Thermophile

Appl Environ Microbiol, 2001 Mar, 67(3), 1128 - 39
Analysis of the genetic switch and replication region of a P335-type bacteriophage with an obligate lytic lifestyle on Lactococcus lactis; Madsen SM et al.; The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage phi31 was determined, and its regulatory elements were investigated . The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that phi31 was derived from a temperate bacteriophage . Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter . The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages . The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression . No detectable cI gene expression could be measured in the presence of cro . cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch . Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue . The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages . Downstream of the primase homologue, an AT-rich noncoding origin region was identified . The characteristics and location of this region and its ability to reduce the efficiency of plaquing of phi31 10(6)-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication . Phage phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments . Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor . Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.

Appl Environ Microbiol, 2001 Mar, 67(3), 1116 - 22
Temperature-driven adaptation of the bacterial community in peat measured by using thymidine and leucine incorporation; Ranneklev SB et al.; The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions . The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures . Increasing the peat incubation temperature from 25 degrees C to 35, 45, or 55 degrees C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures . Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature . Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community . Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures . The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55 degrees C (thermophilic activity) and 25 degrees C (mesophilic activity) . Shifting the peat incubation temperature from 55 to 25 degrees C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity . The availability of substrate for bacterial growth varied over time and among different peat samples . To avoid confounding effects of substrate availability, a temperature adaptation index was calculated . This index consisted of the log(10) ratio of TdR incorporation at 55 and 25 degrees C . The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift . There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55 degrees C 3 or 11 days before incubation at 25 degrees C . Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.

J Biochem (Tokyo), 2001 Mar, 129(3), 477 - 84
Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase; Yasugi M et al.; We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus . Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed . Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types . To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH . The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T . thermophilus wild-type IPMDH and similar to that of the E . coli wild-type IPMDH . The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T . thermophilus wild-type IPMDH . Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes . The two types of cold-adapted mutants employed one of the two strategies of E . coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants . Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T . thermophilus wild-type IPMDH.

J Biochem (Tokyo), 2001 Mar, 129(3), 397 - 402
Monoacylglycerol lipase from moderately thermophilic Bacillus sp . strain H-257: molecular cloning, sequencing, and expression in Escherichia coli of the gene; Kitaura S et al.; Monoacylglycerol lipase {MGLP, EC 3.1.1.23} is produced intracellularly by the moderately thermophilic Bacillus sp . strain H-257 . The gene encoding MGLP was cloned, sequenced, and expressed in Escherichia coli . A genomic library of Bacillus sp . strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino acid sequence of a purified MGLP . The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp . strain H-257 DNA . Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa . The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved among lipases, esterases, and serine proteases . The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology) . When pMGLP31 was introduced into E . coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greater extent than Bacillus sp . strain H-257 . The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.

Environ Microbiol, 2000 Apr, 2(2), 143 - 59
Desulfotomaculum genus- and subgenus-specific 16S rRNA hybridization probes for environmental studies; Hristova KR et al.; Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested hybridization probes was developed and characterized . A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans), and five specific probes target subclusters within the Desulfotomaculum genus . The dissociation temperature of each probe was determined experimentally . Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments . Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized . The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples . Desulfotomaculum rRNA accounted for 0.3-2.1% of the total rRNA in the digesters, 2.6-6.6% in soil, 1.5-3.3% in human faeces and 2.5-6.2% in pig colon samples.

Nat Struct Biol, 2001 Mar, 8(3), 203 - 6
Structural basis for anticodon recognition by discriminating glutamyl-tRNA synthetase; Sekine S et al.; Glutamyl-tRNA synthetases (GluRSs) are divided into two distinct types, with regard to the presence or absence of glutaminyl-tRNA synthetase (GlnRS) in the genetic translation systems . In the original 19-synthetase systems lacking GlnRS, the 'non-discriminating' GluRS glutamylates both tRNAGlu and tRNAGln . In contrast, in the evolved 20-synthetase systems with GlnRS, the 'discriminating' GluRS aminoacylates only tRNAGlu . Here we report the 2.4 A resolution crystal structure of a 'discriminating' GluRS.tRNAGlu complex from Thermus thermophilus . The GluRS recognizes the tRNAGlu anticodon bases via two alpha-helical domains, maintaining the base stacking . We show that the discrimination between the Glu and Gln anticodons (34YUC36 and 34YUG36, respectively) is achieved by a single arginine residue (Arg 358) . The mutation of Arg 358 to Gln resulted in a GluRS that does not discriminate between the Glu and Gln anticodons . This change mimics the reverse course of GluRS evolution from anticodon 'non-dicsriminating' to 'discriminating'.

J Bacteriol, 2001 Mar, 183(6), 2086 - 92
Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8; Yamagata S et al.; A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine) . The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts . The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium . L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase . Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold . A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established . The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction . These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine . Methionine inhibited O-acetylserine sulfhydrylase markedly . The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase . Although it has been reported that homocysteine is directly synthesized in T . thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T . Kosuge, D . Gao, and T . Hoshino, J . Biosci . Bioeng . 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T . thermophilus HB8.

J Bacteriol, 2001 Mar, 183(6), 1974 - 82
Effects of ribosomes and intracellular solutes on activities and stabilities of elongation factor 2 proteins from psychrotolerant and thermophilic methanogens; Thomas T et al.; Low-temperature-adapted archaea are abundant in the environment, yet little is known about the thermal adaptation of their proteins . We have previously compared elongation factor 2 (EF-2) proteins from Antarctic (Methanococcoides burtonii) and thermophilic (Methanosarcina thermophila) methanogens and found that the M . burtonii EF-2 had greater intrinsic activity at low temperatures and lower thermal stability at high temperatures (T . Thomas and R . Cavicchioli, J . Bacteriol . 182:1328-1332, 2000) . While the gross thermal properties correlated with growth temperature, the activity and stability profiles of the EF-2 proteins did not precisely match the optimal growth temperature of each organism . This indicated that intracellular components may affect the thermal characteristics of the EF-2 proteins, and in this study we examined the effects of ribosomes and intracellular solutes . At a high growth temperature the thermophile produced high levels of potassium glutamate, which, when assayed in vitro with EF-2, retarded thermal unfolding and increased catalytic efficiency . In contrast, for the Antarctic methanogen adaptation to growth at a low temperature did not involve the accumulation of stabilizing organic solutes but appeared to result from an increased affinity of EF-2 for GTP and high levels of EF-2 in the cell relative to its low growth rate . Furthermore, ribosomes greatly stimulated GTPase activity and moderately stabilized both EF-2 proteins . These findings illustrate the different physiological strategies that have evolved in two phylogenetically related but thermally distinct methanogens to enable EF-2 to function satisfactorily.

Virology, 2001 Mar 1, 281(1), 6 - 9
The genome of the archaeal virus SIRV1 has features in common with genomes of eukaryal viruses; Blum H et al.; The virus SIRV1 of the extremely thermophilic archaeon Sulfolobus has a double-stranded DNA genome similar in architecture to the genomes of eukaryal viruses of the families Poxviridae, Pycodnaviridae, and Asfarviridae: the two strands of the 32,301 bp long linear genome are covalently connected forming a continuous polynucleotide chain and 2029 kb long inverted repeats are present at the termini . Very likely it also shares with these viruses mechanisms of initiation of replication and resolution of replicative intermediates .

Epidemiol Infect, 2000 Dec, 125(3), 505 - 22
Health burden in the Netherlands due to infection with thermophilic Campylobacter spp; Havelaar AH et al.; Infection with thermophilic Campylobacter spp . usually leads to an episode of acute gastroenteritis . Occasionally, more severe diseases may be induced, notably Guillain Barre syndrome and reactive arthritis . For some, the disease may be fatal . We have integrated available data in one public health measure, the Disability Adjusted Life Year (DALY) . DALYs are the sum of Years of Life Lost by premature mortality and Years Lived with Disability, weighted with a factor between 0 and 1 for the severity of illness . The mean health burden of campylobacter-associated illness in the Dutch population in the period 1990-5 is estimated as 1400 (90% CI 900-2000) DALY per year . The main determinants of health burden are acute gastroenteritis (440 DALY), gastroenteritis related mortality (310 DALY) and residual symptoms of Guillain-Barre syndrome (340 DALY) . Sensitivity analysis demonstrated that alternative model assumptions produced results in the above-mentioned range.

Mol Cell Biochem, 2001 Jan, 216(1-2), 93 - 101
L-asparaginase of Thermus thermophilus: purification, properties and identification of essential amino acids for its catalytic activity; Pritsa AA et al.; L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus . The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer . The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0 . The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM . It is a thermostable enzyme and it follows linear kinetics even at 77 degrees C . Chemical modification experiments implied the existence ofhistidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 187 - 93
Amycolatopsis sacchari sp . nov., a moderately thermophilic actinomycete isolated from vegetable matter; Goodfellow M et al.; The taxonomic position of a group of moderately thermophilic actinomycetes isolated from vegetable matter was determined using a suite of genotypic and phenotypic properties . The organisms were found to share a range of chemical and morphological markers typical of members of the genus Amycolatopsis . A representative of the group, strain K24T, formed a distinct phyletic line within the range of variation occupied by the genus Amycolatopsis in the 16S rDNA tree . The strains have many phenotypic properties in common and some of these distinguish the group from representatives of the validly described species of Amycolatopsis . It is clear from the combined datasets that the strains merit recognition as a new species of Amycolatopsis . The name proposed for the new species is Amycolatopsis sacchari; the type strain is K24T (= DSM 44468T = KCTC 9863T).

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 141 - 9
Carboxydobrachium pacificum gen . nov., sp . nov., a new anaerobic, thermophilic, CO-utilizing marine bacterium from Okinawa Trough; Sokolova TG et al.; A new anaerobic, thermophilic, CO-utilizing marine bacterium, strain JMT, was isolated from a submarine hot vent in Okinawa Trough . Cells of strain JMT were non-motile thin straight rods, sometimes branching, with a cell wall of the Gram-positive type, surrounded with an S-layer . Chains of three to five cells were often observed . The isolate grew chemolithotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O-->CO2+H2) and organotrophically on peptone, yeast extract, starch, cellobiose, glucose, galactose, fructose and pyruvate, producing H2, acetate and CO2 . Growth was observed from 50 to 80 degrees C with an optimum at 70 degrees C . The optimum pH was 6.8-7.1 . The optimum concentration of sea salts in the medium was 20.5-25.5 g l(-1) . The generation time under optimal conditions was 7.1 h . The DNA G+C content was 33 mol % . Growth of isolate JMT was not inhibited by penicillin, but ampicillin, streptomycin, kanamycin and neomycin completely inhibited growth . The results of 16S rDNA sequence analysis revealed that strain JMT belongs to the Thermoanaerobacter phylogenetic group within the Bacillus-Clostridium subphylum of Gram-positive bacteria but represents a separate branch of this group . On the basis of morphological and physiological features and phylogenetic data, this isolate should be assigned to a new genus, for which the name Carboxydobrachium is proposed . The type species is Carboxydobrachium pacificum; the type strain is JMT (= DSM 12653T).

J Dairy Sci, 2001 Jan, 84(1), 74 - 83
Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk; Kim CH et al.; Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats . Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S . aureus mastitis . In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S . aureus (isolates from bovine mastitis) . A specific amplicon of 270 bp was generated as predicted . Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S . aureus detection in milk from experimentally infected cows from 65 to 80% . Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100% . In a random survey involving 100 milk samples from cattle not infected with S . aureus, the test was 100% specific . With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed . It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific . The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S . aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.

J Dairy Sci, 2001 Jan, 84(1), 50 - 9
Isolation, characterization, and influence of native, nonstarter lactic acid bacteria on Cheddar cheese quality; Swearingen PA et al.; To determine whether adventitious nonstarter lactic acid bacteria (NSLAB) might affect cheese flavor and quality, we studied a population of NSLAB present in 30 premium quality Cheddar cheeses (3-mo ripened) produced at a commercial facility in the United States . DNA fingerprinting analysis with a sensitive strategy for arbitrary priming polymerase chain reaction showed that 75 isolates corresponded to at least 18 distinct nonstarter organisms . According to ribotype database comparisons of representatives from the 18 groups, 9 matched Lactobacillus (closest to paracasei species), 8 matched Streptococcus thermophilus, and 1 matched to a Lactococcus species . This finding indicated that among the 75 NSLAB isolates, Lactobacillus made up 64%, S . thermophilus 32%, and Lactococcus 4% . Isolates representing 11 NSLAB groups were characterized for protease, peptidase, and diacetyl production . Based on this phenotypic analysis, two Lactobacillus isolates were evaluated as adjuncts in Cheddar cheese . All of the NSLAB identified from the adjunct cheese at 3 mo by DNA fingerprinting consisted of the adjunct lactobacilli, showing that the adjunct strains predominated throughout the early stages of ripening . The impact of adjunct lactobacilli was evident after 6 mo when free amino acids significantly increased and sensory scores improved in adjunct cheese as compared with a control cheese . The largest impact was found in adjunct cheese containing a blend of both lactobacilli strains . These results show that certain adventitious NSLAB positively contribute to flavor development.

