzygotic development), and resorption of the old macronuclei was observed in the separated cells, without pronuclear exchange . The pronuclei in the separated cells were recognised by the presence of components of the extranuclear cytoskeleton, which were labelled with anti-tubulin and anti-fenestrin antibodies in the same way as in undisturbed conjugants . The apical region of the separated conjugants (the post-junction area), corresponding to the junction area of conjugants was labelled with anti-fenestrin antibody and maintained the properties required for the nuclear development . The results of the genetic study were consistent with a hypothesis that cytogamy (pronuclear fusion) was induced in the separated conjugants . Therefore, the lasting cell contact is not necessary for the successful completion of conjugational nuclear events.

J Dairy Sci, 2000 Mar, 83(3), 395 - 400
Technological properties of milks fermented with thermophilic lactic acid bacteria at suboptimal temperature; Moreira M et al.; In the present work the synergistic relationship between different strains of Lactobacillus delbrueckii subsp . bulgaricus and Streptococcus thermophilus was studied at optimal (44 degrees C) and suboptimal temperatures (30 degrees C) . Acidification, viscosity, whey syneresis, and bacterial concentration of the final product were evaluated on single-strain and mixed cultures after 24 h at 30 degrees C and 6 h at 44 degrees C . Three pairs of strains (LBB + CP2, LBP + CP2, and LBR + CP2) showed synergistic effect, which was reflected by the viscosity and syneresis of the coagulum . These results were more significant when cultures were incubated at 30 degrees C, reaching apparent viscosity values of 19 to 28 mPa x s . On the other hand, lactobacilli cultures enhanced the growth of two streptococci strains (CP2 and CP4) . These results were confirmed by cultures of streptococci supplemented with supernatants of culture of lactobacilli . Those supernatants stimulate the viscosity produced by CP2 and CP4 strains and reduce the syneresis of all cultures of streptococci . Neither the increase of viscosity nor reduction of syneresis could be attributed to a decrease of pH.

J Biol Chem, 2000 Jun 30, 275(26), 19498 - 504
Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling; Declais AC et al.; Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA . Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear . Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide . Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli . We show the following . (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase . (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low . (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase . (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C . The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase . (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide . However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA . These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.

J Biol Chem, 2000 May 26, 275(21), 15820 - 7
Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii; Hack ES et al.; The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli . Only the M . jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity . A purification protocol was devised for the Mj-PyrB and M . jannaschii PyrI (Mj-PyrI) gene products . Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer . Preliminary characterization of the aspartate transcarbamoylase from M . jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E . coli holoenzyme . Kinetic analysis of the M . jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties . The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E . coli catalytic trimer . Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E . coli proteins . The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E . coli aspartate transcarbamoylase were compared.

J Biol Chem, 2000 Jul 7, 275(27), 20652 - 9
Towards structural determination of the water-splitting enzyme . Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium; Kuhl H et al.; A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed . The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days . Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q(B) binding site . The oxygen-evolving activity, 5000-6000 micromol of O(2)/(h.mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 degrees C . The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 A . The space group was determined to be P2(1)2(1)2(1) with four photosystem II dimers per unit cell . Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.

J Appl Microbiol, 2000 Mar, 88(3), 458 - 66
Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium; Donaghy JA et al.; Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses . Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized . This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan . The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction . The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters . The purified enzyme also released ferulic acid from a de-starched wheat bran preparation . At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1 . The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C . At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.

Genetics, 2000 Apr, 154(4), 1587 - 96
RAD51 is required for propagation of the germinal nucleus in Tetrahymena thermophila; Marsh TC et al.; RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks . The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species . This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation . Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation . Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation . rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes . Conjugation of two rad51 nulls leads to an arrest early during prezygotic development (meiosis I) . We discuss the potential usefulness of the ciliates' characteristic nuclear duality for further analyses of the potentially unique roles of Tetrahymena RAD51.

Microbiology, 2000 Mar, 146 ( Pt 3), 749 - 57
Altered patterns of cellular growth, morphology, replication and division in conditional-lethal mutants of the thermophilic archaeon Sulfolobus acidocaldarius; Bernander R et al.; As a basis for studing the essential cellular processes of hyperthermophilic archaea, thermosensitive mutants of Sulfolobus acidocaldarius were isolated and characterized . Exponential-phase liquid cultures were shifted to the nonpermissive temperature and growth, viability, and distributions of cell mass and DNA content were measured as a function of time after the shift . The observed phenotypes demonstrate that chromosome replication, nucleoid organization, nucleoid partition and cell division, which normally are tightly co-ordinated during cellular growth, can be inhibited or uncoupled by mutation in this hyperthermophilic archaeon.

Eur J Clin Nutr, 2000 Apr, 54(4), 288 - 97
Effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases; Agerholm-Larsen L et al.; OBJECTIVE: To investigate the effect of a probiotic milk product containing the culture CAUSIDO(R) and of two alternative products on risk factors for cardiovascular disease in overweight and obese subjects . DESIGN: An 8 week randomized, double-blind, placebo- and compliance-controlled, parallel study . SUBJECTS: Seventy healthy, weight-stable, overweight and obese (25.0<BMI<37.5 kg/m2) males (n=20) and females (n=50), 18-55 y old, were randomly assigned into five groups . INTERVENTION: Four groups consumed 450 ml fermented milk products (yoghurt) daily . Group 1: a yoghurt fermented with two strains of Streptococcus thermophilus and two strains of Lactobacillus acidophilus (StLa) . Group 2: a placebo yoghurt fermented with delta-acid-lactone (PY) . Group 3: a yoghurt fermented with two strains of Streptococcus thermophilus and one strain of Lactobacillus rhamnosus (StLr) . Group 4: a yoghurt fermented with one strain of Enterococcus faecium and two strains of Streptococcus thermophilus (CAUSIDO(R) culture), GAIO(R) (G) . The dietary composition of the yoghurt was otherwise similar . The fifth group was given two placebo pills (PP) daily . RESULTS: When comparing all five treatment groups, unadjusted for changes in body weight, no statistical effects were observed in week 8 in the G-group on low density lipoproteins (LDL)-cholesterol (P=0.29) . After adjustment for small changes in body weight, LDL-cholesterol decreased by 8.4% (0.26+/-0.10 mmol/l; P<0.05) and fibrinogen increased (0.74+/-0.32 mmol/l; P<0.05) after 8 weeks in the G-group . This was significantly different from the group consuming chemically fermented yoghurt and the group consuming placebo pills (P<0.05) . After 8 weeks, systolic blood pressure was significantly more reduced in the StLa and G-group compared to StLr . No other differences were found . CONCLUSION: The CAUSIDO(R) culture reduced LDL-cholesterol and increased fibrinogen in the overweight subjects at a 450 ml consumption daily for 8 weeks . The effect on LDL-cholesterol confirms previous studies . An immunostimulation by one of the strains in the product might explain the effect on fibrinogen in the G-group . SPONSORSHIP: MD Foods A/S, Denmark.

Biotechnol Appl Biochem, 2000 Apr, 31 ( Pt 2), 113 - 8
Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum; Kong KH et al.; Tyrosinase, with an isoelectric point at pH 4.9, was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum . Gel filtration, N-terminal amino acid sequencing and SDS/PAGE analysis indicate that T . roseum tyrosinase is composed of two identical subunits, each with a molecular mass of 43000 Da . The enzyme exhibited high substrate specificity towards catechol, chlorogenic acid, L-3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) and pyrogallol . The K(m) value of the enzyme for L-DOPA was 0.18 mM . beta-Mercaptoethanol and sodium diethyldithiocarbamate notably inhibited the enzymic activity . The activity of the enzyme was optimal at pH 9.5 and 70 degrees C, and was increased by addition of 1 mM Mg(2+), K(+) or Cu(2+) . The enzyme was highly stable against high temperature and guanidine hydrochloride . The N-terminal amino acid sequence of the enzyme was determined to be Asp-Ile-Asn-Gly-Gly-Gly-Ala-Thr-Leu-Pro-Gln-Lys-Leu-Tyr . These facts indicate that T . roseum tyrosinase appears to be distinct from the tyrosinases so far purified from other sources.

J Biol Chem, 2000 Apr 7, 275(14), 10057 - 63
Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides; Ren H et al.; In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J . P., Leslie, A . G . W., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations . In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen-bonded to the carboxylate of betaGlu(199) . Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP . These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively . Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant . These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis.

RNA, 2000 Mar, 6(3), 402 - 8
A hierarchy of RNA subdomains in assembly of the central domain of the 30 S ribosomal subunit; Agalarov SC et al.; Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus . The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way junctions at both ends composed of portions of helices 20, 21, and 23 . Addition of the remaining portion of helix 23 to this construct results in the minimal site for S11 . Surprisingly, almost half of the central domain (helices 24, 25, and 26) is dispensable for binding the central domain proteins . Thus, at least two classes of RNA elements can be identified in ribosomal RNA . A protein-binding core element (such as helices 20, 21, 22, and 23) is required for the association of ribosomal proteins, whereas secondary binding elements (such as helices 24, 25, and 26) associate only with the preformed core RNP complex . Apparently, there may be a hierarchy of ribosomal RNA elements similar to the hierarchy of primary, secondary, and tertiary binding ribosomal proteins.

