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 s