Yi Chuan Xue Bao, 2000, 27(12), 1100 - 7
{Studies of the active site, thermostability and thermophilicity of the thermostable alkaline phosphatase by site-directed mutagenesis}; Ji CN et al.; Through PCR-mediated mutagenesis, three mutants E68S, S70A and E68SS70A around active site S(69) were obtained . Their enzymatic characteristics was determined . It was found that the specific activity of E68S ascended 8 times while its optional reactive temperature climbed 20 degrees C and its Tm descended 3 degrees C; the specific activity of S70A ascended 1 time while its optional reactive temperature climbed 5 degrees C and its Tm descended 2 degrees C; the specific activity of E68SS70A descended 50% while its optional reactive temperature climbed 5 degrees C and its Tm descended 19 degrees C . These result implied that the amino acids, beside the active site, were contributed not only to enzymatic activity but also to its thermostability and thermophilicity . The work provided the direction for mutation to improve enzymatic specific activity and studying the mechanism of thermostability and thermophilicity.

Antonie Van Leeuwenhoek, 2000 Aug, 78(2), 141 - 51
Characterization by molecular cloning and sequencing of the gene encoding an aminopeptidase from Listeria monocytogenes; Winters DK et al.; The pepC gene of Listeria monocytogenes encodes aminopeptidase C that is predicted to share 72% amino acid sequence similarity and 53% sequence identity with the cysteine aminopeptidase PepC from Lactococcus lactis . The gene product also shows strong similarity to aminopeptidase C from Streptococcus thermophilus and Lactobacillus helveticus, and to a cysteine proteinase/bleomycin hydrolase from Saccharomyces cerevisiae . The enzyme from L . monocytogenes displayed broad N-terminal hydrolytic activity, with a similar substrate specificity to its lactic acid bacterial counterpart . The inhibition spectrum shows a great deal of similarity with enzymes from the family of lactic acid bacteria . In addition, one of the clones studied contained DNA sequences that could encode a regulatory protein of the deoR helix-turn-helix DNA binding protein family . The organization of the locus, designated pep, is presented along with the characterization of the gene products of the pep locus.

Tsitologiia, 2000, 42(11), 1103 - 10
{Mode of serotype inheritance in exoconjugant progeny of the ciliate Dileptus anser}; Uspenskaia ZI et al.; Two clones of Dileptus anser, originally isolated from natural reservoirs and referred to below as B and D clones, were found to display different serotypes, when cultured under identical laboratory conditions . On being tested with two different polyclonal rabbit immune sera against each particular clone (the classic immobilization test) these clones showed no cross-reaction . At a standard dilution (1:50) and at a standard exposure time (4 h), either of the two immune sera immobilized 100% or commonly 0% of homologous and heterologous clone cells, respectively . In addition, the difference in serotypes was confirmed by the immunofluorescence analysis . By crossing (conjugation) between B (mating type I) and D (mating type III) cells, exconjugant F1 clones were obtained . Their serotypes were then tested (the same immobilization test) with antisera against both the "parental" clones: some clones were tested before their sexual maturation in ca . one month after conjugation, while others were examined in approximately 4 months after conjugation, i.e . after reaching maturity . Each of the F1 clones could react with both immune sera, which means that they possessed the intermediate, "hybrid" phenotype . Five different F1 clones were selected, and each of them was back-crossed to both "parental" clones, B and D . We succeeded in raising 25 exconjugant F2 (B1, to be more exact) clones from F1 x B crosses and 26 clones from F1 x D crosses . The conventional testing of these clones in 5-10 weeks after conjugation provided quite unexpected results, since among them no segregation for "parental" serotypes was observed . Each of the 51 tested clones demonstrated the "hybrid" serotype--seemingly the same as that of F1 clones . Such a non-Mendelian inheritance of the character is hardly to explain from the standard, canonical assumptions on the genetic control of serotype difference between original "parental" clones (different alleles in one locus? different loci?) . Also it does not seem likely that the absence of segregation could result from differential survival of various phenotypes in F2 (although the total viability of exconjugant clones appeared rather low) . The above data obviously need further confirmations and experimental analyses . We attempt to discuss the obtained results in terms of the epigene hypothesis (Tchuraev, 1975) and in relation to the epigenetic control of serotype expression in species of the Paramecium aurelia complex and in Tetrahymena thermophila, which are "the chosen few" subjects in ciliate genetics.

Photochem Photobiol, 2001 Jan, 73(1), 90 - 5
Equal-quantum action spectra indicate fluence-rate-selective action of multiple photoreceptors for photomovement of the thermophilic cyanobacterium Synechococcus elongatus; Kondou Y et al.; Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55 degrees C . Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source . The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 mumol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 mumol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red) . At high fluence rates (30-90 mumol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700-740 nm (far-red) newly appeared . These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates . Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively . These results indicate involvement of some phytochrome(s).

Biocell, 2000 Dec, 24(3), 223 - 32
Gut mucosal immunostimulation by lactic acid bacteria; Vitini E et al.; The beneficial properties of lactic acid bacteria (LAB) on human health have been frequently demonstrated . The interaction of LAB with the lymphoid cells associated to the gut to activate the mucosal immune system and the mechanisms by which they can exert an adjuvant effect is still unclear, as well as if this property is common for all the LAB . We studied the influence of the oral administration of different geneous of LAB such as Lactobacillus casei, L . acidophilus, L . rhamnosus, L . delbrueckii subsp . bulgaricus, L . plantarum, Lactococcus lactis and Streptococcus thermophilus . We determined if the LAB assayed were able to stimulate the specific, the non-specific immune response (inflammatory response), or both . We demonstrated that all the bacteria assayed were able to increase the number of IgA producing cells associated to the lamina propria of small intestine . This effect was dose dependent . The increase in IgA+ producing cells was not always correlated with an increase in the CD4+ T cell number, indicating that some LAB assayed only induced clonal expansion of B cells triggered to produce IgA . Most of them, induced an increase in the number of cells involved in the inflammatory immune response . CD8+ T cell were diminished or not affected, with exception of L . plantarum that induced an increase at low dose . This fact would mean that LAB are unable to induce cytotoxicity mechanisms . We demonstrated the importance in the selection of LAB to be used as gut mucosal adjuvant . The different behaviours observed among them on the gut mucosal immune response, specially those that induce inflammatory immune response, show that not all the LAB can be used as oral adjuvant and that the beneficial effect of them can not generalized to genous or specie . The immunoadjuvant capacity would be a property of the strain assayed.

J Food Prot, 2001 Jan, 64(1), 81 - 6
The effects of cultivating lactic starter cultures with bacteriocin-producing lactic acid bacteria; Oumer A et al.; The effects of bacteriocins produced by six strains of lactic acid bacteria on 9 mesophilic and 11 thermophilic commercial starter cultures were investigated in mixed cultures of commercial starters with bacteriocin-producing strains in milk . The bacteriocins produced by the test organisms were nisin A, nisin Z, lacticin 481, enterocin AS-48, a novel enterocin, and a novel plantaricin . Mesophilic commercial starters were in most cases tolerant of bacteriocins, with only two of the starters being partially inhibited, one by four and the other by two bacteriocins . The aminopeptidase activities of mesophilic starters were generally low, and only one of the combinations of mesophilic starter-bacteriocin producer gave double the aminopeptidase activity of the starter culture without the bacteriocin producer . Thermophilic commercial starters were more sensitive to bacteriocins than mesophilic starters, with six thermophilic starters being partially inhibited by at least one of the bacteriocins . Their aminopeptidase activities were generally higher than those of the mesophilic starters . The aminopeptidase activities of seven thermophilic starters were increased in the presence of bacteriocins, by factors of up to 9.0 as compared with the corresponding starter cultures alone . Bacteriocin-producing strains may be used as adjunct cultures to mesophilic starters for the inhibition of pathogens in soft and semihard cheeses, because mesophilic starters are rather tolerant of bacteriocins . Bacteriocin producers may also be used as adjunct cultures to thermophilic starters of high aminopeptidase activity, more sensitive to lysis by bacteriocins than mesophilic starters, for the acceleration of ripening in semihard and hard cheeses.

Inorg Chem, 2000 Jul 10, 39(14), 3020 - 8
Mechanism of hydrogen peroxide dismutation by a dimanganese catalase mimic: dominant role of an intramolecular base on substrate binding affinity and rate acceleration; Boelrijk AE et al.; Several modifications of the manganese coordination environment and oxidation states of a family of synthetic dimanganese complexes have been introduced in search of the structural features that promote high rates of hydrogen peroxide dismutation (catalase activity) . The X-ray structure of reduced catalase (T thermophilus) reveals a dimanganese(II,II) site linked by three bridges: mu 13-glutamate-, mu-OH-, and mu-OH2 . The roles of a bridging hydroxide vs mu-aqua and the carboxylate have been examined in the reduced Mn2(II,II) complexes, {(L1,2)Mn2(mu-O2CCH3)(mu-X)}2+ for X- = OH- (7A) or X = H2O (1-4), and their oxidized Mn2(III,III) analogues, {(L1,2)Mn2(mu-O)(O2CCH3)(OH)}+ (6) (L1 is N,N,N',N'-tetrakis(2-methylenebenzamidazolyl)-1,3-diaminopropan- 2-ol, and L2 is the tetrakis-N-ethylated analogue of L1, which has all amine protons replaced by ethyl groups) . The steady-state catalase rate is first-order in concentration of both substrate and reduced catalyst and saturates at high peroxide concentrations in all cases, confirming peroxide/catalyst complex formation . No catalyst decomposition is seen after > 2000 turnovers . Catalysis proceeds via a ping-pong mechanism between the Mn2(II,II/III,III) redox states, involving complexes 6 and 7A/7A' . The Mn2(III,IV) oxidation state was not active in catalase activity . Replacement of the mu-aqua bridge by mu-hydroxide eliminates a kinetic lag phase in production of the O2 product, increases the affinity for substrate peroxide in the rate-limiting step as seen by a 5-fold . decrease in the Michaelis constant (KM), and accelerates the maximum rate (kcat) by 65-fold The kinetic and spectroscopic data are consistent with substrate deprotonation by the hydroxide bridge, yielding a hydroperoxyl bridge coordinated between the Mn ions (mu, eta 2 geometry, "end-on") as the basis for catalysis: mu-OH- + H2O2-->mu-O2H- + H2O . Binding of a second hydroxide ion to 7A causes a further increase in kcat by 4-fold with no further change in substrate affinity (KM) . By contrast, free (noncoordinating) bases in solution have no effect on catalysis, thus establishing intramolecular sites for both functional hydroxide anions . Solution structural studies indicate that the presence of 2-5 equiv of hydroxide in solution leads to formation of a bishydroxide species, {(L1,2)Mn2(mu 13-O2CCH3)(OH)2}, which in the presence of air or oxygen auto-oxidizes to yield complex 6, a Mn2(III,III)(mu-O) species . Complex 6 oxidizes H2O2 to O2 without a kinetic lag phase and is implicated as the active form of the oxidized catalyst . A maximum increase by 240-fold in catalytic efficiency (kcat/KM = 700 s-1 M-1) is observed with the bishydroxide species versus the aquo complex 1, or only 800-fold less efficient than the enzyme . Deprotonation of the amine groups of the chelate ligand L was shown not to be involved in the hydroxide effects because identical results were obtained using the catalyst with tetrakis(N-ethylated)-L . Uncoupling of the Mn(II) spins by protonation of the alkoxyl bridge (LH) was observed to lower the catalase activity . Comparisons to other dimanganese complexes reveals that the Mn2(II,II)/Mn2(III,III) redox potential is not the determining factor in the catalase rate of these complexes . Rather, rate acceleration correlates with the availability of an intramolecular hydroxide for substrate deprotonation and with binding of the substrate at the bridging site between Mn ions in the reductive O-O bond cleavage step that forms water and complex 6.