Appl Environ Microbiol, 2000 Apr, 66(4), 1749 - 53
Characterization of a novel integrative element, ICESt1, in the lactic acid bacterium Streptococcus thermophilus; Burrus V et al.; The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3' end of a gene encoding a putative fructose-1,6-biphosphate aldolase . This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252 . The integrase was found to be involved in a site-specific excision of a circular form . ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916 . Therefore, ICESt1 could be or could be derived from an integrative conjugative element.

Appl Environ Microbiol, 2000 Apr, 66(4), 1259 - 65
Molecular diversity within Lactobacillus helveticus as revealed by genotypic characterization; Giraffa G et al.; Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium that is used in the manufacture of Swiss type and long-ripened Italian cheeses, such as Emmental, Grana, and Provolone cheeses . Substantial differences in several technologically important characteristics are found among L . helveticus strains isolated from natural dairy starter cultures . In the present study we investigated the genotypic diversity of 74 strains isolated from different dairy cultures used for manufacturing Grana and Provolone cheeses and six collection strains . A restriction fragment length polymorphism analysis of both total genomic DNA and the 16S rRNA gene (ribotyping) was used as genotypic fingerprinting . A multivariate statistical analysis of the data enabled us to identify significant genotypic heterogeneity in L . helveticus . We found that genotypic fingerprinting could be used to distinguish strains; in particular, it was possible to associate the presence of specific strain genotypes with dairy ecosystem sources (e.g., Grana or Provolone cheese) . Our data contribute to the description of microbial heterogeneity in L . helveticus and provide a more solid basis for understanding the functional and ecological significance of the presence of different L . helveticus biotypes in natural dairy starter cultures.

Extremophiles, 2000 Feb, 4(1), 23 - 33
The structure of the alpha-galactosidase gene loci in Thermus brockianus ITI360 and Thermus thermophilus TH125; Fridjonsson O et al.; The Thermus thermophilus TH125 alpha-galactosidase gene, agaT, and flanking sequences were cloned in Escherichia coli and sequenced as well as flanking sequences of the previously cloned agaT from Thermus brockianus ITI360 . Different structures of putative alpha-galactosidase operons in the two Thermus strains were revealed . Downstream of and overlapping with the alpha-galactosidase genes of both strains, a gene was identified that is similar to the galactose-1-phosphate uridylyltransferase gene (galT) of E . coli and Streptomyces lividans . Upstream of the agaT of T . brockianus ITI360, four open reading frames were observed . The deduced translation products displayed similarity to components of bacterial binding protein-dependent transport systems and a beta-galactosidase . No galactoside utilization genes were identified upstream of agaT in T . thermophilus TH125 . The inactivation of the alpha-galactosidase genes of both strains by insertional mutagenesis led to an inability to use melibiose or galactose as a single carbohydrate source . An attempt was made to isolate a gene encoding the enzyme responsible for para-nitrophenyl-(pNP-) beta-galactoside hydrolyzing activity in T . thermophilus TH125 . A gene designated bglT was cloned and expressed in E . coli . The inactivation of the bglT gene led to 55% reduction of the pNP-beta-galactoside hydrolyzing activity in the mutant strain in comparison to the wild type.

Extremophiles, 2000 Feb, 4(1), 9 - 17
Thermosipho japonicus sp . nov., an extremely thermophilic bacterium isolated from a deep-sea hydrothermal vent in Japan; Takai K et al.; A novel barophilic, extremely thermophilic bacterium was isolated from a deep-sea hydrothermal vent chimney at the Iheya Basin, in the Okinawa area, Japan . The cells were found to be rod shaped and surrounded by a sheath-like outer structure; the organism did not possess flagella and was not motile . Growth was observed between 45 degrees and 80 degrees C (optimum, 72 degrees C, 45 min doubling time), pH 5.3 and 9.3 (optimum, pH 7.2-7.6), 6.6 and 79g/l sea salts (optimum, 40g/l), and 0.1 and 60MPa (optimum, 20MPa) . Strain IHB1 was found to be a strictly anaerobic chemoorganotroph capable of utilizing yeast extract and proteinaceous substrates such as peptone and tryptone . Elemental sulfur or thiosulfate acted as electron acceptors improving growth . The isolate was able to utilize casein as a sole carbon and energy source in the presence of thiosulfate . The G + C content of the genomic DNA was 31.4 mol% . Phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization analysis indicated that the isolate is closely related to Thermosipho africanus; however, it represents a species distinct from the previously described members of the genus Thermosipho . On the basis of the physiological and molecular properties, we propose that the new isolate represents a new species, which we name Thermosipho japonicus sp . nov . (type strain: IHB1; JCM10495).

J Biochem (Tokyo), 2000 Apr, 127(4), 617 - 25
Engineering subtilisin E for enhanced stability and activity in polar organic solvents; Takagi H et al.; We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I . Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis . The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B . subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants . However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form . These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E . Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195) . Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity . A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).

J Biochem (Tokyo), 2000 Apr, 127(4), 551 - 7
Energy-yielding properties of SoxB-type cytochrome bo(3) terminal oxidase: analyses involving Bacillus stearothermophilus K1041 and its mutant strains; Sone N et al.; We isolated a K17q8 mutant from K17 mutant cells of Bacillus stearothermophilus which contain SoxB-type cytochrome bo(3) as well as cytochrome bd but not SoxM-type cytochrome caa(3), which is the main terminal oxidase in B . stearothermophilus K1041 . The respiration of K17q8 was highly sensitive to as little as 10 microM cyanide, indicating that the main terminal oxidase is cytochrome bo(3) . The aerobic growth yield of K17q8 was lower than that of wild-type K1041, but higher than that of parental K17 . The H(+)/O ratio of K17q8 was about 5, i.e . a little lower than the 6.1-6.5 of K1041, but higher than the 2.9-3.1 of K17 {Sone et al . (1999) J . Biosci . Bioeng . 87, 495-499} . Analyses of membrane fragments indicated that K17q8 contains about 0.2 nmol cytochrome bo(3) per mg membrane protein, and scarcely any subunits of cytochromes caa(3) and bd . From the membrane fraction of K17q8, cytochrome bo(3) was purified and shown to be composed of two subunits with apparent molecular masses of 56 and 19 kDa . The enzyme contained protoheme IX and heme O, as the main low-spin heme and high-spin heme . Analysis of the substrate specificity indicated that the high-affinity site is very specific to cytochrome c-551, a cytochrome c which is a membrane-bound lipoprotein of thermophilic Bacillus . The I(50) of purified cytochrome bo(3) was determined to be 4 microM, indicating that cytochrome bo(3) among the three terminal oxidases in B . stearothermophilus was most susceptible to cyanide . The respiration of K17q8 was mostly inhibited by the addition of cyanide at this concentration.

Z Naturforsch {C}, 2000 Jan-Feb, 55(1-2), 66 - 9
Extracellular xylanase production by two thermophilic alkali-tolerant Bacillus strains in batch and continuous cultures; Emanuilova EI et al.; Xylanase production of newly isolated thermophilic alkali-tolerant Bacillus sp . strain SP and strain BC was investigated in batch and continuous cultures . Enzyme synthesis was inducible with both strains and was observed only in xylan-containing media . Xylan from oat spelt is a better inducer than xylan from birch for strain Bacillus sp . BC while such difference was not observed for strain SP . Compared with batch cultures xylanase production of both strains increased about two times and its rate became more than four times faster in continuous cultures at a dilution rate of 0.2 h(-1).

Proc R Soc Lond B Biol Sci, 2000 Mar 7, 267(1442), 485 - 9
Three energy variables predict ant abundance at a geographical scale; Kaspari M et al.; Energy theory posits three processes that link local abundance of ectotherms to geographical gradients in temperature . A survey of 49 New World habitats found a two order of magnitude span in the abundance (nests m(-2)) of ground nesting ants (Formicidae) . Abundance increased with net primary productivity (r2=0.55), a measure of the baseline supply of harvestable energy . Abundance further increased with mean temperature (r2=0.056), a constraint on foraging activity for this thermophilic taxon . Finally for a given mean temperature, ants were more abundant in seasonal sites with longer, colder winters (r2 = 0.082) that help ectotherm taxa sequester harvested energy in non-productive months . All three variables are currently changing on a global scale . All should be useful in predicting biotic responses to climate change.

J Mol Biol, 2000 Apr 7, 297(4), 1015 - 26
Directed evolution study of temperature adaptation in a psychrophilic enzyme; Miyazaki K et al.; We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures . A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity . The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins . The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability . The temperature optimum of the activity of 3-2G7 is shifted upward by approximately 10 degrees C . Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised . The catalytic efficiency, k(cat)/K(M), was enhanced approximately threefold over a wide temperature range (10 to 60 degrees C) . The activation energy for catalysis, determined by the temperature dependence of k(cat)/K(M) in the range 15 to 35 degrees C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability . These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously . The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins .

J Mol Biol, 2000 Apr 7, 297(4), 975 - 88
Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein; Mueller U et al.; The bacterial cold shock proteins are small compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors . Bc-Csp, the cold shock protein from the thermophile Bacillus caldolyticus shows a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB from the mesophile Bacillus subtilis, although the two proteins differ by only 12 out of 67 amino acid residues . This pair of cold shock proteins thus represents a good system to study the atomic determinants of protein thermostability . Bs-CspB and Bc-Csp both unfold reversibly in cooperative transitions with T(M) values of 49.0 degrees C and 77.3 degrees C, respectively, at pH 7.0 . Addition of 0.5 M salt stabilizes Bs-CspB but destabilizes Bc-Csp . To understand these differences at the structural level, the crystal structure of Bc-Csp was determined at 1.17 A resolution and refined to R=12.5% (R(free)=17.9%) . The molecular structures of Bc-Csp and Bs-CspB are virtually identical in the central beta-sheet and in the binding region for nucleic acids . Significant differences are found in the distribution of surface charges including a sodium ion binding site present in Bc-Csp, which was not observed in the crystal structure of the Bs-CspB . Electrostatic interactions are overall favorable for Bc-Csp, but unfavorable for Bs-CspB . They provide the major source for the increased thermostability of Bc-Csp . This can be explained based on the atomic-resolution crystal structure of Bc-Csp . It identifies a number of potentially stabilizing ionic interactions including a cation-binding site and reveals significant changes in the electrostatic surface potential .

Cell Biol Int, 2000, 23(11), 729 - 38
Ribosome synthesis in Tetrahymena: a quantitative analysis; Larsen LK et al.; We have performed a detailed quantitative analysis of the transcription and accumulation of ribosomal RNA and ribosomal protein mRNA in the ciliated protozoan Tetrahymena thermophila during changes in growth conditions, and found that: (1) nutritional downshifts lead to a rapid decrease in transcriptional activity whereas nutritional upshifts lead to rapid restoration of transcriptional activity, (2) starvation leads to decreased translation of ribosomal protein mRNA and (3) the rate of ribosomal protein mRNA degradation decreases after a nutritional upshift . We present evidence that the proximal promoters of two ribosomal protein genes and the ribosomal RNA gene compete for binding of nuclear factor(s) in vitro, suggesting that the coordinated regulation of these genes may involve a common set of transcriptional regulators .

Cell Biol Int, 2000, 23(11), 719 - 28
The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum; Neve S et al.; The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation . Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum . In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains . In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles . Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention .

Biochemistry, 2000 Apr 4, 39(13), 3671 - 7
Identification of essential arginines in the acetate kinase from Methanosarcina thermophila; Singh-Wissmann K et al.; Site-directed mutagenesis is a powerful tool for identifying active-site residues essential for catalysis; however, this approach has only recently become available for acetate kinase . The enzyme from Methanosarcina thermophila has been cloned and hyper-produced in a highly active form in Escherichia coli (recombinant wild-type) . The role of arginines in this acetate kinase was investigated . Five arginines (R91, R175, R241, R285, and R340) in the M . thermophila enzyme were selected for individual replacement based on their high conservation among sequences of acetate kinase homologues . Replacement of R91 or R241 with alanine or leucine produced variants with specific activities less than 0.1% of the recombinant wild-type enzyme . The circular dichroism spectra and other properties of these variants were comparable to those of recombinant wild-type, indicating no global conformational changes . These results indicate that R91 and R241 are essential for activity, consistent with roles in catalysis . The variant produced by conservative replacement of R91 with lysine had approximately 2% of recombinant wild-type activity, suggesting a positive charge is important in this position . The K(m) value for acetate of the R91K variant increased greater than 10-fold relative to recombinant wild-type, suggesting an additional role for R91 in binding this substrate . Activities of both the R91A and R241A variants were rescued 20-fold when guanidine or derivatives were added to the reaction mixture . The K(m) values for ATP of the rescued variants were similar to those of recombinant wild-type, suggesting that the rescued activities are the consequence of replacement of important functional groups and not changes in the catalytic mechanism . These results further support roles for R91 and R241 in catalysis . Replacement of R285 with alanine, leucine, or lysine had no significant effect on activity; however, the K(m) values for acetate increased 6-10-fold, suggesting R285 influences the binding of this substrate . Phenylglyoxal inhibition and substrate protection experiments with the recombinant wild-type enzyme and variants were consistent with the presence of one or more essential arginine residues in the active site as well as with roles for R91 and R241 in catalysis . It is proposed that R91 and R241 function to stabilize the previously proposed pentacoordinate transition state during direct in-line transfer of the gamma-phosphate of ATP to acetate . The kinetic characterization of variants produced by replacement of R175 and R340 with alanine, leucine, or lysine indicated that these residues are not involved in catalysis but fulfill important structural roles.

J Appl Microbiol, 2000 Jan, 88(1), 124 - 31
The production of freeze-dried immobilized cultures of Streptococcus thermophilus and their acidification properties in milk; Champagne CP et al.; The aim of this study was to prepare alginate-immobilized freeze-dried cultures of Streptococcus thermophilus and to compare the acidifying activities of these rehydrated cultures with classical free cell liquid inoculants . Streptococcus thermophilus BT1 grew in alginate beads and the population reached 10(10) cfu g(-1) after 6 h incubation . Re-inoculation of the beads in fresh medium with a further 6 h incubation did not improve the biomass level, but extending the incubation at 42 degrees C to 24 h caused significant death . The rehydrated immobilized cell technology (ICT) starter contained 13% free cells . In acidifying activity tests, the ICT culture had a similar acidification curve to that of a classical milk-grown free cell culture, except that it reached lower final pH values . Although the differences between the ICT and liquid cultures were not important, there were significant effects of inoculation level on lag time, maximum acidification rate and on the pH and time at which the acidification rate was at its highest.

Acta Biol Hung, 1999, 50(4), 413 - 24
Stress-responsive gene expression in Tetrahymena; Nakashima S et al.; Cells properly respond to extracellular stimuli and circumstantial environment . The unicellular eukaryotic protozoan Tetrahymena is a potentially useful animal cell model system for studying the molecular mechanism of adaptation to environment . Tetrahymena is exposed to fluctuations in temperature, pH, amounts of nutrients and concentration of dissolved gases in natural habitat . For example, the cells adapt to cold environment by increase in unsaturated fatty acids in membrane phospholipids to maintain proper membrane fluidity . To accomplish this modification, the activity of fatty acid desaturase is increased upon a down-shift in temperature . We have cloned delta9 fatty acid desaturase which is involved in this process and shown evidence that its mRNA level increased in response to cold environment . Moreover, in order to examine other genes responsive to clod stress, we have adopted mRNA differential display technique to temperature shift-down of T . thermophila . We have cloned two kinase genes, NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk) and MAP kinase-related kinase (MRK) . Interestingly, these genes were also shown to be expressed by the osmotic stress.

Biochem Biophys Res Commun, 2000 Apr 2, 270(1), 81 - 8
Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp . A11-2; Ishii Y et al.; Paenibacillus A11-2 can efficiently cleave two carbon&bond;sulfur bonds in dibenzothiophene (DBT) and alkyl DBTs, which are refractory by conventional petroleum hydrodesulfurization, to remove sulfur atom at high temperatures . An 8.7-kb DNA fragment containing the genes for the DBT desulfurizing enzymes of A11-2 was cloned in Escherichia coli and characterized . Heterologous expression analysis of the deletion mutants identified three open reading frames that were required for the desulfurization of DBT to 2-hydroxybiphenyl (2-HBP) . The three genes were designated tdsA, tdsB, and tdsC (for thermophilic desulfurization) . Both the nucleotide sequences and the deduced amino acid sequences show significant homology to dszABC genes of Rhodococcus sp . IGTS8, but there are several local differences between them . Subclone analysis revealed that the product of tdsC oxidizes DBT to DBT-5,5'-dioxide via DBT-5-oxide, the product of tdsA converts DBT-5,5'-dioxide to 2-(2-hydroxyphenyl) benzene sulfinate, and the product of tdsB converts 2-(2-hydroxyphenyl)benzene sulfinate to 2-HBP . Cell-free extracts of a recombinant E . coli harboring all the three desulfurization genes converted DBT to 2-HBP at both 37 and 50 degrees C . In vivo and in vitro exhibition of desulfurization activity of the recombinant genes derived from a Paenibacillus indicates that an E . coli oxidoreductase can be functionally coupled with the monooxygenases of a gram-positive thermophile .

Nucleic Acids Res . 2000 Apr 15;28(8):E36.
One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA; Sachadyn P et al.; A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed . The examined and reference DNA fragments are PCR amplified . The PCR products are purified, mixed, heated and cooled to form heteroduplexes . In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded . The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR-Gold) selectively binding DNA . The Thermus thermophilus recombined His-tagged MutS and 3'-5' exonuclease activity of T4 DNA polymerase were used in the assay.

J Biochem (Tokyo), 2000 Mar, 127(3), 419 - 25
Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp . H-257; Imamura S et al.; A thermostable monoacylglycerol lipase {MGLP, EC 3.1.1.23} was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp . H-257 . The enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-Sepharose CL-4B, Q-Sepharose FF, and Superose 12 columns . The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS-PAGE, suggesting a monomeric protein . The isoelectric point was determined to be 4.66 by isoelectric focusing . The MGLP retained its full activity upon incubation at 60 degrees C for 10 min (pH 7 . 3), and was stable at pH 7-10 . The optimal temperature for activity at pH 7.5 was 75 degrees C, and the maximum activity was observed from pH 6-8 . This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol . Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme . The K(m) values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 mM, respectively . The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate . The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined.