Avian Dis, 2000 Oct-Dec, 44(4), 993 - 9
National surveillance of Campylobacter in broilers at slaughter in Denmark in 1998; Wedderkopp A et al.; A surveillance study for thermophilic Campylobacter spp . in broiler flocks was carried out for the year 1998 in Denmark . The study included examinations of 4286 broiler flocks comprising samples from 57,000 birds . Overall, a flock prevalence of 46.0% was recorded . The species distribution was Campylobacter jejuni 86%, Campylobacter coli 11%, Campylobacter lari 1%, other not further diagnosed species 2% . The prevalence was significantly higher in the period from June to October (3.2 < odds ratio {OR} <1.8, P < 0.0002) and was significantly associated with abattoir (OR < 2.8, P < 0.0001) and the length of the period the broiler houses were left empty between flocks (download period; 6 days or more) (OR = 1.6, P < 0.0198) . No association between Campylobacter colonization and the age at slaughter was found . Separating the flocks into batches for slaughter elevated the flock prevalence from 0.41 after the first batch had been slaughtered to 0.46 after all batches had been slaughtered.

Arch Microbiol, 2000 Dec, 174(6), 415 - 21
Hydrogen production by methanogens under low-hydrogen conditions; Valentine DL et al.; Hydrogen production was studied in four species of methanogens (Methanothermobacter marburgensis, Methanosaeta thermophila, Methanosarcina barkeri, and Methanosaeta concilii) under conditions of low (sub-nanomolar) ambient hydrogen concentration using a specially designed culture apparatus . Transient hydrogen production was observed and quantified for each species studied . Methane was excluded as the electron source, as was all organic material added during growth of the cultures (acetate, yeast extract, peptone) . Hydrogen production showed a strong temperature dependence, and production ceased at temperatures below the growth range of the organisms . Addition of polysulfides to the cultures greatly decreased hydrogen production . The addition of bromoethanesulfonic acid had little influence on hydrogen production . These experiments demonstrate that some methanogens produce excess reducing equivalents during growth and convert them to hydrogen when the ambient hydrogen concentration becomes low . The lack of sustained hydrogen production by the cultures in the presence of methane provides evidence against "reverse methanogenesis" as the mechanism for anaerobic methane oxidation.

Arch Microbiol, 2000 Dec, 174(6), 406 - 14
A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii; Mukhopadhyay B et al.; The pyruvate carboxylase (PYC) of the hyperthermophilic, strictly hydrogenotrophic, autotrophic and marine methanarchaeon Methanococcus jannaschii was purified to homogeneity . Optimal activity was at pH 8.5, > or = 80 degrees C, and a KCl concentration of 0.175 M . This enzyme is the most thermophilic PYC so far studied . Unlike the Methanobacterium thermoautotrophicum enzyme, Mc . jannaschii PYC was expressed in cells grown without an external source of biotin and in the purified form was stable during storage at 4, -20 and -80 degrees C . However, it was rapidly inactivated at 80 degrees C . The enzyme was insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and was strongly inhibited by ATP and ADP (apparent Km, for ATP, 0.374 +/- 0.039 mM; apparent Ki for ATP, 5.34 +/- 2.14 mM; Ki for ADP, 0.89 +/- 0.18 mM) . It was also strongly inhibited when the Mg2+ concentration in the assay exceeded that of ATP . Thus, this stable PYC could serve as a model for mechanistic studies on archaeal PYCs . It was apparently an alpha4beta4-type PYC composed of a non-biotinylated 55.5-kDa subunit (PYCA) and a 64.2-kDa biotinylated subunit (PYCB) . The determined NH2-terminal sequences for these subunits provided additional support for our earlier proposal to rename the ORFs MJ1229 and MJ1231 in the NCBI Mc . jannaschii genome sequence database as PYCA and PYCB, respectively; even very recently, these have been misidentified as a subunit of acetyl-CoA carbxoylase (AccC) and the alpha-subunit of ion-pumping oxaloacetate decarboxylase (OADalpha), respectively.

Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2360 - 7
Purification and characterization of thermostable pectate lyase with protopectinase activity from thermophilic Bacillus sp . TS 47; Takao M et al.; A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated . It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60 degrees C on a medium containing polygalacturonate (PGA) . The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies . The molecular mass of the enzyme was 50 kDa by SDS-PAGE . The isoelectric point of the enzyme was pH 5.3 . The enzyme had a half-life of 13 and 1 h at 65 and 70 degrees C, respectively, and showed optimal activity around at 70 degrees C and pH 8.0 . It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers . The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity.

J Vet Med Sci, 2000 Dec, 62(12), 1291 - 5
Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter; Chuma T et al.; The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined . The multiplex PCR was able to detect type strains of the three species . All results of identification of wild strains (30 strains of C . jejuni, 20 strains of C . coli, and 4 strains of C . lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C . jejuni, C . coli, and C . lari from wild strains of campylobacters easily and rapidly . Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C . jejuni . Three out of 13 strains of C . jejuni isolated from sparrow feces showed quinolone resistance . From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C . jejuni in sparrows must have been originated from those industrial animals . Sparrows that have quinolone-resistant C . jejuni were considered to have contacted with industrial animals or thier feed . It may be presumed, on the contrary, that C . jejuni in sparrows could be a potential source of contamination of broilers.

Wei Sheng Wu Xue Bao, 1997 Oct, 37(5), 378 - 84
{Features of nine strains of thermophilic methanogens}; Gong G et al.; Nine strains of thermophilic bacteria producing methane from hydrogen and carbon dioxide or formate were isolated and purified from four samples of anaerobic digester feeding with family waste water . They are similar with each other on cell morphology and physiological characteristics . The cells are straight or slightly curved with rounded ends, 0.3-0.4 x 1-3 microns; single or in pairs, sometimes joined into long chains (> 10 microns); Gram-positive, non-mo-tile, non-spore-forming . The organisms under fluorescence microscope shine green fluorescence specific for methanogens and are chemoautotrophs . The temperature range for growth is 30-75 degrees C, optimum 55-65 degrees C . The initial pH range for growth is 5.8-9.0, optimum 6.9-7.6 . The G + C content of DNA of the strain 602B3 is 44.6 mol% . According to the results of identification, the strain 602B3 is considered of belonging to species Methanobacterium thermoformicicum 602B3.

Genome Biol . 2000;1(6):REVIEWS1029 . Epub 2000 Dec 04.
Extreme genomes; DeLong EF; The complete genome sequence of Thermoplasma acidophilum, an acid- and heat-loving archaeon, has recently been reported . Comparative genomic analysis of this 'extremophile' is providing new insights into the metabolic machinery, ecology and evolution of thermophilic archaea.

Gene, 2001 Jan 10, 262(1-2), 161 - 8
The ciliate Euplotes octocarinatus expresses two polypeptide release factors of the type eRF1; Liang A et al.; Amplification of macronuclear DNA of the ciliate Euplotes octocarinatus revealed the presence of two genes encoding putative polypeptide release factors (RFs) of the codon specific class-I type . They are named eRF1a and eRF1b, respectively . cDNA amplification revealed that both eRF1 genes are expressed . Determination of their copy numbers showed that they are similarly amplified to a level of about 27,000 . The deduced protein sequences of the two genes are 57 and 58% identical with human eRF1 and 79% identical to each other . The gene encoding eRF1b possesses three in-frame UGA codons . This codon is known to encode cysteine in Euplotes; only UAA and UAG are used as stop codons in this organism . The primary structure of the two release factors is analyzed and compared with the primary structure of other eukaryotic release factors including the one of Tetrahymena thermophila which uses only UGA as a stop codon . eRF1a and eRF1b of Euplotes as well as eRF1 of Tetrahymena differ from human eRF1 and other class-I release factors of eukaryotes in a domain recently proposed to be responsible for codon recognition . Based on the changes which we observe in this region and the differential use of the stop codons in these two ciliates we predict the amino acids participating in stop codon recognition in eRF1 release factors.

Infect Immun, 2001 Mar, 69(3), 1373 - 80
Genetic loci of Streptococcus mitis that mediate binding to human platelets; Bensing BA et al.; The direct binding of bacteria to platelets is a postulated major interaction in the pathogenesis of infective endocarditis . To identify bacterial components that mediate platelet binding by Streptococcus mitis, we screened a Tn916deltaE-derived mutant library of S . mitis strain SF100 for reduced binding to human platelets in vitro . Two distinct loci were found to affect platelet binding . The first contains a gene (pblT) encoding a highly hydrophobic, 43-kDa protein with 12 potential membrane-spanning segments . This protein resembles members of the major facilitator superfamily of small-molecule transporters . The second platelet binding locus consists of an apparent polycistronic operon . This region includes genes that are highly similar to those of Lactococcus lactis phage r1t and Streptococcus thermophilus phage 01205 . Two genes (pblA and pblB) encoding large surface proteins are also present . The former encodes a 107-kDa protein containing tryptophan-rich repeats, which may serve to anchor the protein within the cell wall . The latter encodes a 121-kDa protein most similar to a tail fiber protein from phage 01205 . Functional mapping by insertion-duplication mutagenesis and gene complementation indicates that PblB may be a platelet adhesin and that expression of PblB may be linked to that of PblA . The combined data indicate that at least two genomic regions contribute to platelet binding by S . mitis . One encodes a probable transmembrane transporter, while the second encodes two large surface proteins resembling structural components of lysogenic phages.

J Mol Biol, 2001 Feb 23, 306(3), 513 - 25
A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL apical domain with implications for substrate interactions; Hua Q et al.; A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus . The domains share 70 % sequence identity (101 out of 145 residues) . The thermal stability of the T . thermophilus apical domain (Tm>100 degrees C as evaluated by circular dichroism) is at least 35 degrees C greater than that of the E . coli apical domain (Tm=65 degrees C) . The crystal structure of a selenomethione-substituted apical domain from T . thermophilus was determined to a resolution of 1.78 A using multiwavelength-anomalous-diffraction phasing . The structure is similar to that of the E . coli apical domain (root-mean-square deviation 0.45 A based on main-chain atoms) . The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds . Only one of the additional salt bridges would face the "Anfinsen cage" in GroEL . High temperatures were exploited to map sites of interactions between the apical domain and molten globules . NMR footprints of apical domain-protein complexes were obtained at elevated temperature using 15N-1H correlation spectra of 15N-labeled apical domain . Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50 degrees C) are essentially the same and consistent with the peptide-binding surface previously defined in E . coli GroEL and its apical domain-peptide complexes . An additional part of this surface comprising a short N-terminal alpha-helix is observed . The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the "classical" peptide-binding site . Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.

J Mol Biol, 2001 Feb 9, 306(1), 47 - 67
Crystal structure of auracyanin, a "blue" copper protein from the green thermophilic photosynthetic bacterium Chloroflexus aurantiacus; Bond CS et al.; Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloroflexus aurantiacus, crystallizes in space group P6(4)22 (a=b=115.7 A, c=54.6 A) . The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was refined at 1.55 A resolution . The molecular model comprises 139 amino acid residues, one Cu, 247 H(2)O molecules, one Cl(-) and two SO(4)(2-) . The final residual and estimated standard uncertainties are R=0.198, ESU=0.076 A for atomic coordinates and ESU=0.05 A for Cu---ligand bond lengths, respectively . The auracyanin B molecule has a standard cupredoxin fold . With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin . As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8 . The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands . The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins .

Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 310 - 3
Crystallization and preliminary X-ray analysis of native and selenomethionine fructose-1,6-bisphosphate aldolase from Thermus aquaticus; Sauve V et al.; Fructose-1,6-bisphosphate aldolase (E.C . 4.1.2) catalyses the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in the glycolytic pathway of prokaryote and eukaryote organisms . The enzyme was obtained from the extreme thermophile Thermus aquaticus and, in contrast to mesophilic aldolases, expresses maximal activity in the presence of Co(2+) as cofactor instead of Zn(2+) . The purified recombinant protein was monodisperse according to dynamic light-scattering measurements . Crystals of recombinant native class II fructose-1,6-bisphosphate aldolase from T . aquaticus were obtained from two different starting conditions at low protein concentrations . Condition I, using the sitting-drop vapour-diffusion method, yielded monoclinic crystals having space group P2 and unit-cell parameters a = 99.5, b = 57.5, c = 138.6 A, beta = 90.25 degrees . Diffraction data were collected to 2 A resolution at beamline X8-C of the NSLS synchrotron-radiation source . Native and selenomethionine-substituted protein crystals were obtained from condition II by hanging-drop vapor diffusion . The tetragonal crystals of the native protein belong to the space group P4(1), with unit-cell parameters a = b = 88.8, c = 163.1 A, while those of the SeMet protein have space group I4(1), with unit-cell parameters a = b = 88.6, c = 164.1 A . A data set suitable for MAD phasing was collected to 2.6 A resolution at beamline X8-C of the NSLS synchrotron source.

Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 272 - 5
Gene cloning, expression, crystallization and preliminary X-ray analysis of Thermus thermophilus arginyl-tRNA synthetase; Shimada A et al.; The gene encoding the highly thermostable arginyl-tRNA synthetase (ArgRS) from Thermus thermophilus was cloned and overexpressed in Escherichia coli under the control of the T7 promoter . The recombinant ArgRS was purified by two chromatographic steps and was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 and ethylene glycol as precipitants . The crystals belong to the hexagonal space group P6(5), with unit-cell parameters a = b = 156.04 (7), c = 87.17 (4) A . X-ray data to 2.8 A resolution were collected at room temperature from a native crystal using an in-house X-ray source . Uranium, platinum and selenomethionine derivatives were found to be useful for phasing by the multiple isomorphous replacement method with anomalous scattering . The flash-frozen crystals diffracted beyond 2.3 A resolution using synchrotron radiation from the beamline 41XU at SPring-8 (Harima).

Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 225 - 32
Design, X-ray crystallography, molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3-isopropylmalate dehydrogenase from Thermus thermophilus; Qu C et al.; The relationship between the structure and the thermostability of the 3-isopropylmalate dehydrogenase from Thermus thermophilus was studied by site-directed mutation of a single Ala residue located at the domain interface . The crystal structures of three mutant enzymes, replacing Ala172 with Gly, Val and Phe, were successfully determined at 2.3, 2.2 and 2.5 A resolution, respectively . Substitution of Ala172 by relatively 'short' residues (Gly, Val or Ile) enlarges or narrows the cavity in the vicinity of the C(beta) atom of Ala172 and the thermostablity of the enzyme shows a good correlation with the hydrophobicity of the substituted residues . Substitution of Ala172 by the 'longer' residues Leu or Phe causes a rearrangement of the domain structure, which leads to a higher thermostability of the enzymes than that expected from the hydrophobicity of the substituted residues . Mutation of Ala172 to negatively charged residues gave an unexpected result: the melting temperature of the Asp mutant enzyme was reduced by 2.7 K while that of the Glu mutant increased by 1.8 K . Molecular-modelling studies indicated that the glutamate side chain was sufficiently long that it did not act as a buried charge as did the aspartate, but instead protruded to the outside of the hydrophobic cavity and contributed to the stability of the enzyme by enhancing the packing of the local side chains and forming an extra salt bridge with the side chain of Lys175.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1442 - 7 Epub 2001 Feb 06.
Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8; Yamada K et al.; We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination . RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis . DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain . The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.

J Exp Biol, 2001 Feb, 204(Pt 4), 741 - 50
Anaerobic sulfur metabolism in thiotrophic symbioses; Arndt C et al.; Hydrogen sulfide is generally accepted to be the energy source for the establishment of sulfur-oxidizing symbiotic communities . Here, we show that sulfur-storing symbioses not only consume but also produce large amounts of hydrogen sulfide . The prerequisite for this process appears to be the absence of oxygen . Anaerobic sulfide production is widespread among different thiotrophic symbioses from vent and non-vent sites (Riftia pachyptila, Calyptogena magnifica, Bathymodiolus thermophilus, Lucinoma aequizonata and Calyptogena elongata) . The extent of H2S generation correlates positively with the amount of elemental sulfur stored in the symbiont-bearing tissues of the hosts . Sulfide production starts a few hours after anoxia sets in, with H2S initially accumulating in the circulatory system before it is excreted into the surrounding environment . We propose that not sulfate but the elemental sulfur deposited in the symbionts serves as a terminal electron acceptor during anoxia and is reduced to sulfide . In anoxia-tolerant symbioses such as L . aequizonata, anaerobic sulfur respiration may be important for producing maintenance energy to help the species survive several months without oxygen . The increased levels of cysteine in the gills of L . aequizonata may be caused by a lack of reoxidation due to the absence of oxygen.

Clin Infect Dis, 2001 Jan 15, 32(2), 295 - 6 Epub 2001 Jan 15.
Thermophilic multidrug-resistant Campylobacter fetus infection with hypersplenism and histiocytic phagocytosis in a patient with acquired immunodeficiency syndrome; Anstead G et al.; We present a case report of a patient who had acquired immunodeficiency syndrome (AIDS) and Campylobacter fetus infection with a number of unusual clinical and microbiological features . The patient had prominent gastrointestinal symptoms, splenic infarction, splenomegaly with hypersplenism, and hemophagocytic histiocytosis in the spleen and lymph nodes; the organism displayed growth on Campy-selective blood agar, thermotolerance, and resistance to quinolones, piperacillin/tazobactam, ceftazidime, and erythromycin.

Biotechnol Prog, 2001 Jan-Feb, 17(1), 118 - 25
Salt accumulation resulting from base added for pH control, and not ethanol, limits growth of Thermoanaerobacteriumthermosaccharolyticum HG-8 at elevated feed xylose concentrations in continuous culture; Lynd LR et al.; Thermoanaerobacter thermosaccharolyticum HG-8 was grown in continuous culture to characterize growth limitation at high feed substrate and product concentrations . Continuous fermentation of 50 and 73 g/L xylose at a dilution rate based on the feed flow, D(f), of 0.053 h(-)(1) and with the pH controlled at 7.0 by addition of KOH resulted in steady state utilization of >99% of the xylose fed and production of ethanol and acetic acid at a mass ratio of about 2:1 . Continuous cultures of T . thermosaccharolyticum growing at D(f) = 0.053 h(-)(1) achieved complete utilization of 75 g/L xylose in the presence of 19.1 g/L K(+) (0.49 M) and an ethanol concentration of 22.4 g/L ethanol . When the feed to a culture initially at steady state with a 75 g/L xylose feed and D(f) = 0.053 h(-)(1) was increased to 87.5 g/L xylose, limitation of growth and xylose utilization was observed . This limitation was not relieved by repeating this feed upshift experiment in the presence of increased nutrient levels and was not reproduced by addition of ethanol to a steady-state culture fed with 75 g/L xylose . By contrast, addition of KCl to a steady-state culture fed with 75 g/L xylose reproduced the K(+) concentration, limitation of growth and xylose utilization, and product concentration profiles observed in the feed upshift experiment . The maximum concentration at which growth of batch cultures was observed was 0.43 M for KCl, NaCl, and equimolar mixtures of these salts, suggesting that the observed limitation is not ion-specific . These data support the interpretation that inhibition salt accumulation resulting from addition of KOH for pH control is the limiting factor manifested in the feed upshift experiment and that both nutrient limitation and ethanol inhibition played little or no role as limiting factors . More generally, salt inhibition would appear to be a possible explanation for the discrepancy between the tolerance to added ethanol and the maximum concentration of produced ethanol reported in the literature for fermentation studies involving thermophilic bacteria.

Biochemistry, 2001 Feb 6, 40(5), 1144 - 9
Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase; Chen JF et al.; Leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that catalyzes leucylation of tRNA(Leu) . Several mutants in the CP1 domain of Escherichia coli LeuRS were obtained by introduction of restriction endonuclease sites into its gene, leuS . Of these mutants, only LeuRS-A293F had decreased activity (46%) compared to the native enzyme . To investigate the effect of A293 on enzyme function, A293 was mutated to Y, G, I, R, or D . The mutants were impaired in activity and editing function to varying extents . The decrease in K(m) values for three substrates showed that the binding of ATP to these mutants became much stronger . The inhibition of ATP binding to most of the mutants was also stronger . In particular, LeuRS-A293D had the lowest activity, the strongest ATP binding, and the most impaired editing function . A red shift of the fluorescence emission maximum of LeuRS-A293D indicated a less hydrophobic chromophore environment and a relatively more flexible dynamic conformation . The change in T(m) of LeuRS-A293D was higher than that of all other substitutions . Evidence from sequence alignment and crystal structure of LeuRS from Thermus thermophilus shows that A293 was conserved as R (K) or A and is located at a small helix in the editing domain of the enzyme facing the active site . Hence, any amino acid substitution of A293 may affect the stability of the helix, which may lead to impaired editing function and aminoacylation activity and may be indirectly involved in ATP binding.

Lett Appl Microbiol, 2001 Jan, 32(1), 31 - 5
Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans; Narang S et al.; AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions . METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks . Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively . Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium . The increase in enzyme production was achieved using vitamins and amino acids . When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h . CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.

J Pept Res, 2001 Jan, 57(1), 39 - 47
NMR and CD conformational studies of the C-terminal 16-peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes; Bouchayer E et al.; The 16-amino acid sequences of the C-terminal helices of the homologous bacterial cytochromes c551 from Pseudomonas aeruginosa and C552 from Hydrogenobacter thermophilus were synthesized and their solution structure studied . Circular dichroism and NMR experiments in aqueous solution have shown the presence of alpha-helices and 3(10)-helices . The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c551 and 20% for c552, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2-trifluoroethanol . An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2-trifluoroethanol . NMR spectra in phosphate buffer show the presence of both alpha- and 3(10)-helical structures . In water/2,2,2-trifluoroethanol (50:50) alpha-helices are predominant . CD temperature-dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2-trifluoroethanol (50:50) . The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c552 C-terminal helix at the local level . Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.

J Pept Res, 2001 Jan, 57(1), 19 - 28
The metal binding properties of the CCCH motif of the 50S ribosomal protein L36 from Thermus thermophilus; Boysen RI et al.; The TthL36 protein of the 50S ribosomal proteins from Thermus thermophilus has been found to contain the rare C(Xaa)2C(Xaa)12C(Xaa)4H (CCCH) sequence motif, a zinc finger binding motif, which for other zinc finger proteins is known to cleave RNA hairpins . In order to investigate the metal-binding properties of this T . thermophilus TthL36 protein, the core 26-mer polypeptide containing this CCCH motif was prepared by solid-phase peptide synthesis methods using Fmoc chemistry, purified by preparative RP-HPLC and characterized by circular dichroism, high-performance capillary zone electrophoresis and electrospray ionization mass spectrometry . Reaction of the acetamidomethyl (Acm)-protected polypeptide with iodine under acidic conditions resulted in the formation of the fully de-protected polypeptide . Of interest, the results demonstrate that the standard Acm-deprotection method with the synthetic TthL36 polypeptide using mercuric acetate in the presence of a large excess of 2-mercaptoethanol resulted in preferential formation of a very stable mercuro-polypeptide complex . The properties of the Acm-deprotected polypeptide in the presence of different metal ions were also investigated by spectroscopic methods . The results confirm that this TthL36 polypeptide containing the CCCH motif binds metal ions with different affinities, namely in the order Co(II)>Hg(II)>Zn(II).

J Appl Microbiol, 2001 Feb, 90(2), 256 - 67
The effects of UVB and temperature on the survival of natural populations and pure cultures of Campylobacter jejuni, Camp . coli, Camp . lari and urease-positive thermophilic campylobacters (UPTC) in surface waters; Obiri-Danso K et al.; AIMS: To determine whether diurnal and seasonal variations in campylobacters in surface waters result from the effects of temperature and u.v . radiation, and whether natural populations of Campylobacter lari and urease-positive thermophilic campylobacters (UPTC) from birds survive better in surface waters than Camp . jejuni from sewage . METHODS AND RESULTS: Natural populations of Camp . lari and UPTC in sea water, and Camp . jejuni in river water, were exposed to artificial sunlight (equivalent to a sunny day in June) . Both populations became non-culturable within 30 min, with T90s of 15 min and 25 min, respectively . Cultures of Camp . jejuni became non-culturable within 40 min and those of Camp . coli, Camp . lari and UPTC, within 60 min . In darkness, survival was temperature-dependent . Natural populations took 12 h at 37 degrees C and 5 days at 4 degrees C to become non-culturable in sea water, and slightly less in river water . Cultures of Camp . lari and UPTCs survived for significantly longer than Camp . jejuni and Camp . coli . Loss of culturability for all isolates was most rapid at 37 degrees C and slowest at 4 degrees C . Newly isolated strains from sea water and river water behaved in an almost identical manner to NCTC strains . CONCLUSION: Campylobacter lari and UPTCs survive for longer in surface waters than Camp . jejuni and Camp . coli, particularly in the dark . Low Campylobacter numbers in coastal waters in the summer, especially in the afternoon, are due to the combined effects of higher temperatures and higher levels of u.v . radiation . SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter lari and UPTCs from birds predominate in bathing waters in Morecambe Bay because they are better able to survive; they also originate from closer to the shore than Camp . jejuni and Camp . coli in sewage effluent, which survive poorly and die before the incoming tide reaches the shore . The predominance of Camp . jejuni in river water results from its dominance of the inputs and not from its ability to survive.

J Appl Microbiol, 2001 Feb, 90(2), 151 - 7
The growth of Bacillus stearothermophilus on stainless steel; Flint S et al.; AIMS: To determine the potential for Bacillus stearothermophilus cells to form biofilms of significance in dairy manufacture . METHODS AND RESULTS: The ability of isolates of B . stearothermophilus from dairy manufacturing plants to attach to stainless steel surfaces was demonstrated by exposing stainless steel samples to suspensions of spores or vegetative cells and determining the numbers attaching using impedance microbiology . Spores attached more readily than vegetative cells . The attachment of cells to stainless steel was increased 10-100-fold by the presence of milk fouling the stainless steel . The growth of B . stearothermophilus as a biofilm on stainless steel surfaces was determined using a continuously flowing experimental reactor . Vegetative cells were released in greater numbers than spores from biofilms of most strains studied . Biofilms of one strain (B11) were studied in detail . Biofilms of > 106 cells cm-2 formed in the reactor and released approximately 106 cells ml-1 into milk passing over the biofilm . A doubling time of 25 min was calculated for this organism grown as a biofilm . CONCLUSION: The formation of biofilms of thermophilic Bacillus species within the plant appears to be a likely cause of contamination of manufactured dairy products . Methods to control the formation of biofilms in dairy manufacturing plants are required to reduce the contamination of dairy products with thermophilic bacilli . SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms of B . stearothermophilus growing in dairy manufacturing plants can explain the contamination of dairy products with these bacteria.

Curr Opin Biotechnol, 2001 Feb, 12(1), 99 - 104
Enzyme fluorescence as a sensing tool: new perspectives in biotechnology; D'Auria S et al.; The technology for fluorescence protein-sensing is advancing rapidly owing to the continued introduction of new concepts, new fluorophores, and proteins engineered for sensing-specific analytes . Concerns about the reversibility and selectivity of engineered proteins are being addressed by developing biosensors that are based on the utilisation of coenzyme-depleted enzymes . Such biomolecules do not consume the substrate and can exhibit conformational changes upon the binding of the analyte, which can be easily detected as fluorescence change . In addition, concerns about the stability of biosensors can be overcome by using thermostable enzymes isolated from thermophilic microorganisms . Finally, the development of new techniques such as polarization-based sensing, anisotropy-based sensing and lifetime-based sensing, all of which can be accomplished with light-emitting diodes as the light source, is prompting the design of a new class of specific and stable biosensors, as has occurred with blood glucose measurement . These biosensors represent a valid alternative to the conventional clinical chemistry diagnostics.

Gene, 2000 Dec 31, 261(2), 299 - 303
Identification and characterization of the dnaA upstream region of Thermus thermophilus; Nardmann J et al.; The gene order in the dnaA region of Thermus thermophilus was determined . Previously, we showed that the putative oriC of T . thermophilus is located in the dnaA-dnaN intergenic region . In the 4 kb region upstream of the dnaA gene four ORFs were found, all orientated in the same direction which is opposite to that of dnaA . The ORFs were identified as T . thermophilus homologs of gidA, gidB, soj and spo0J of Bacillus subtilis . The gene order spo0J-soj-gidB-gidA-dnaA-dnaN resembles that of B . subtilis, Pseudomonas putida, Coxiella burnetii, Streptomyces coelicolor, Mycobacterium leprae, and Mycobacterium tuberculosis . We identified the transcriptional start point of the dnaA gene . The -10 region shows significant homology to the Escherichia coli -10 consensus sequence . The putative -35 region shows homology neither to the E . coli -35 consensus sequence nor to known -35 sequences of T . thermophilus . There are no DnaA boxes in the promoter region, and consequently dnaA transcription is not repressed by DnaA protein in vitro, i.e . the dnaA gene of T . thermophilus is not autoregulated.

FEMS Microbiol Lett, 2001 Feb 5, 195(1), 47 - 51
Selective extraction of subunit D of the Na(+)-translocating methyltransferase and subunit c of the A(1)A(0) ATPase from the cytoplasmic membrane of methanogenic archaea by chloroform/methanol and characterization of subunit c of Methanothermobacter thermoautotrophicus as a 16-kDa proteolipid; Ruppert C et al.; Chloroform/methanol was applied to cytoplasmic membranes of the thermophilic methanogens Methanothermobacter thermoautotrophicus and Methanothermobacter marburgensis as well as to the mesophile Methanosarcina mazei Go1 . In any case, the chloroform/methanol extraction yielded only two proteins, subunit D (MtrD) of the Na(+)-translocating methyltetrahydromethanopterin:coenzyme M methyltransferase and the proteolipid of the A(1)A(0) ATPase . Both polypeptides are assumed to be directly involved in ion translocation in their respective enzymes, but have not been studied in detail due to lack of simple isolation procedures . The rapid and selective isolation by chloroform/methanol offers a new way to obtain the large quantities of material required for biochemical analyses . As a first result, molecular and biochemical data suggest that the proteolipid from M . thermoautotrophicus is a duplication of the 8-kDa proteolipid usually present in other archaea, but it retained the conserved glutamate involved in proton translocation in every copy . This is the first 16-kDa proteolipid found in archaea.

FEMS Microbiol Lett, 2001 Feb 5, 195(1), 17 - 22
Cloning and expression of thermophilic catechol 1,2-dioxygenase gene (catA) from Streptomyces setonii; An HR et al.; Streptomyces setonii (ATCC 39116) degrades various single aromatic compounds such as phenol or benzoate via an ortho-cleavage pathway using catechol 1,2-dioxygenase (C12O) . A PCR using degenerate primers based on the conserved regions of known C12O-encoding genes amplified a 0.45-kbp DNA fragment from S . setonii total DNA . A Southern hybridization analysis and size-selected DNA library screening using the 0.45-kbp PCR product as a probe led to the isolation of a 6.4-kbp S . setonii DNA fragment, from which the C12O-encoding genetic locus was found to be located within a 1.4-kbp DNA fragment . A complete nucleotide sequencing analysis of the 1.4-kbp DNA fragment revealed a 0.84-kbp open reading frame, which showed a strong overall amino acid similarity to the known high-G+C Gram-positive (but significantly less to the Gram-negative) bacterial mesophilic C12Os . The heterologous expression of the cloned 1.4-kbp DNA fragment in Escherichia coli demonstrated that this C12O possessed a thermophilic activity within a broad temperature range (up to 65 degrees C) and showed a higher activity against 3-methylcatechol than catechol or 4-methylcatechol, but no activity against protocatechuate.

Trends Microbiol, 2001 Jan, 9(1), 39 - 43
Viruses of the extremely thermophilic archaeon Sulfolobus; Prangishvili D et al.; Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence . Certain characteristics of the replication strategies of these viruses and the virus-host interactions suggest relationships with eukaryal and bacterial viruses, and the primeval existence of common ancestors . Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms . The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future.

FEMS Microbiol Lett, 2001 Jan 15, 194(2), 201 - 6
Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius; Gueguen Y et al.; We report the molecular characterization and the detailed study of the recombinant maltooligosyl trehalose synthase mechanism from the thermoacidophilic archaeon Sulfolobus acidocaldarius . The mts gene encoding a maltooligosyl trehalose synthase was overexpressed in Escherichia coli using the T7-expression system . The purified recombinant enzyme exhibited optimum activity at 75 degrees C and pH 5 with citrate-phosphate buffer and retained 60% of residual activity after 72 h of incubation at 80 degrees C . The recombinant enzyme was active on maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose and maltoheptaose . Investigation of the enzyme action on maltooligosaccharides has brought much insight into the reaction mechanism . Results obtained from thin-layer chromatography suggested a possible mechanism of action for maltooligosyl trehalose synthase: the enzyme, after converting the alpha-1,4-glucosidic linkage to an alpha-1,1-glucosidic linkage at the reducing end of maltooligosaccharide glc(n) is able to release glucose and maltooligosaccharide glc(n-1) residues . And then, the intramolecular transglycosylation and the hydrolytic reaction continue, with the maltooligosaccharide glc(n-1) until the initial maltooligosaccharide is reduced to maltose . An hypothetical mechanism of maltooligosyl trehalose synthase acting on maltooligosaccharide is proposed.

Gene, 2000 Dec 30, 261(1), 189 - 95
The late stage of genetic code structuring took place at a high temperature; Di Giulio M; The correlation between the optimal growth temperature of organisms and a thermophily index based on the propensity of amino acids to enter more frequently into (hyper)thermophile proteins is used to conduct an analysis aiming to establish whether genetic code structuring took place at a low or a high temperature . If the number of codons attributed to the various amino acids in the genetic code constitutes an estimate of the mean amino acid composition of proteins produced when the genetic code was definitively structured, then the thermophily index can also be associated to the genetic code . This value and the sampling of the variable thermophily index of different alignments of protein sequences from mesophile, thermophile and hyperthermophile species make it possible to establish, with an extremely high statistical confidence, that the late stage of genetic code structuring took place in a hyperthermophile (or thermophile) 'organism' . Moreover the 95% confidence interval of the temperature at which the genetic code was fixed turned out to be 91+/-24 degrees C . These observations seem to support the hypothesis that the origin of life might have taken place at a high temperature.

J Mol Biol, 2001 Feb 2, 305(5), 1173 - 83
Regulation of ATPase and chaperone cycle of DnaK from Thermus thermophilus by the nucleotide exchange factor GrpE; Groemping Y et al.; The nucleotide binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ, also called co-chaperones . The concerted action of the nucleotide exchange factor GrpE and the ATPase-stimulating factor DnaJ determines the ratio of the two nucleotide states of DnaK, which differ in their mode of interaction with unfolded proteins . In the Escherichia coli system, the stimulation by these two antagonists is comparable in magnitude, resulting in a balance of the two nucleotide states of DnaK(Eco) in the absence and the presence of co-chaperones.The regulation of the DnaK chaperone system from Thermus thermophilus is apparently substantially different . Here, DnaJ does not stimulate the DnaK-mediated ATP hydrolysis and thus does not appear to act as an antagonist of the nucleotide exchange factor GrpE(Tth) . This raises the question of whether T . thermophilus GrpE stimulates nucleotide exchange to a smaller degree as compared to the E . coli system and how the corresponding rates relate to intrinsic ATPase and ATP binding as well as luciferase refolding kinetics of T . thermophilus DnaK.We determined dissociation constants as well as kinetic constants that describe the interactions between the T . thermophilus molecular chaperone DnaK, its nucleotide exchange factor GrpE and the fluorescent ADP analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine-5'-diphosphate by isothermal equilibrium titration calorimetry and stopped-flow kinetic experiments and investigated the influence of T . thermophilus DnaJ on the DnaK nucleotide cycle.The interaction of GrpE with the DnaK.ADP complex versus nucleotide-free DnaK can be described by a simple equilibrium system, where GrpE reduces the affinity of DnaK for ADP by a factor of about 10 . Kinetic experiments indicate that the maximal acceleration of nucleotide release by GrpE is 80,000-fold at a saturating GrpE concentration.Our experiments show that in T . thermophilus, although the thermophilic DnaK system displays no stimulation of the DnaK-ATPase activity by DnaJ, nucleotide exchange is still efficiently stimulated by GrpE . This indicates that two counteracting factors are not absolutely necessary to maintain a functional and regulated chaperone cycle . This conclusion is corroborated by data that show that the slower ATPase cycle of the DnaK system as well as of heterologous T . thermophilus DnaK/E . coli DnaK systems is directly reflected in altered refolding kinetics of firefly luciferase but not necessarily in refolding yields.

J Mol Biol, 2001 Jan 26, 305(4), 785 - 803
Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15; Serganov A et al.; Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions . Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites . The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting . The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E . coli complex and facilitated interpretation of biochemical data . Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking . Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry . The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated . Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction . Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation . Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites . In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18 . Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly .

Anal Biochem, 2001 Feb 15, 289(2), 103 - 15
Determination of macromolecular folding and structure by synchrotron x-ray radiolysis techniques; Maleknia SD et al.; Radiolysis of water by synchrotron X-rays generates oxygen-containing radicals that undergo reactions with solvent accessible sites of macromolecules inducing stable covalent modifications or cleavage on millisecond time scales . The extent and site of these reactions are determined by gel electrophoresis and mass spectrometry analysis . These data are used to construct a high-resolution map of solvent accessibility at individual reactive sites . The experiments can be performed in a time-resolved manner to provide kinetic rate constants for dynamic events occurring at individual sites within macromolecules or can provide equilibrium parameters of binding and thermodynamics of folding processes . The application of this synchrotron radiolysis technique to the study of lysozyme protein structure and the equilibrium urea induced unfolding of apomyoglobin are described . The Mg2+-induced folding of Tetrahymena thermophila group I ribozyme shows the capability of the method to study kinetics of folding .