J Biochem (Tokyo), 2000 Jan, 127(1), 9 - 11
Crystallization and preliminary X-ray crystallographic studies of Thermus thermophilus HB8 MutM protein involved in repairs of oxidative DNA damage; Sugahara M et al.; MutM protein, which removes the oxidatively damaged DNA base product, 8-oxoguanine (GO), has been crystallized by means of a hanging-drop vapor-diffusion procedure using polyethyleneglycol monomethylether 2000 as a precipitant in 2-(cyclohexylamino) ethanesulfonic acid (CHES) buffer, pH 9.8 . The diffraction data derived from oscillation photographs indicate that the crystals belong to the monoclinic system and space group P2(1) . The crystals have unit-cell dimensions of a = 45.4 A, b = 62.0 A, c = 99.7 A, and beta = 90.8 degrees . Assuming that the asymmetric unit contains two molecules, the Vm value was calculated to be 2.35 A(3).Da(-1) . The crystals diffracted X-rays to at least 2.1 A resolution and were suitable for high-resolution X-ray crystal structure determination.

Mikrobiol Z, 1999 Nov-Dec, 61(6), 22 - 8
{The role of the carbohydrate composition of the glycocalyx in some species of lactobacilli in the manifestation of their adhesive properties}; Onyshchenko AM et al.; Availability of certain monosaccharides in the composition of glycocalyx of lactic acid bacteria (Lactobacillus plantarum--strains 337D and 11/16; Streptococcus thermophilus--strains S1 (nonmucous race) and S5 (mucous race), Enterococcus faecium (K-50) has been investigated with the help of plant lectins with certain carbohydrate specificity labelled by colloid gold . All the microorganisms under investigation were characterized by the presence of N-acetyl-D-galactosamine and N-acetyl-D-glucosamine in rather insignificant amounts . Glycocalyx of lactic acid bacteria was also characterized by availability of essential amount of L-fructose and low amount of sialic acid (except for S . thermophilus S5 (mucous race) . Presence of alpha-N-acetyl-D-galactosamine, alpha-D, beta-D-galactose, alpha-D-glucose, alpha-D-mannose in the composition of the lactic acid bacteria glycocalyx composition evidences for the additional role of these monosaccharides in the process of the microorganism adhesion on the human and animal intestine mucosa . It has been confirmed that availability of certain monosaccharides in the composition of surface glycopolymers of lactic acid bacteria was connected with adhesive properties of cells and their existence conditions.

Mol Gen Genet, 2000 Feb, 263(1), 119 - 30
Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana; Dimova D et al.; The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria . We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga . The two-domain ArgR proteins encoded by T . neapolitana and T . maritima share a low degree of sequence similarity with other bacterial arginine repressors . The ArgR protein from T . neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated . The protein has extremely high intrinsic thermostability and tolerance to urea . Moreover, its binding to target DNA increases the melting temperature by approximately 15 degrees C . The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations . In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine . However, the affinity of T . neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers . Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host.

J Med Assoc Thai, 1999 Nov, 82 Suppl 1, S43 - 8
Reduction of rotavirus infection in children receiving bifidobacteria-supplemented formula; Phuapradit P et al.; This study was conducted at Pakkred Babies Home, Bangkok, Thailand; with the hypothesis that children receiving probiotic-supplemented milk-based formula may be protected from developing diarrheal diseases . Salivary rotavirus-specific IgA antibody was used as an indicator of rotavirus infection . One hundred and seventy-five children, aged 6-36 months, were enrolled in the study . They were divided into 3 groups according to the type of formula given . There were 81 episodes of diarrhea during an 8-month study period, most of which were caused by bacterial enteropathogens . Ninety-seven pairs of salivary samples were adequate for the analysis of rotavirus antibody . Among 23 children receiving milk-based follow-up formula and serving as control group, 30.4 per cent of them had > or = 4-fold increase in the antibody titre, indicating subclinical rotavirus infection . The majority of children in the other 2 study groups, receiving the same formula supplemented with either Bifidobacterium Bb12 alone or together with Streptococcus thermophilus, had no significant change in the antibody titres between the two time points . The results of this study support our hypothesis that children receiving bifidobacteria-supplemented milk-based formula may be protected against symptomatic rotavirus infection.

Lett Appl Microbiol, 2000 Jan, 30(1), 85 - 9
Quantification of micro-organisms in binary mixed populations by Fourier transform infrared (FT-IR) spectroscopy; Oberreuter H et al.; Fourier Transform Infrared (FT-IR) spectroscopy was used for the first time to determine the ratios of different microorganisms in mixtures . Exemplarily, systems composed of two food-associated yeast species (Saccharomyces cerevisiae/Hanseniaspora uvarum) and two yoghurt lactic acid bacteria (Lactobacillus acidophilus/Streptococcus salivarius ssp . thermophilus) were investigated . Determination of the cell number ratio in the lactic acid bacteria system was possible with a minimal prediction accuracy of +/- 16 ratio percentage points while the minimum accuracy of prediction in the yeast two-component system was +/- 4% (both at a 95% confidence level) . These results show that FT-IR spectroscopy is potentially a rapid method for the quantification of cell ratios in mixtures of two different microorganisms, provided that the cell ratio does not drop below a certain, system-specific threshold.

Cell Biol Int, 1999, 23(9), 619 - 28
Cell pairing and methylation in Tetrahymena thermophila are altered by exogenous homocysteine; Wolfe J et al.; Homocysteine is causally associated with birth defects such as spina bifida, and with premature vascular disease . We have investigated the effects of homocysteine on a cell-cell interaction in a fundamental eukaryotic system, the free-living ciliate Tetrahymena . Exogenously added homocysteine inhibits cell pairing in a dose-dependent manner . These effects are exacerbated by adenosine, which by itself has little demonstrable influence on pairing . S-adenosylhomocysteine (SAH) is a product of the reaction between adenosine and homocysteine, and is an inhibitor of methyl transferases . We therefore predicted that protein methylation would be significantly inhibited by homocysteine . A direct test of that hypothesis involved a demonstration that incorporation of an isotopically labeled methyl group from methionine into proteins was significantly reduced by homocysteine . The undermethylated proteins are of low molecular weight, and might correspond to known methylatable signaling proteins . We show that vanadate, an inhibitor of protein phosphatase, also inhibits cell pairing, and that the effects of vanadate and homocysteine are additive . This is the first demonstration that methylation and possibly phosphorylation play a regulatory role in cell-cell interactions in ciliates.

Biochemistry, 2000 Mar 28, 39(12), 3216 - 30
Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase; Becker HD et al.; Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes . Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs . The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences . AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies . Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation . These properties relate functionally AspRS2 to archaebacterial AspRSs . The structural basis of the dual specificity of T . thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity . It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing . The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed . This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs . The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T . thermophilus to eubacteria and archaebacteria.

J Biotechnol, 2000 Mar 10, 78(2), 93 - 113
A critical review of cellobiose dehydrogenases; Henriksson G et al.; Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi . It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors including quinones, phenoxyradicals, Fe(3+), Cu(2+) and triiodide ion . Monosaccharides, maltose and molecular oxygen are poor substrates . CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis . The FAD-containing fragment carries all known catalytic and cellulose binding properties . One-electron acceptors, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer . Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ) . The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain . The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase . The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase . A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding . The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin . CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested.

J Agric Food Chem, 2000 Mar, 48(3), 724 - 31
Vicinal diketone formation in yogurt: (13)C precursors and effect of branched-chain amino acids; Ott A et al.; Addition of branched-chain amino acids (BCAA) or an inhibitor of the BCAA biochemical pathways during fermentation of milk with a lac(-) mutant of Lactobacillus delbrueckii subsp . bulgaricus and Streptococcus thermophilus strongly influenced the formation of two aroma-impact compounds, 2,3-butanedione and 2,3-pentanedione, as well as their direct precursors 2-acetolactate and 2-acetohydroxybutyrate . This suggests a connection between vicinal diketone formation and BCAA biosynthesis in yogurt bacteria . A recently developed static-and-trapped headspace technique combined with gas chromatography-mass spectrometry demonstrated incorporation of (13)C from {U-(13)C(6)}-D-glucose and {U-(13)C(4)}-L-threonine into both vicinal diketones . For 2,3-butanedione, glucose is the major precursor via pyruvate and activated acetaldehyde . For 2, 3-pentanedione, L-threonine is a precursor via 2-ketobutyrate, but glucose is the major contributor via activated acetaldehyde and, possibly, also via 2-ketobutyrate, which is a degradation product of 3-methylaspartate, an intermediate in glutamate synthesis.

FEBS Lett, 2000 Mar 17, 470(1), 65 - 9
Elucidation of determinants of protein stability through genome sequence analysis; Chakravarty S et al.; Sequences of putative soluble proteins from complete genomes of eight thermophiles and 12 mesophiles were analyzed to gain insight into determinants of protein thermostability . The predator algorithm was used to assign secondary structures to each protein sequence . Based on simple statistical tests, a set of stabilizing factors was identified . These include reduced protein size, increases in number of residues involved in hydrogen bonding, beta-strand content and helix stabilization through ion pairs . There are also significant increases in the relative amounts of charged and hydrophobic beta-branched amino acids and decreases in uncharged polar amino acids in proteins from thermophiles relative to mesophilic organisms . Factors such as the relative proportion of residues in loops, proline and glycine content and helix capping do not appear to be important.