Protein Eng, 2000 Nov, 13(11), 753 - 61
Aromatic clusters: a determinant of thermal stability of thermophilic proteins; Kannan N et al.; A number of factors have been elucidated as responsible for the thermal stability of thermophilic proteins . However, the contribution of aromatic interactions to thermal stability has not been systematically studied . In the present investigation we used a graph spectral method to identify aromatic clusters in a dataset of 24 protein families for which the crystal structures of both the thermophilic and their mesophilic homologues are known . Our analysis shows a presence of additional aromatic clusters or enlarged aromatic networks in 17 different thermophilic protein families, which are absent in the corresponding mesophilic homologue . The additional aromatic clusters identified in the thermophiles are smaller in size and are largely found on the protein surface . The aromatic clusters are found to be relatively rigid regions of the surface and often the additional aromatic cluster is located close to the active site of the thermophilic enzyme . The residues in the additional aromatic clusters are preferably mutated to Leu, Ser or Ile in the mesophilic homologue . An analysis of the packing geometry of the pairwise aromatic interaction in the additional aromatic clusters shows a preference for a T-shaped orthogonal packing geometry . The present study also provides new insights for protein engineers to design thermostable and thermophilic proteins.

Nucleic Acids Res, 2001 Feb 15, 29(4), 904 - 13
Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein; Lipps G et al.; There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles . Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus . orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein . The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements . Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56 . Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication . By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein . Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site . CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA . ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range . Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of -6.2 kJ/mol . Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85 degrees C.

Nucleic Acids Res, 2001 Feb 15, 29(4), 880 - 5
Characterization of RNase P from Thermotoga maritima; Paul R et al.; The protein subunit of RNase P from a thermophilic bacterium, Thermotoga maritima, was overexpressed in and purified from Escherichia coli . The cloned protein was reconstituted with the RNA subunit transcribed in vitro . The temperature optimum of the holoenzyme is near 50 degrees C, with no enzymatic activity at 65 degrees C or above . This finding is in sharp contrast to the optimal growth temperature of T.maritima, which is near 80 degrees C . However, in heterologous reconstitution experiments in vitro with RNase P subunits from other species, we found that the protein subunit from T.maritima was responsible for the comparative thermal stability of such complexes.

Nucleic Acids Res, 2001 Feb 1, 29(3), 644 - 51
Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations; Chopin A et al.; We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403 . The three larger prophages (36-42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13-15 kb) are most probably satellites relying on helper phage(s) for multiplication . These data give a new insight into the genetic structure of lactococcal phage populations . P335 temperate phages have variable genomes, sharing homology over only 10-33% of their length . In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length . Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, MYCOBACTERIUM: and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life . This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues . We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.

J Bacteriol, 2001 Mar, 183(5), 1792 - 5
New host-vector system for Thermus spp . based on the malate dehydrogenase gene; Kayser KJ et al.; A Thermus thermophilus HB27 strain was constructed in which the malate dehydrogenase (mdh) gene was deleted . The Deltamdh colonies are recognized by a small-colony phenotype . Wild-type phenotype is restored by transformation with Thermus plasmids or integration vector containing an intact mdh gene . The wild-type phenotype provides a positive selection tool for the introduction of plasmid DNA into Thermus spp., and because mdh levels can be readily quantified, this host-vector system is a convenient tool for monitoring gene expression.

J Bacteriol, 2001 Mar, 183(5), 1560 - 7
Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin; Das A et al.; A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced . Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin) . Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub . The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified . The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms . Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C . thermoaceticum, indicating that this protein was expressed in the native organism . Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C . thermoaceticum.

Mol Cell Biol, 2001 Feb, 21(4), 990 - 1000
RNA binding domain of telomerase reverse transcriptase; Lai CK et al.; Telomerase is a ribonucleoprotein reverse transcriptase that extends the ends of chromosomes . The two telomerase subunits essential for catalysis in vitro are the telomerase reverse transcriptase (TERT) and the telomerase RNA . Using truncations and site-specific mutations, we identified sequence elements of TERT and telomerase RNA required for catalytic activity and protein-RNA interaction for Tetrahymena thermophila telomerase . We found that the TERT amino and carboxyl termini, although evolutionarily poorly conserved, are nonetheless important for catalytic activity . In contrast, high-affinity telomerase RNA binding requires only a small region in the amino terminus of TERT . Surprisingly, the TERT region necessary and sufficient for telomerase RNA binding is completely separable from the reverse transcriptase motifs . The minimal Tetrahymena TERT RNA binding domain contains two sequence motifs with ciliate-specific conservation and one TERT motif with conservation across all species . With human TERT, we demonstrate that a similar region within the TERT amino terminus is essential for human telomerase RNA binding as well . Finally, we defined the Tetrahymena telomerase RNA sequences that are essential for TERT interaction . We found that a four-nucleotide region 5' of the template is critical for TERT binding and that the 5' end of telomerase RNA is sufficient for TERT binding . Our results reveal at least one evolutionarily conserved molecular mechanism by which the telomerase reverse transcriptase is functionally specialized for obligate use of an internal RNA template.

J Clin Microbiol, 2001 Feb, 39(2), 720 - 4
Peritonitis due to Thermoascus taitungiacus (Anamorph Paecilomyces taitungiacus); Korzets A et al.; The first case of human disease due to the thermophilic ascomycete Thermoascus taitungiacus (the teleomorph of Paecilomyces taitungiacus) is presented . T . taitungiacus was recovered from four dialysate fluid specimens of a 57-year-old patient undergoing chronic peritoneal dialysis . Identification was based upon cylindrical conidia, reddish orange nonostiolate ascomata, lack of growth at 20 degrees C, thermotolerance, and ascospores that appeared pale yellow, elliptical, thick walled, and predominately echinulate by light microscopy but irregularly verrucose by scanning electron microscopy.

J Bacteriol, 2001 Feb, 183(4), 1491 - 4
Multiple regulatory mechanisms act on the 5' untranslated region of the S-layer gene from Thermus thermophilus HB8; Castan P et al.; The role of the 5' untranslated region (5'UTR) of the S-layer gene from Thermus thermophilus was analyzed through the isolation of Delta 5'UTR mutants . In these mutants the half-life of splA mRNA was strongly reduced and slpA transcription was no longer subjected to growth phase-dependent repression . Overproduction and detachment of the external envelopes of the mutants were observed in stationary phase.

J Bacteriol, 2001 Feb, 183(4), 1184 - 94
Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus; Vaughan EE et al.; Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar . Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes . These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ . Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302 . However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose . In both CNRZ 302 and the Gal(+) mutant NZ302G, the transcription of the galR gene was induced by growth on lactose . Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression . Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal(+) mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions -9 and -15 as well as a single-base-pair insertion at position -37 with respect to the main transcription initiation point . Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations . We propose that poor expression of the gal genes in the galactose-negative S . thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.

EMBO J, 2001 Feb 1, 20(3), 562 - 9
The crystal structure of the ttCsaA protein: an export-related chaperone from Thermus thermophilus; Kawaguchi S et al.; The CsaA protein was first characterized in Bacillus subtilis as a molecular chaperone with export-related activities . Here we report the 2.0 Angstrom-resolution crystal structure of the Thermus thermophilus CsaA protein, designated ttCsaA . Atomic structure and experiments in solution revealed a homodimer as the functional unit . The structure of the ttCsaA monomer is reminiscent of the well known oligonucleotide-binding fold, with the addition of extensions at the N- and C-termini that form an extensive dimer interface . The two identical, large, hydrophobic cavities on the protein surface are likely to constitute the substrate binding sites . The CsaA proteins share essential sequence similarity with the tRNA-binding protein Trbp111 . Structure-based sequence analysis suggests a close structural resemblance between these proteins, which may extend to the architecture of the binding sites at the atomic level . These results raise the intriguing possibility that CsaA proteins possess a second, tRNA-binding activity in addition to their export-related function.

Am J Clin Nutr, 2001 Feb, 73(2 Suppl), 451S - 455S
Protective role of probiotics and prebiotics in colon cancer; Wollowski I et al.; Ingestion of viable probiotics or prebiotics is associated with anticarcinogenic effects, one mechanism of which is the detoxification of genotoxins in the gut . This mechanism was shown experimentally in animals with use of the rat colon carcinogen 1,2-dimethylhydrazine and by determining endpoints that range from tumorigenesis to induction of DNA damage . Because of the complexity of cancer initiation, cancer progression, and the exposure of cancer in the gut, many types of interactions may be envisaged . Notably, some of our newer studies showed that short-lived metabolite mixtures isolated from milk that was fermented with strains of Lactobacillus bulgaricus and Streptococcus thermophilus are more effective in deactivating etiologic risk factors of colon carcinogenesis than are cellular components of microorganisms . Ingestion of prebiotics results in a different spectrum of fermentation products, including the production of high concentrations of short-chain fatty acids . Gut flora, especially after the ingestion of resistant starch, induces the chemopreventive enzyme glutathione transferase pi in the colon of the rat . Together, these factors lead to a reduced load of genotoxic agents in the gut and to an increased production of agents that deactivate toxic components . Butyrate is one such protective agent and is associated with lowering cancer risk . It was recently shown that buytrate may inhibit the genotoxic activity of nitrosamides and hydrogen peroxide in human colon cells . In humans, the ingestion of probiotics leads to the excretion of urine with low concentrations of components that are genotoxic in human colon cells and high concentrations of components that induce oxidized DNA bases.

Appl Environ Microbiol, 2001 Feb, 67(2), 827 - 33
Discovery and description of giant submarine smectite cones on the seafloor in Eyjafjordur, northern Iceland, and a novel thermal microbial habitat; Marteinsson VT et al.; With the submersible JAGO and by scuba diving we discovered three remarkable geothermal cones, rising 33, 25, and 45 m from the seafloor at a depth of 65 m in Eyjafjordur, northern Iceland . The greatest geothermal activity was on the highest cone, which discharged up to 50 liters of freshwater per s at 72 degrees C and pH 10.0 . The cones were built up from precipitated smectite, formed by mixing of the hot SiO2-rich geothermal fluid with the cold Mg-rich seawater . By connecting a rubber hose to one outflow, about 240 liters of pure geothermal fluids was concentrated through a 0.2-microm-pore-size filter . Among 50 thermophilic isolates, we found members of Bacillus and Thermonema and a new unidentified low-G+C gram-positive member of the Bacteria as well as one member of the Archaea, Desulfurococcus mobilis . Analysis of small-subunit rRNA genes PCR amplified and cloned directly from environmental DNA showed that 41 out of 45 Bacteria sequences belonged to members of the Aquificales, whereas all of the 10 Archaea sequences belonged to the Korarchaeota . The physiological characteristics of isolates from different parts of the cones indicate a completely freshwater habitat, supporting the possibility of subterranean transmittance of terrestrial organisms.

Appl Environ Microbiol, 2001 Feb, 67(2), 673 - 9
Novel bifunctional hyperthermostable carboxypeptidase/aminoacylase from Pyrococcus horikoshii OT3; Ishikawa K et al.; Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus . The gene from P . horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues . However, some of the proposed active-site residues for carboxypeptidase were not found in this gene . The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures . The enzyme was stable at 90 degrees C, with the highest activity above 95 degrees C . The enzyme contained one bound zinc ion per one molecule that was essential for the activity . The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme . The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity . From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase . Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.

Bioresour Technol, 2001 Jan, 76(2), 99 - 106
Co-composting of soybean residues and leaves in Hong Kong; Wong JW et al.; The goal of this project was to evaluate the feasibility of co-composting of soybean residues and leaves and the effects of turning frequency on compost quality . Soybean residues were mixed with leaves and sawdust in 1:1:3 (w/w wet weight) for achieving a C/N ratio of about 30 . Three heaps of about 4 m3 of compost mixtures were prepared receiving a turning frequency of daily (pile A), 3-day (pile B) and weekly (pile C) turning . Different turning frequencies did not significantly affect the changes in pH and volatile solids throughout the composting period . High turning frequency caused a lower electrical conductivity and NH4-N contents as well as a shorter duration of thermophilic phase, because of a high heat loss by evaporation and volatilization of ammonia in the pile . The highest C decomposition of 4% occurred in the pile with a 3-day turning period, which coincided with the higher-nitrogen content in this treatment . All treatments with different turning frequencies reached maturation at 63 days as indicated by the soluble organic carbon, soluble NH4-N, C/N ratio and cress seed germination index . However, increasing the aeration during composting period was beneficial in accelerating the maturation process . Taking into consideration less labour and lower operation costs as compared to daily turning, it can be suggested that a 3-day turning frequency would be more appropriate for reaching acceptable quality of compost and ease in operation.