FEMS Microbiol Ecol, 2000 Mar 1, 31(3), 225 - 229
Growth kinetics of thermophilic Methanosarcina spp . isolated from full-scale biogas plants treating animal manures; Mladenovska Z et al.; This study determines the growth kinetics of thermophilic strains of Methanosarcina spp . from full-scale thermophilic biogas plants . The complete set of kinetic parameters, including maximum specific growth rate micro(max), half saturation constant K(S), acetate threshold concentration and cell growth yield Y(X/S), were determined for six Methanosarcina strains newly isolated from full-scale reactors and the type strain Methanosarcina thermophila TM-1(T) . The kinetic experiments were performed in media supplemented with acetate and activated carbon at the optimum growth temperatures of the individual strains, 50-55 degrees C . The micro(max) values of the isolates were in the range of 0.044-0.064 h(-1), the K(S) ranged from 6.5 to 24.7 mM acetate and the threshold for acetate utilization from 0.11 to 0.40 mM . The cell growth yields of the strains were between 0.78 and 2.97 g dry weight cells mol(-1) acetate . The six isolates exhibited significantly higher micro(max) and had higher affinity to acetate than the type strain M . thermophila TM-1(T) . Generally, the affinities of thermophilic Methanosarcina strains tested in this study cover a similar range to those reported in the literature for mesophilic Methanosarcina spp . with acetate as substrate . The strains isolated from plants treating mixtures of animal manures and industrial organic wastes had higher affinity for acetate and lower thresholds than strains isolated from reactors operating solely on manures.

J Theor Biol, 2000 Apr 7, 203(3), 203 - 13
The universal ancestor lived in a thermophilic or hyperthermophilic environment; Di Giulio M; Galtier et al . (Science 1999, 283, 220-221) exploit the correlation between the optimal growth temperature in prokaryotes and the G+C content of rRNAs and establish that the last universal common ancestor (LUCA) lived in a mesophilic environment . This result was achieved by estimating the G+C content of the ancestral sequences of the rRNAs of the LUCA through use of a complex Markov model . I have re-analysed their alignments of the rDNAs with maximum parsimony and I have found that their result is not robust and is, in all likelihood, incorrect . In particular, the rRNA ancestral sequences reconstructed with maximum parsimony from these rDNA alignments as well as those reconstructed after eliminating all the sites that turn out to be ambiguous to the parsimony algorithm and to a site-by-site inspection of these alignments, are such as to suggest that the LUCA lived in a thermophilic or hyperthermophilic environment . This finding is also supported by some tRNA ancestral sequences . The main conclusion of this analysis is that if the LUCA was a progenote then the origin of life might have taken place at a high temperature .

Biochem, Educ. . 2000 Jan 1, 28(1), 47 - 49
A simple and rapid method for screening amylolytic bacteria; Chimoy Effio P et al.; A laboratory class was designed for the study of the ecology of amylolytic bacteria in soil, although other sources may be equally suitable for this purpose . Groups of three students carried out the following: (a) preparation and sterilization of medium and plates, (b) collection and preparation of soil samples, spreading the samples on the plates, (c) incubation of the plates at 37 degrees C overnight, a further 1 h incubation at 60 degrees C to observe amylolytic activity due to thermophilic bacteria, and (d) interpretation and discussion of the results . These tasks are accomplished in two periods of 4h on consecutive days . No sophisticated instruments are required for these experiments, which can be carried out in three classes of 4h each . On the first day the students prepare culture media, buffers and reagents, as well as collect and grow soil samples . The second day is spent for both taxonomic identification of colonies and the HAI determination.

J Food Prot, 2000 Mar, 63(3), 299 - 303
Detection of thermophilic Campylobacter spp . in blood-free enriched samples of inoculated foods by the polymerase chain reaction; Thunberg RL et al.; The detection of thermophilic Campylobacter spp., as represented by Campylobacter jejuni, by the polymerase chain reaction (PCR) was investigated and compared with the selective agar isolation (SAI) method . Stationary-phase cultures of C . jejuni were inoculated into either blood-free enrichment broth (BFEB) or BFEB that contained 10% broccoli, crabmeat, mushroom, raw milk, and raw oyster rinses . Following a 48-h enrichment period, aliquots of food test portions were removed for simultaneous analysis by PCR and SAI . It was determined that the presence of charcoal and iron in the enrichment broth interfered with the PCR assay . Therefore, three DNA extraction techniques were developed and evaluated using a 16S rRNA primer pair in the PCR assay . The 50% end point (DL50) values (determined upon six initial C . jejuni spiking levels) were used to assess the frequency of isolation utilizing PCR versus SAI for the detection of this organism in the enrichment matrices . There were virtually no differences in detection of C . jejuni among enriched samples analyzed by PCR and SAI . Mean DL50 values (n = 3) for plain BFEB, broccoli, crabmeat, mushroom, raw milk, and raw oyster were, respectively, 0.02 (PCR) versus 0.01 (SAI), 0.01 versus 0.06, 0.07 versus 0.04, 0.03 versus 0.08, 0.01 versus 0.01, and 0.01 versus 0.01 CFU/5 g food . Significant variability in the detection limit of C . jejuni by PCR in the food enrichments was observed among DNA extraction techniques . Using 48-h enrichment cultures followed by PCR analysis could save 1 day of the time required for the presumptive identification of C . jejuni in suspected foods.

J Mol Biol, 2000 Mar 24, 297(2), 481 - 500
The crystal structure of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 A resolution; Charron C et al.; The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A resolution by molecular replacement . Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP(+) over NAD(+) . The present structure is the first archaeal GAPDH crystallized with NADP(+) . GAPDH from M . fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts . Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group . Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidoreductases . Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes . Within the catalytic site, the reported crystal structure definitively confirms the essential role previously assigned to Cys140 by site-directed mutagenesis studies . In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism . M . fervidus grows optimally at 84 degrees C with a maximal growth temperature of 97 degrees C . The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms . Among the various phenomena related to protein thermostabilization, reinforcement of electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha-helices in the archaeal GAPDHs . The first one, named alpha4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level . The second one, named alphaJ, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly .

J Dairy Sci, 2000 Feb, 83(2), 255 - 63
Effect of administration of fermented milk containing whey protein concentrate to rats and healthy men on serum lipids and blood pressure; Kawase M et al.; The effect of fermented milk supplemented with whey protein concentrate on the serum lipid level of rats was investigated . The serum total cholesterol level for the group fed fermented milk with both Lactobacillus casei TMC0409 and Streptococcus thermophilus TMC 1543 was significantly lower than that of the control group (P<0.05) in rats . Furthermore, the effect of the longterm intake of this fermented milk on the serum lipid level of twenty healthy adult men was investigated . During the 8-wk study, the volunteers consumed 200 ml of fermented milk or placebo in the morning and evening . Blood samples were drawn for analysis three times, just before taking the experimental diet, and after 4 wk and 8 wk of consumption . After 8 wk, the high density lipoprotein cholesterol level for the fermented milk group showed a significant rise after 4 wk (P<0.05), whereas that of the placebo group showed no change even after 4 wk (P>0.05) . The triglyceride level for the fermented milk group lowered significantly after 4 wk (<0.05), whereas that of the placebo group showed no change even after 4 wk (P>0.05) . The atherogenic index {(total cholesterol - high density lipoprotein cholesterol)/high-density lipoprotein cholesterol} for the fermented milk group decreased significantly from 4.24 to 3.52 (P<0.05) . The systolic blood pressure lowered significantly by the intake of fermented milk (P<0.05) On the other hand, such effect was not observed in the placebo group (P>0.05) . These results indicate potential of the development of fermented milk with multiple therapeutic effects.

J Inorg Biochem, 2000 Jan 15, 78(1), 35 - 41
Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens; Wittung-Stafshede P et al.; The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric protein containing two iron-sulfur centres, one {3Fe-4S}(1+/0) and one {4Fe-4S}(2+/1+) . It is an intrinsically hyperstable protein, being expressed at the organism's extreme optimal growth temperature: 80 degrees C . Using spectroscopic methods we have investigated the unfolding reaction of the Acidianus ambivalens ferredoxin . No unfolding of the oxidised ferredoxin was observed at pH 7.0, even in the presence of 8 M GuHCl . Upon increasing the pH to 10.0, the unfolding transition showed a midpoint at 6.3 M GuHCl and an unfolding-free energy of 70 kJ mol(-1) in buffer (pH 10) was estimated . Kinetic-unfolding experiments showed that the polypeptide unfolding correlated with rearrangement of the iron-sulfur centres to new ones which had strong absorption maxima at 520 and 610 nm . These new, possibly linear three-iron, clusters were coordinated to the unfolded protein but degraded slowly . From thermal experiments in the presence of GuHCl we estimated the melting temperature for the Acidianus ambivalens ferredoxin in buffer (at pH 7) to be 122 degrees C . Possible structural properties that contribute to the large thermal stability of the Acidianus ambivalens ferredoxin are discussed using a three-dimensional protein model.

Bioorg Med Chem Lett, 2000 Feb 21, 10(4), 365 - 8
A novel thermophilic glycosynthase that effects branching glycosylation; Trincone A et al.; A novel thermophilic glycosynthase that effects branching glycosylation has been obtained by mutation of the nucleophile in the active site of the glycosidase from Sulfolobus solfataricus . Two methods for the use of this mutant are reported.