Bioresour Technol, 2001 Jan, 76(2), 107 - 12
Integrating composting and vermicomposting in the treatment and bioconversion of biosolids; Ndegwa PM et al.; Traditional thermophilic composting is commonly adopted for treatment of organic wastes or for production of organic/natural fertilizers . A related technique, called vermicomposting (using earthworms to breakdown the organic wastes) is also becoming popular . These two techniques have their inherent advantages and disadvantages . The integrated approach suggested in this study borrows pertinent attributes from each of these two processes and combines them to enhance the overall process and improve the products qualities . Two approaches investigated in this study are: (1) pre-composting followed by vermicomposting, and (2) pre-vermicomposting followed by composting . The substrate was biosolids (activated sewage sludge) with mixed paper-mulch as the carbon base . Eisenia fetida (red wigglers) was the species of earthworms used in the vermicomposting processes . The results indicate that, a system that combines the two processes not only shortens stabilization time, but also improves the products quality . Combining the two systems resulted in a product that was more stable and consistent (homogenous), had less potential impact on the environment and for compost-vermicomposting (CV) system, the product met the pathogen reduction requirements.

Curr Microbiol, 2000 Jul, 41(1), 27 - 32
Characterization of the cryptic plasmid pSBO2 isolated from Streptococcus bovis JB1 and construction of a new shuttle vector; Nakamura M et al.; A cryptic plasmid designated pSBO2 (3582 bp) was isolated from Streptococcus bovis JB1 . The pSBO2 contained putative sites for a double-strand origin (dso), a small transcriptional repressor protein (Cop), countertranscribed RNAs (ctRNAs), and a replication protein (Rep), which were similar to those from pMV158 and pLS1, which were isolated from S . agalactiae, and pWVO1, isolated from Lactococcus lactis . The putative single-strand origin (sso) of pSBO2 was similar to pER341 and pST1, which were isolated from S . thermophilus . Recombinant plasmid designated pSBE2 was constructed to bind pECM184 vector and the DNA fragment containing sso, dso, Cop, ctRNAs, and Rep of pSBO2 . When pSBE2 was introduced into S . bovis 12-U-1 and no8, the plasmids in the transformants had deleted the 160-bp fragment between sso and dso . This plasmid, designated pSBE2A, was capable of transforming Escherichia coli and S . bovis strains 12-U-1 and no8 on high frequency; therefore, pSBE2A is an effective shuttle vector.

J Biol Chem, 2000 Apr 14, 275(15), 11147 - 53
Structural, kinetic, and calorimetric characterization of the cold-active phosphoglycerate kinase from the antarctic Pseudomonas sp . TACII18; Bentahir M et al.; The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp . TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase . The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli . The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast . These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK . The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK . Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms . In the free form, a heat-labile and a thermostable domain unfold independently . It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

Science, 2000 Apr 7, 288(5463), 107 - 13
Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain; Agalarov SC et al.; The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution . The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18 . S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18 . The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.

Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2141 - 9
Thermoanaerobacter subterraneus sp . nov., a novel thermophile isolated from oilfield water; Fardeau ML et al.; A new thermophilic, anaerobic glucose-fermenting, Gram-positive, rod-shaped bacterium, designated strain SEBR 7858T, was isolated from an oilfield water sample . Under optimal conditions on a glucose-containing medium (3% NaCl, 65 degrees C and pH 7.5), the generation time was 2.5 h . No growth occurred at 35 or 80 degrees C, nor at pH 5..5 or 9.0 . Strain SEBR 7858T possessed lateral flagella . Spores were undetected but heat-resistant forms were present . Strain SEBR 7858T fermented a range of carbohydrates to acetate, L-alanine, lactate, H2 and CO2 . The isolate reduced thiosulfate and elemental sulfur, but not sulfate or sulfite to sulfide . In the presence of thiosulfate, the ratio of acetate produced per mole of glucose consumed increased, suggesting a shift in the use of electron acceptors during carbohydrate metabolism . The DNA G+C content was 41 mol% . Based on 16S rRNA gene sequence analysis, the strain was almost equidistantly related to all members of the genus Thermoanaerobacter (mean similarity 92%) . Based on phenotypic, genomic and phylogenetic characteristics, strain SEBR 7858T was clearly different from all members of the genus Thermoanaerobacter and was therefore designated as a new species, Thermoanaerobacter subterraneus sp . nov . The type strain is SEBR 7858T (= CNCM 1-2383T, DSM 13054T).

Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2109 - 17
Anoxybacillus pushchinensis gen . nov., sp . nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavitherms comb . nov; Pikuta E et al.; A new strictly anaerobic, alkaliphilic, moderately thermophilic, fermentative, spore-forming bacterium, strain K1T, was isolated from manure samples (pH 6-8) . Cells were Gram-positive, straight, non-motile rods that grew at temperatures of 37-66 degrees C (optimum at 62 degrees C) and in a pH range of 8.0-10.5 (optimum at 9.5-9.7) . The bacterium fermented D-glucose, sucrose, D-fructose, D-trehalose and starch as carbon and energy sources . It required vitamins and its growth is stimulated by yeast extract . The major metabolic products were H2 and acetate . Cells were catalase-negative and could reduce nitrate to nitrite . The G+C content of the DNA was 42.2 mol% . Based on the phenotypic properties and 16S rDNA sequencing and DNA-DNA hybridization data, strain K1T (= DSM 12423T = ATCC 700785T = VKM B-2193T) was assigned to the new genus Anoxybacillus gen . nov., as a representative of a new species, Anoxybacillus pushchinensis sp . nov . 'Bacillus flavothermus' strain d.y., which was found to be closely related to strain K1T, is described as Anoxybacillus flavithermus comb . nov . (type strain = d.y.T = DSM 2641T).

Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2009 - 12
Bacillus vulcani sp . nov., a novel thermophilic species isolated from a shallow marine hydrothermal vent; Caccamo D et al.; A thermophilic spore-forming bacterium was isolated from sediment of a shallow hydrothermal vent at Vulcano Island (Italy) . After phenotypic and molecular analyses, it was identified as a novel Bacillus species, for which the name Bacillus vulcani is proposed . The type strain is strain 3s-1T (= DSM 13174T = CIP 106305T).

J Appl Microbiol, 2001 Jan, 90(1), 123 - 30
Characterization of cell envelope-associated proteinases of thermophilic lactobacilli; Fira D et al.; The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact . delbrueckii BGPF1, and Lact . acidophilus CH2 (Chr . Hansen's strain) and Lact . acidophilus V74 (Visby's strain), were compared . Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases . Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein . The BGPF1 proteinase completely hydrolysed only beta-casein . The BGRA43 proteinase completely hydrolysed all three casein fractions . The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases . DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene . Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.

J Appl Microbiol, 2001 Jan, 90(1), 96 - 105
Molecular characterization and diversity of thermophilic iron-reducing enrichment cultures from deep subsurface environments; Zhou J et al.; AIMS: The objectives of this work were to explore the diversity in Fe (III)-reducing enrichment cultures from the deep subsurface and to identify strains involved in metal reduction . METHODS AND RESULTS: Analyses of 16S ribosomal RNA (rRNA) of enrichments, supplemented with hydrogen, acetate or pyruvate as an electron donor, identified three dominant operational taxonomic units (OTUs) . All cultures exhibited considerable diversity (36-24 OTUs), even after being transferred at least nine times . Two OTUs were present in all three cultures, constituting about 65% of the total clones examined . CONCLUSION: Dominant OTUs appeared to be most closely related to Thermoanaerobacter ethanolicus or T . kivui . One OTU, which is potentially responsible for autotrophic Fe (III) reduction, was only about 95% similar to T . ethanolicus and may represent a new species . SIGNIFICANCE AND IMPACT OF THE STUDY: An unexpectedly high diversity was found in these enrichments and this diversity may be a feature that can be exploited.

Prev Vet Med, 2001 Jan 29, 48(2), 85 - 99
A trial of biosecurity as a means to control Campylobacter infection of broiler chickens; Gibbens JC et al.; We ran a controlled intervention trial to assess whether the risk of a broiler flock becoming infected with Campylobacter could be reduced by biosecurity measures . These were a standard method of cleansing and disinfecting the poultry house prior to stocking, and a standard hygiene protocol followed by all personnel who entered the study house during the flock's life . Thirty-nine flocks were allocated to intervention or control groups in a ratio of 1:2 . Intervention flocks were asked to follow the specified biosecurity measures; all flocks were monitored weekly for Campylobacter infection . Analysis of infection at 42 days of age and over the life of the flock showed that the risk of thermophilic Campylobacter infection of broilers was reduced by over 50% in intervention flocks . Parts of the intervention identified as significant in the univariable analysis included twice weekly replenishment of boot dip disinfectant; potential independent risk factors identified included the location of ventilation fans and daily sanitisation of the water supply . The non-random allocation of 10 flocks to the control group may have introduced some study bias (the effect of which is discussed in the paper).

Biol Chem, 2000 Nov, 381(11), 1079 - 87
Identification of the 50S ribosomal proteins from the Eubacterium Thermus thermophilus; Katsani KR et al.; The total protein mixture from the 50S subunit (TP-50) of the eubacterium Thermus thermophilus was characterized after blotting onto PVDF membranes from two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and sequencing . The proteins were numbered according to their primary structure similarity with their counterparts from other species . One of them has been marked with an asterisk, namely L*23, because unlike the other known ribosomal proteins it shows a very low degree of homology . A highly acidic 5S rRNA binding protein, TL5, was characterized and compared with the available primary structure information . Proteins L1 and L4 migrate similarly on 2D-PAGE . Protein L4, essential for protein biosynthesis, is N-terminally blocked and shows a strikingly low homology to other L4 proteins . In addition to L4, two other proteins, namely L10 and L11, were found to be N-terminally blocked . In conclusion, 33 proteins from the large subunit were identified, including TL5 . Homologs to rpL25 and rpL26 were not found.

Ann Agric Environ Med, 2000, 7(2), 133 - 9
Occupational biohazards in agricultural dusts from India; Pande BN et al.; Sixteen samples of settled dusts deposited during handling of various granular plant materials (green gram, red gram, amaranth, rice, pearl millet, sorghum, wheat, maize) in small food storing and processing facilities (godowns) were collected in the region of Aurangabad (Southern India) . The samples were examined by the dilution plating method for the concentration and species composition of Gram-positive mesophilic bacteria, Gram-negative mesophilic bacteria, thermophilic actinomycetes and fungi . They were also examined by Limulus test for the concentration of bacterial endotoxin . The total concentration of microorganisms (bacteria + fungi) in examined samples varied within a wide range of 1.4 x 10(5) - 8.45 x 10(8) cfu/g (median 8.36 x 10(6) cfu/g) . On average, the most common were Gram-positive bacteria (87.84% of all isolates) followed by Gram-negative bacteria (11.12%) . Less common were fungi (1.24%) and thermophilic actinomycetes (0.01%) . Among isolated bacteria and fungi, there were many species known as causative agents of allergic alveolitis, asthma and organic dust toxic syndrome . The concentration of bacterial endotoxin in the examined samples ranged between 12.5 - 62500 microg/g (median 781.25 microg/g), being particularly large in the samples of dust from maize (6250 microg/g and 62500 microg/g) and pearl millet (6250 microg/g and 12500 microg/g) . The results of the present work indicate that the agricultural dusts from India represent a potential hazard for the workers because of high concentrations of allergenic microorganisms and bacterial endotoxin . The particular risk is associated with handing of maize and pearl millet . Further studies on this subject with the use of aerobiological methods are highly desirable

J Mol Biol, 2001 Jan 19, 305(3), 451 - 8
DNA-binding of phenylalanyl-tRNA synthetase is accompanied by loop formation of the double-stranded DNA; Dou X et al.; The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus has previously been shown to bind DNA . We demonstrate that the "winged" helix-turn-helix motifs in the duplicate domains B5 are the relevant structural elements for this DNA-binding property . By altering particular amino acids in the "wing", the affinity of the FRS to DNA was significantly reduced . Based on experimental data, which indicate that the FRS prefers a certain DNA structure rather than a particular consensus sequence, we propose a novel loop model for the DNA-binding mode of the FRS . In our model we assume that two segments of the same DNA molecule are bound simultaneously by both B5 domains and are aligned in parallel, while the intervening DNA forms a loop . Due to the limited flexibility of the DNA, loop formation is only possible if the respective intervening DNA stretch exceeds a certain length . Several lines of evidence support this model . (1) We demonstrate by gel retardation assays that the DNA requires a minimal number of ca 80 base-pairs to be bound by the FRS . (2) In the presence of the FRS, DNA longer than ca 80 base-pairs has a significantly increased DNase I accessibility . This agrees well with its known preferential cleavage at positions where the minor grove is on the outside of looped-out DNA molecules . (3) The initial cleavage by DNase I of >80 bp long DNA occurs in the middle of the fragment . In a looped molecule this is the position with the highest accessibility to DNase I.The function of the FRS related to DNA binding is still unknown . Since the FRS exists in the nucleus of rapidly growing mammalian cells, and protein-induced DNA bending or looping contributes to several transcription, replication, and recombination systems in both prokaryotes and eukaryotes, it is likely that the FRS, in addition to its aminoacylation function, influences common cellular processes via DNA binding .