Biochemistry (Mosc), 2000 Feb, 65(2), 244 - 9
Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents; Rechkunova NI et al.; The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs . The 5;-{32P}-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5, 6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP) . Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP) . The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis . The enzyme was protected from the photosensitized modification by dNTP . To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture . In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled . The suggested method enables highly selective affinity modification of DNA-polymerases.

Enzyme Microb Technol, 2000 Mar 1, 26(5-6), 459 - 465
Isolation and properties of a cellulosome-type multienzyme complex of the thermophilic Bacteroides sp . strain P-1; Ponpium P et al.; The extracellular form of cellulosome-type multienzyme complex of thermophilic Bacteroides sp . strain P-1 which was isolated from the anaerobic digester, is described . Multienzyme complex was isolated from the culture supernatant by an adsorption-desorption affinity chromatography on microcrystalline cellulose . The isolated multienzyme complex was found to form a complex that exhibited a high molecular weight (estimated at more than 1400 kDa) and was quite stable, requiring strong denaturing condition for dissociation . Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved multienzyme complex into at least 12 subunits with the molecular weight range of 49 to 209 kDa, respectively . The isolated multienzyme complex showed cellulose-binding ability, cellulase and xylanase activities and effected the hydrolysis of crystalline cellulose and lignocellulosic materials in the form of corncob, corn hull, rice straw, and sugarcane bagasse.

Enzyme Microb Technol, 2000 Mar 1, 26(5-6), 421 - 430
Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit; Hiol A et al.; We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae . Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation . The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography . The specific activity of the purified enzyme was 8800 U/mg . The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration . The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6 . Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation . Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus . The optimum pH for enzyme activity was 7.5 . Lipase was stable in the pH range from 4.5 to 7.5 . The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min . The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+) . Lipase activity against triolein was enhanced by sodium cholate or taurocholate . The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity . It showed a good stability in organic solvents and especially in long chain-fatty alcohol . The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate . About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.

Enzyme Microb Technol, 2000 Mar 1, 26(5-6), 368 - 373
Nitrile biotransformations using free and immobilized cells of a thermophilic Bacillus spp; Graham D et al.; A thermophilic Bacillus spp . capable of transforming aliphatic nitriles, cyclic nitriles and dinitriles was used as a free cell suspension and immobilized in alginate beads to study the utilization of acetonitrile and acrylonitrile in a buffered biotransformation medium . The cells grew optimally at 65 degrees C and contained a nitrile hydratase-amidase enzyme system that transformed nitrile compounds stoichiometrically to the corresponding carboxylic acids . In the presence of urea or chloroacetone, amidase activity was inhibited and the amide intermediate was accumulated . Mass transfer limitation of nitrile utilization rates was observed with immobilized cells, but the alginate afforded the cells some degree of additional thermal stability and potential advantage in re-use . In vitro inhibition of the partially purified amidase was confirmed and the use of whole cells of this organism in a continuous bioreactor to generate amide products from nitrile substrates was demonstrated.

Biotechnol Bioeng, 2000 Apr 20, 68(2), 184 - 90
Preparation of Thermus thermophilus holo-chaperonin-immobilized microspheres with high ability to facilitate protein refolding; Teshima T et al.; Carboxylated poly(styrene/acrylamide) (P(St/AAm)-H) microspheres with different acrylamide contents were prepared by emulsifier-free emulsion polymerization . Thermus thermophilus holo-chaperonin (cpn) was covalently immobilized onto these microspheres with high yield . The T . thermophilus holo-cpn-immobilized microspheres were used for refolding of guanidine hydrochloride (Gdn-HCl)-denatured enzymes and showed sufficiently high ability to facilitate refolding of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PD) and pig heart lactate dehydrogenase (LDH) at 30 degrees C and Bacillus stearothermophilus LDH at 60 degrees C . The specific ability of T . thermophilus holo-cpn-immobilized microspheres increased with increasing immobilization amount and reached plateau at around 10-15 mg/m(2) . When the immobilization amounts of T . thermophilus holo-cpn were approximately 10 mg/m(2), the microspheres retained sufficiently high ability to facilitate protein refolding during repeated use . Therefore, the P(St/AAm)-H microspheres on which approximately 10 mg/m(2) of T . thermophilus holo-cpn is immobilized are very effective for refolding of various (Gdn-HCl)-denatured enzymes over a wide temperature range .

Appl Microbiol Biotechnol, 2000 Feb, 53(2), 173 - 9
Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC); Nemati M et al.; The effect of mineral particle size on the bioleaching of pyrite by the acidophilic thermophile Sulfolobus metallicus was investigated in a batch bioreactor . Decreasing the particle size from a mean diameter of 202 micron (size fraction: 150-180 micron) to a mean diameter of 42.5 micron (size fraction: 25-45 micron) enhanced the bioleaching rate from 0.05 kg m(-3) h(-1) to 0.098 kg m(-3) h(-1) . The particle size distribution of the mineral in this range did not influence the morphology and growth kinetics of the cells . The values of specific growth rate (mu) and yield factor (Y) were 0.018-0.025 h(-1) and 0.67x10(11)-1.45x10(11) cells (g iron)(-1), respectively . Decreasing the particle size of the mineral to a mean diameter of 6.40 micron (size fraction <25 micron) adversely influenced the activity of the cells . The presence of fine particles apparently damaged the structure of the cells, resulting in their inability to oxidise pyrite.

Indian J Exp Biol, 1999 Aug, 37(8), 750 - 7
Phototoxicity evaluation--Tetrahymena thermophila as an alternative model; Misra RB et al.; Interest in utilizing an alternative to animal method for toxicological evaluation has received considerable attention due to cost effectiveness and the ethical issues involving animal experimentation . Alternative methods for phototoxicity evaluation are significant because of growing concern over increasing health effects due to stratospheric ozone depletion resulting in an increasing penetration of ultraviolet light-B radiation (UVB, 290-320 nm) which contributes to activation of chemical and biological molecules to potential phototoxic agents . The classic rabbit eye-irritancy test referred to as Draize test has been the subject of severe criticism by animal welfare groups . Dermal toxicity test using guinea pigs and mouse tail phototoxicity test is time consuming and requires a large number of laboratory animals . In photohaemolysis assay some of the phototoxic agents (such as riboflavin) react with the membrane proteins of the erythrocyte . However, in vitro test system using protozoa offers a promising alternative means of phototoxicity evaluation . Our previous studies have demonstrated that synergistic action of photochemically reactive agents and sunlight produces lethal effects to Paramecium but the protozoan has not received serious consideration for use as an alternative model for phototoxicity evaluation . In the present communication we have described the potential application of Tetrahymena as an alternative model to study the radiation-induced changes both in the presence or absence of photoreactive chemical agents . This model is likely to provide scope for studying the biological effects of environmental UVB radiation, DNA damage and defence against oxidative stress.

Proteins, 2000 Mar 1, 38(4), 368 - 83
Electrostatic strengths of salt bridges in thermophilic and mesophilic glutamate dehydrogenase monomers; Kumar S et al.; Here we seek to understand the higher frequency of occurrence of salt bridges in proteins from thermophiles as compared to their mesophile homologs . We focus on glutamate dehydrogenase, owing to the availability of high resolution thermophilic (from Pyrococcus furiosus) and mesophilic (from Clostridium symbiosum) protein structures, the large protein size and the large difference in melting temperatures . We investigate the location, statistics and electrostatic strengths of salt bridges and of their networks within corresponding monomers of the thermophilic and mesophilic enzymes . We find that many of the extra salt bridges which are present in the thermophilic glutamate dehydrogenase monomer but absent in the mesophilic enzyme, form around the active site of the protein . Furthermore, salt bridges in the thermostable glutamate dehydrogenase cluster within the hydrophobic folding units of the monomer, rather than between them . Computation of the electrostatic contribution of salt bridge energies by solving the Poisson equation in a continuum solvent medium, shows that the salt bridges in Pyrococcus furiosus glutamate dehydrogenase are highly stabilizing . In contrast, the salt bridges in the mesophilic Clostridium symbiosum glutamate dehydrogenase are only marginally stabilizing . This is largely the outcome of the difference in the protein environment around the salt bridges in the two proteins . The presence of a larger number of charges, and hence, of salt bridges contributes to an electrostatically more favorable protein energy term . Our results indicate that salt bridges and their networks may have an important role in resisting deformation/unfolding of the protein structure at high temperatures, particularly in critical regions such as around the active site.

Proteins, 2000 Mar 1, 38(4), 351 - 60
The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature; D'Auria S et al.; The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa . The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C . We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy . The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets . This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet . The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact . The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays . The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics . Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments . The relationship between enzyme activity and protein structure is discussed.

Curr Microbiol, 2000 May, 40(5), 333 - 40
Multidomain and multifunctional glycosyl hydrolases from the extreme thermophile Caldicellulosiruptor isolate Tok7B.1; Gibbs MD et al.; DNA sequencing techniques have revealed widespread molecular diversity of the genomic organization of apparently closely related bacteria (as judged from SSU rDNA sequence similarity) . We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in possessing multi-catalytic, multidomain arrangements for the majority of its glycosyl hydrolases . We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp . strain Tok7B.1 . These clusters are not closely linked, and each is different in its organization from any described for Cs . saccharolyticus . The catalytic domains of the enzymes belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48 . The cellulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp . Tok7B.1 are types IIIb, IIIc, or VI . A number of individual catalytic and binding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE.

Cell Biol Int, 1999, 23(8), 551 - 60
Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila . Evidence for differential codon usage in highly expressed genes; Larsen LK et al.; We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila . The r-protein L3 is encoded by a single copy gene interrupted by one intron . The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena . The codon usage of the L3 gene is highly biased . A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias . Class A comprises r-protein genes and a number of other highly expressed genes . Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1 . Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases . Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions . The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena .

Biochemistry, 2000 Mar 14, 39(10), 2484 - 98
Homology modeling, molecular dynamics simulations, and analysis of CYP119, a P450 enzyme from extreme acidothermophilic archaeon Sulfolobus solfataricus; Chang YT et al.; The recent characterization of a thermophilic and barophilic CYP119 from Sulfolobus solfataricus offers a new opportunity to identify the origin of its stability by comparing it with mesophilic P450s with known structures . Since the three-dimensional structure of CYP119 is not yet available, homology modeling techniques were used to build model structures for this enzyme . The overall quality and stability of the models were assessed using three protein analysis programs and by monitoring structural stability during 1 ns of molecular dynamics simulations at 300 and 390 K . The results show the CYP119 models to be of good quality . Possible origins of the thermo- and barostability of CYP119 were then investigated by examining the amino acid compositions and the three-dimensional structure of CYP119 compared with the five mesophilic templates . Three possible factors were identified that could contribute to the enhanced stability of CYP119 . The first was the higher relative population of salt bridges and the presence of a few unique salt bridges found in CYP119 that were absent in all five template CYP450s . The second factor was a decreased population of Ala and an increased population of Ile found in the interior of CYP119, which are likely to improve packing in CYP119 . The third factor was a more extensive aromatic cluster seen in CYP119 which was not found in all five template P450s . In addition, the model CYP119 three-dimensional structures were also used to determine key properties related to its function . Specifically, binding site residues and surface residues for redox partner interactions were identified . These residues identified together with those residues found that might contribute to the increased stability are suggested for future mutagenesis studies . The results obtained from these experimental studies shall then provide further validation of the suggested origins of stability and the structure-function relationships derived from the model structures of this enzyme.

Genome, 2000 Feb, 43(1), 116 - 36
Gene content and organization of a 281-kbp contig from the genome of the extremely thermophilic archaeon, Sulfolobus solfataricus P2; Charlebois RL et al.; The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed . Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences . The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes.

Appl Environ Microbiol, 2000 Mar, 66(3), 1038 - 49
Highly ordered vertical structure of Synechococcus populations within the one-millimeter-thick photic zone of a hot spring cyanobacterial mat; Ramsing NB et al.; A variety of contemporary techniques were used to investigate the vertical distribution of thermophilic unicellular cyanobacteria, Synechococcus spp., and their activity within the upper 1-mm-thick photic zone of the mat community found in an alkaline siliceous hot spring in Yellowstone National Park in Wyoming . Detailed measurements were made over a diel cycle at a 61 degrees C site . Net oxygenic photosynthesis measured with oxygen microelectrodes was highest within the uppermost 100- to 200-microm-thick layer until midmorning, but as the day progressed, the peak of net activity shifted to deeper layers, stabilizing at a depth of 300 microm from midday throughout the afternoon . Examination of vertical thin sections by bright-field and autofluorescence microscopy revealed the existence of different populations of Synechococcus which form discrete bands at different vertical positions . Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments from horizontal cryosections obtained at 100-microm-thick vertical intervals also suggested vertical stratification of cyanobacterial, green sulfur bacterium-like, and green nonsulfur bacterium-like populations . There was no evidence of diel migration . However, image analysis of vertical thin sections revealed the presence of a narrow band of rod-shaped Synechococcus cells in which the cells assumed an upright position . These upright cells, located 400 to 800 microm below the surface, were observed only in mat samples obtained around noon . In mat samples obtained at other time points, the cells were randomly oriented throughout the mat . These combined observations reveal the existence of a highly ordered structure within the very thin photic zone of this hot spring microbial mat, consisting of morphologically similar Synechococcus populations that are likely to be differentially adapted, some co-occurring with green sulfur bacterium-like populations, and all overlying green nonsulfur bacterium-like populations.

Appl Environ Microbiol, 2000 Mar, 66(3), 995 - 1000
High-affinity maltose binding and transport by the thermophilic anaerobe Thermoanaerobacter ethanolicus 39E; Jones CR et al.; Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch . Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose . Filter-binding assays revealed that cell membranes bound maltose with high affinity . Metabolic labeling of T . ethanolicus maltose-grown cells with {(14)C}palmitic acid showed that this protein was posttranslationally acylated . A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM . Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact . In whole-cell transport assays, the affinity for maltose was approximately 40 nM . Maltotriose and alpha-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect . Based on these results, it appears that T . ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and alpha-trehalose.

Biochem J, 2000 Mar 15, 346 Pt 3, 583 - 6
The thermostabilizing domain, XynA, of Caldibacillus cellulovorans xylanase is a xylan binding domain; Sunna A et al.; We show that the N-terminal 'thermostabilizing domain' (TSD) of the xylanase, XynA, from the thermophilic bacterium Caldibacillus cellulovorans also acts as a xylan binding domain . Affinity electrophoresis experiments show that this TSD selectively binds soluble xylan and binds weakly to hydroxyethylcellulose . Based on this, and previously reported evidence, we propose that xylanase-associated TSDs are xylan binding domains.

J Biomol Struct Dyn, 2000 Feb, 17(4), 617 - 28
Genetic and biochemical manipulations of the small ribosomal subunit from Thermus thermophilus HB8; Auerbach T et al.; Crystals of the small ribosomal subunit from Thermus thermophilus diffract to 3A and exhibit reasonable isomorphism and moderate resistance to irradiation . A 5A MIR map of this particle shows a similar shape to the part assigned to this particle within the cryo-EM reconstructions of the whole ribosome and contains regions interpretable either as RNA chains or as protein motifs . To assist phasing at higher resolution we introduced recombinant methods aimed at extensive selenation for MAD phasing . We are focusing on several ribosomal proteins that can be quantitatively detached by chemical means . These proteins can be modified and subsequently reconstituted into depleted ribosomal cores . They also can be used for binding heavy atoms, by incorporating chemically reactive binding sites, such as -SH groups, into them . In parallel we are co-crystallizing the ribosomal particles with tailor made ligands, such as antibiotics or cDNA to which heavy-atoms have been attached or diffuse the latter compounds into already formed crystals.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1683 - 8
Nonlinearity in genetic decoding: homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting; Larsen B et al.; The tau and gamma subunits of DNA polymerase III are both encoded by a single gene in Escherichia coli and Thermus thermophilus . gamma is two-thirds the size of tau and shares virtually all its amino acid sequence with tau . E . coli and T . thermophilus have evolved very different mechanisms for setting the approximate 1:1 ratio between tau and gamma . Both mechanisms put ribosomes into alternate reading frames so that stop codons in the new frame serve to make the smaller gamma protein . In E . coli, approximately 50% of initiating ribosomes translate the dnaX mRNA conventionally to give tau, but the other 50% shift into the -1 reading frame at a specific site (A AAA AAG) in the mRNA to produce gamma . In T . thermophilus ribosomal frameshifting is not required: the dnaX mRNA is a heterogeneous population of molecules with different numbers of A residues arising from transcriptional slippage on a run of nine T residues in the DNA template . Translation of the subpopulation containing nine As (or +/- multiples of three As) yields tau . The rest of the population of mRNAs (containing nine +/- nonmultiples of three As) puts ribosomes into the alternate reading frames to produce the gamma protein(s) . It is surprising that two rather similar dnaX sequences in E . coli and T . thermophilus lead to very different mechanisms of expression.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1530 - 5
A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase; Chong JP et al.; The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes . Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication . Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770) . This gene is most similar to MCM4 in eukaryotic cells . Here we have expressed and purified the M . thermoautotrophicum MCM protein . The purified protein forms a complex that has a molecular mass of approximately 850 kDa, consistent with formation of a double hexamer . The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs . The 3' to 5' helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site . Moreover, the double hexamer form is the active helicase . It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea . The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.

Prep Biochem Biotechnol, 2000 Feb, 30(1), 61 - 7
Purification of the ADP-ribosylating enzyme from S . solfataricus by SDS-polyacrylamide gel electrophoresis and electroelution; Faraone-Mennella MR et al.; The ADPribosylating enzyme from the thermophilic archaeon S . solfataricus was purified by a simple procedure which included preparative electrophoresis on a 0.1% SDS- polyacrylamide gel . The gel slice containing the enzymatic protein was cut out and the enzyme was solubilized by electroelution . The pure enzyme was obtained by chromatography of the electroeluted sample on a DNA-Sepharose column . The purified enzyme retained both its full activity and the structuring ability as a function of temperature increase.

Biochemistry, 2000 Mar 7, 39(9), 2283 - 96
Kinetics and mechanism of the citrate synthase from the thermophilic archaeon Thermoplasma acidophilum; Kurz LC et al.; The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS) . All discrete steps in the mechanistic sequence of PCS can be identified in TpCS . The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS . Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes . However, significant differences exist between the two enzymes . PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible . TpCS is a less efficient catalyst . Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis renders this step nearly rate-limiting for the forward reaction of TpCS . Further, excessive stabilization of the citryl-CoA intermediate renders the condensation step nearly irreversible . Values of substrate and solvent deuterium isotope effects are consistent with the kinetic model . Near its temperature optimum (70 degrees C), there is a modest increase in the reversibility of the condensation step for TpCS, but reversibility still falls short of that shown by PCS at 37 degrees C . The root cause of the catalytic inefficiency of TpCS may lie in the lack of protein flexibility imposed by the requirement for thermal stability of the protein itself or its temperature-labile substrate, oxaloacetate.

Biochemistry, 2000 Mar 7, 39(9), 2269 - 75
Engineering of Sulfolobus solfataricus HMG-CoA reductase to a form whose activity is regulated by phosphorylation and dephosphorylation; Kim DY et al.; There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria . The activity of the class I Syrian hamster HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Ser871 . Phosphorylation apparently prevents the active site histidine, His865, from protonating the inhibitory coenzyme A thioanion prior to its release from the enzyme . Structural evidence for this hypothesis is, however, lacking . The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus, whose stability recommends it for physical studies, lacks both a phosphoacceptor serine and a protein kinase recognition motif . Consequently, its activity is not regulated by phosphorylation . We therefore employed site-directed mutagenesis to engineer an appropriately located phosphoacceptor serine and cAMP-dependent protein kinase recognition motif . Substitution of serine for Ala406, the apparent cognate of hamster Ser871, and replacement of Leu403 and Gly404 by arginine created S . solfataricus mutant enzyme L403R/G404R/A406S . The general properties of enzyme L403R/G404R/A406S (K(m) values, V(max), optimal pH and temperature) were essentially those of the wild-type enzyme . Exposure of enzyme L403R/G404R/A406S to {gamma-(32)P}ATP and cAMP-dependent protein kinase was accompanied by incorporation of (32)P(i) and by a parallel decrease in catalytic activity . Subsequent treatment with a protein phosphatase released enzyme-bound (32)P(i) and restored activity to pretreatment levels . The regulatory properties of enzyme L403R/G404R/A406S thus match those of the hamster enzyme . Solution of the three-dimensional structures of the phospho and dephospho forms of this mutant enzyme thus should reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.

Nutr Cancer, 1999, 35(2), 153 - 9
A probiotic strain of L . acidophilus reduces DMH-induced large intestinal tumors in male Sprague-Dawley rats; McIntosh GH et al.; Probiotic bacteria strains were examined for their influence on 1,2-dimethylhydrazine (DMH)-induced intestinal tumors in 100 male Sprague-Dawley rats . Lactobacillus acidophilus (Delvo Pro LA-1), Lactobacillus rhamnosus (GG), Bifidobacterium animalis (CSCC1941), and Streptococcus thermophilus (DD145) strains were examined for their influence when added as freeze-dried bacteria to an experimental diet based on a high-fat semipurified (AIN-93) rodent diet . Four bacterial treatments were compared: L . acidophilus, L . acidophilus + B . animalis, L . rhamnosus, and S . thermophilus, the bacteria being added daily at 1% freeze-dried weight (10(10) colony-forming units/g) to the diet . Trends were observed in the incidence of rats with large intestinal tumors for three treatments: 25% lower than control for L . acidophilus, 20% lower for L . acidophilus + B . animalis and L . rhamnosus treatments, and 10% lower for S . thermophilus . Large intestinal tumor burden was significantly lower for treated rats with L . acidophilus than for the control group (10 and 3 tumors/treatment group, respectively, p = 0.05) . Large intestinal tumor mass index was also lower for the L . acidophilus treatment than for control (1.70 and 0.10, respectively, p < 0.05) . Other treatments showed no statistically significant change from control for these indexes of tumorigenesis . For rats fed L . acidophilus, no adenocarcinomas were present in the colons . Pulsed-field gel electrophoresis of bacterial chromosomal DNA fragments was used to differentiate introduced (exogenous) bacterial strains from indigenous bacteria of the same genera present in the feces . Survival during gut passage and displacement of indigenous lactobacilli occurred with introduced L . acidophilus and L . rhamnosus GG during the probiotic treatment period . However, introduced strains of B . animalis and S . thermophilus were not able to be isolated from feces . It is concluded that this strain of L . acidophilus supplied as freeze-dried bacteria in the diet was protective, as seen by a small but significant inhibition of tumors within the rat colon.

J Bacteriol, 2000 Mar, 182(6), 1616 - 23
Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus; Studholme DJ et al.; The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five potential sigma(N)-dependent transcriptional activators . Although A . aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria . We identified five putative sigma(N)-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity . We cloned, overexpressed (in Escherichia coli), and purified A . aeolicus sigma(N) and the NifA homologue, AQ_218 . Purified A . aeolicus sigma(N) bound to E . coli core RNA polymerase and bound specifically to a DNA fragment containing E . coli promoter glnHp2 and to several A . aeolicus DNA fragments containing putative sigma(N)-dependent promoters . When combined with E . coli core RNA polymerase, A . aeolicus sigma(N) supported A . aeolicus NifA-dependent transcription from the glnHp2 promoter . The E . coli activator PspFDeltaHTH did not stimulate transcription . The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A . aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism . These results argue that the sigma(N) enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.

FEBS Lett, 2000 Feb 18, 468(1), 48 - 52
Mirror image mutations reveal the significance of an intersubunit ion cluster in the stability of 3-isopropylmalate dehydrogenase; Nemeth A et al.; The comparison of the three-dimensional structures of thermophilic (Thermus thermophilus) and mesophilic (Escherichia coli) 3-isopropylmalate dehydrogenases (IPMDH, EC 1.1.1.85) suggested that the existence of extra ion pairs in the thermophilic enzyme found in the intersubunit region may be an important factor for thermostability . As a test of our assumption, glutamine 200 in the E . coli enzyme was turned into glutamate (Q200E mutant) to mimic the thermophilic enzyme at this site by creating an intersubunit ion pair which can join existing ion clusters . At the same site in the thermophilic enzyme we changed glutamate 190 into glutamine (E190Q), hereby removing the corresponding ion pair . These single amino acid replacements resulted in increased thermostability of the mesophilic and decreased thermostability of the thermophilic enzyme, as measured by spectropolarimetry and differential scanning microcalorimetry.

Plant Cell Physiol, 1999 Dec, 40(12), 1219 - 31
Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43; Sugiura M et al.; The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex . The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography . The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor . The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes . These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs . The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d . These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.

Protein Eng, 2000 Jan, 13(1), 9 - 13
Protein thermal stability: insights from atomic displacement parameters (B values); Parthasarathy S et al.; The factors contributing to the thermal stability of proteins from thermophilic origins are matters of intense debate and investigation . Thermophilic proteins are thought to possess better packed interiors than their mesophilic counterparts, leading to lesser overall flexibility and a corresponding reduction in surface-to-volume ratio . These observations prompted an analysis of B values reported in high-resolution X-ray crystal structures of mesophilic and thermophilic proteins . In this analysis, the following aspects were addressed: (1) frequency distribution of normalized B values (B' factors) over all the proteins and for individual amino acids; (2) amino acid compositions in high B value regions of polypeptide chains; (3) variation in the B values from core to the surface of proteins in terms of their radius of gyration; and (4) degree of dispersion of normalized B values in spheres around the Calpha atoms . The analysis revealed that (1) Ser and Thr have lesser flexibility in thermophiles than in mesophiles, (2) the proportion of Glu and Lys in high B value regions of thermophiles is higher and that of Ser and Thr is lower and (3) the dispersion of B values within spheres at Calpha atoms is similar in mesophiles and thermophiles . These observations reflect plausible differences in the dynamics of thermophilic and mesophilic proteins and suggest amino acid substitutions that are likely to change thermal stability.

Curr Opin Chem Biol, 2000 Feb, 4(1), 110 - 9
Enzyme-catalyzed synthesis of carbohydrates; Wymer N et al.; Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year . Additionally, the utility of several well studied enzymes has been expanded . Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community . Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 151 - 60
Improved purification of the membrane-bound hydrogenase-sulfur-reductase complex from thermophilic archaea using epsilon-aminocaproic acid-containing chromatography buffers; Laska S et al.; A hydrogenase-sulfur reductase (SR) complex was purified from membrane preparations of the extremely thermophilic, acidophilic archaeon Acidianus ambivalens using a combination of sucrose density gradient centrifugation and column chromatography (FPLC) . All chromatographic steps were performed in the presence of 0.5% epsilon-aminocaproic acid resulting in the elution of the SR complex as a sharp peak . In contrast, chromatography using buffers without epsilon-aminocaproic acid, or in the presence of detergents, were not successful . The purified A . ambivalens SR complex consisted of at least four subunits with relative molecular masses of 110000, 66000, 39000 and 29000, respectively . A similar procedure was applied to purify the membrane-bound hydrogenase from Thermoproteus neutrophilus, a non-related extremely thermophilic but neutrophilic archaeon, which consisted of only two subunits with relative molecular masses of 66000 and 39000, respectively.

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