Protein Sci, 2000 Nov, 9(11), 2074 - 84
Integrity of thermus thermophilus cytochrome c552 synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH: biochemical, spectral, and structural characterization of the recombinant protein; Fee JA et al.; We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene . Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH) . Approximately 1 mg of purified protein is obtained from 1 L of culture medium . The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme-copper oxidase . Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H-NMR spectroscopies . The 1.7 A resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644) . This approach may be generally useful for expression of alien cytochrome c genes in E . coli.

Can J Microbiol, 2000 Dec, 46(12), 1123 - 7
Production and monomer composition of exopolysaccharides by yogurt starter cultures; Frengova GI et al.; As components of starter cultures for Bulgarian yogurt, Streptococcus salivarius subsp . thermophilus and Lactobacillus delbrueckii subsp . bulgaricus revealed extensive exopolysaccharide (EPS) production activity when cultivated in whole cow's milk . The polymer-forming activity of thermophilic streptococci was lower (230-270 mg EPS/L) than that of the lactobacilli (400-540 mg EPS/L) . Mixed cultures stimulated EPS production in yogurt manufacture, and a maximum concentration of 720-860 mg EPS/L was recorded after full coagulation of milk . The monomer structure of the exopolysaccharides formed by the yogurt starter cultures principally consists of galactose and glucose (1:1), with small amounts of xylose, arabinose, and/or mannose.

J Eukaryot Microbiol, 2000 Jul-Aug, 47(4), 412 - 8
Genetic and environmental factors affecting mating type frequency in natural isolates of Tetrahymena thermophila; Arslanyolu M et al.; In Tetrahymena thermophila mating type alleles specify temperature sensitive frequency distributions of multiple mating types . A-like alleles specify mating types I, II, III, V and VI, whereas B-like alleles specify mating types II through VII . We have characterized the mating type distributions specified by several A- and B-like genotypes segregated by genomic exclusion from cells isolated from a pond in northwestern Pennsylvania . The B-like genotypes are alike in specifying very low frequencies of mating type III, but differ with respect to the frequencies of other mating types, particularly II and VII . An A-like genotype specifies a high frequency of mating type III and is unstable in successive generations for the expression of mating type II, suggesting a possible modifier . Inter se crosses performed at 18 degrees C, 28 degrees C and 34 degrees C showed that each genotype specifies a frequency distribution that is uniquely affected by temperature . No mating type was affected the same way by temperature in all genotypes . In A/B heterozygotes, the B-like genotype exhibited partial dominance . The genotypes described here differ significantly from previously described genotypes from the same pond, indicating that there are numerous mating type alleles . For frequency-dependent selection to equalize mating type frequencies, it must act not only on complex multiple alleles but also on the response of mating type alleles to temperature.

J Eukaryot Microbiol, 2000 Jul-Aug, 47(4), 328 - 33
Toward sequencing the Tetrahymena genome: exploiting the gift of nuclear dimorphism; Orias E; Important scientific discoveries have utilized the unique advantages of Tetrahymena thermophila as a research organism . Recently developed molecular genetic manipulations allow full exploitation of the many scientific dividends that would result from having its genome sequenced . As a typical ciliated protozoan, Tetrahymena exhibits "nuclear dimorphism" . It possesses two differentiated forms of its nuclear genome: a globally repressed, diploid germline or micronuclear genome, and a polyploid, site-specifically fragmented somatic or macronuclear genome . The macronuclear genome is, in effect, a natural, large-insert library of the micronuclear genome . This presentation describes how the gifts of nuclear dimorphism are being exploited in the experimental analysis of molecular and cell biology . Mechanisms present in humans that are either absent in other eukaryotic microbial model systems, or not as readily accessible in them as in Tetrahymena, are especially relevant . This presentation also reviews unique tools generated by nuclear dimorphism that are being used for genetically and physically mapping the Tetrahymena genome.

Nucleic Acids Res, 2001 Jan 15, 29(2), 488 - 98
Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila; Fillingham JS et al.; During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs) . Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein . We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries . A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47-81 bp from the right side of mse2.9 . The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs . In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing . Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

Extremophiles, 2000 Dec, 4(6), 365 - 71
Isolation and characterization of lipid-degrading Bacillus thermoleovorans IHI-91 from an icelandic hot spring; Markossian S et al.; An efficient lipid-degrading thermophilic aerobic bacterium was isolated from an icelandic hot spring and classified as Bacillus thermoleovorans IHI-91 . The aerobic bacterium grows optimally at 65 degrees C and pH 6.0 and secretes a high level of lipase (300 Ul(-1)) . The newly isolated strain utilizes several lipids such as palmitic acid, stearic acid, lanolin, olive oil, sunflower seed oil, soya oil, and fish oil as sole carbon and energy source without an additional supply of growth factors . The degradation of about 93% of triolein, which is present in olive oil, was observed after only 7h of fermentation at a maximal growth rate of 1.0 h(-1) . During growth at optimal conditions on yeast extract, the doubling time was only 15 min . Based on 16S rDNA studies, DNA-DNA hybridization and morphological and physiological properties, the isolate IHI-91 was identified as Bacillus thermoleovorans IHI-91 sp . nov . Because of its production of high concentrations of thermoactive lipases and esterases and the capability of degrading a wide range of lipids at high temperatures, the isolated strain is an ideal candidate for application in various biotechnological processes such as wastewater treatment.

Extremophiles, 2000 Dec, 4(6), 321 - 31
Cold stress response in Archaea; Cavicchioli R et al.; We live on a cold planet where more than 80% of the biosphere is permanently below 5 degrees C, and yet comparatively little is known about the genetics and physiology of the microorganisms inhabiting these environments . Based on molecular probe and sequencing studies, it is clear that Archaea are numerically abundant in diverse low-temperature environments throughout the globe . In addition, non-low-temperature-adapted Archaea are commonly exposed to sudden decreases in temperature, as are other microorganisms, animals, and plants . Considering their ubiquity in nature, it is perhaps surprising to find that there is such a lack of knowledge regarding low-temperature adaptation mechanisms in Archaea, particularly in comparison to what is known about archaeal thermophiles and hyperthermophiles and responses to heat shock . This review covers what is presently known about adaptation to cold shock and growth at low temperature, with a particular focus on Antarctic Archaea . The review highlights the similarities and differences that exist between Archaea and Bacteria and eukaryotes, and addresses the potentially important role that protein synthesis plays in adaptation to the cold . By reviewing the present state of the field, a number of important areas for future research are identified.

Curr Microbiol, 2001 Feb, 42(2), 122 - 8
Molecular properties of Streptococcus thermophilus plasmid pER35 encoding a restriction modification system; Solow BT et al.; Bacteriophage attack on lactic fermentation bacteria (LFB) is costly to the dairy industry because it results in product loss . One mechanism used by LFB to protect themselves from bacteriophage attack is restriction of foreign DNA . Three plasmids, pER16, pER35, and pER36, from three different strains of the thermotolerant dairy fermentation bacterium Streptococcus thermophilus were sequenced . One of these plasmids, pER35, isolated from S . thermophilus ST135, encoded a type IC restriction-modification (R-M) system very similar to those encoded on plasmids pIL2614 in Lactococcus lactis subsp . lactis and pND861 in Lactococcus lactis biovar diacetylactis . The high degree of identity between the R-M systems encoded on pER35, pIL2614, and pND861 indicated the potential for horizontal transfer of these genes between different species of lactic fermentation bacteria . Similar to the functional R-M system encoded on pIL2614 that protects the mesophilic L . lactis subsp . lactis against phage attack, the R-M system on pER35 most likely functions in the same role in S . thermophilus ST135 . The plasmid pER16 was found to encode the specificity subunit of the R-M system, but not the R or M subunits . In addition, all three plasmids encoded proteins that are present on other S . thermophilus plasmids, including a protein for rolling-circle replication (RepA) and a low-molecular-weight stress protein (Hsp) . The presence of a complete R-M system encoded on a plasmid in S . thermophilus, a species that often lacks plasmids, is novel and may be beneficial for protecting S . thermophilus from bacteriophage attack under dairy fermentation conditions.

J Biochem (Tokyo), 2001 Jan, 129(1), 173 - 8
Temperature dependence of the enzyme-substrate recognition mechanism; Ura H et al.; We determined the crystal structure of the liganded form of alpha-aminotransferase from a hyperthermophile, Pyrococcus horikoshii . This hyperthermophilic enzyme did not show domain movement upon binding of an acidic substrate, glutamate, except for a small movement of the alpha-helix from Glu16 to Ala25 . The omega-carboxyl group of the acidic substrate was recognized by Tyr70* without its side-chain movement, but not by positively charged Arg or Lys . Compared with the homologous enzymes from Thermus thermophilus HB8 and Escherichia coli, it was suggested that the more thermophilic the enzyme is, the smaller the domain movement is . This rule seems to be applicable to many other enzymes already reported.

J Bacteriol, 2001 Jan, 183(2), 800 - 3
Identification of the outer membrane porin of Thermus thermophilus HB8: the channel-forming complex has an unusually high molecular mass and an extremely large single-channel conductance; Maier E et al.; The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation . Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl . The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis . It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.

J Bacteriol, 2001 Jan, 183(2), 680 - 6
Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases; Buss KA et al.; Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate . We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography . As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin . Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily . The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases . Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the alpha-phosphate of ADP . Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

J Inorg Biochem, 2000 Nov, 82(1-4), 65 - 72
Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus; Uchida T et al.; Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies . This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center . EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04 . However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences . RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state . The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases . On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.

Mol Biotechnol, 2000 Oct, 16(2), 109 - 15
PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus; Ozturk ZN et al.; The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s) . The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi . The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature . Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers . PCR amplification of T . thermophila genomic DNA with these primers resulted in a specific amplification . The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T . reesei as probe . This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.

Appl Microbiol Biotechnol, 2000 Nov, 54(5), 715 - 8
Identification of interacting mixed cultures of lactic acid bacteria by their exclusion from a model predicting the acidifying activity of non-interacting mixed cultures; Sodini I et al.; A model predicting the acidifying activity of mixed cultures of lactic acid bacteria and based on the lack of interaction between the strains has been investigated to identify interacting cultures . Three mixed cultures with Streptococcus thermophilus TH3 and ST7 and Lactobacillus delbrueckii ssp . bulgaricus LB10 were grown on milk . The acidifying activities of the two mixed cultures TH3/LB10 and TH3/ST7 were predicted accurately by the model, with mean prediction errors of 7.7% and 14.1%, respectively . However, the model underestimated the acidifying activity of the mixed culture ST7/LB10, with a mean prediction error of 43.5%, which provides evidence of positive interaction between the strains ST7 and LB10 during acidification.

Cell Struct Funct, 2000 Aug, 25(4), 243 - 51
Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis; Gonda K et al.; The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex . The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed . The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus . Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood . In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis . Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus . Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI . In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis . The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility . Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.

J Eukaryot Microbiol, 2000 Nov-Dec, 47(6), 590 - 6
The role of cortical geometry in the nuclear development of Tetrahymena thermophila; Gaertig J et al.; Vegetative cells were subjected to electrofusion and the resulting heteropolar doublets were then mated to normal single cells and followed throughout conjugation using cytological and genetic techniques . The unique cyto-geometry created in a heteropolar doublet--a continuous cytoplasmic compartment bounded by two anterior poles and sharing a fused posterior pole at midbody, and the potential for two conjugal exchange junctions--resulted in instructive perturbations of nuclear behavior . Our results indicate that the course of nuclear development is strongly dependent on the cortical geometry of conjugating cells . Specifically, 1) continuation of development after meiosis requires an established conjugal junction; 2) after pronuclear exchange, pronuclei are subjected to attractive forces; and 3) products of the second postzygotic division are actively positioned near the posterior region of the cell cortex where they develop into micronuclei.

J Eukaryot Microbiol, 2000 Nov-Dec, 47(6), 561 - 8
MYO1, a novel, unconventional myosin gene affects endocytosis and macronuclear elongation in Tetrahymena thermophila; Williams SA et al.; Targeted gene disruption was used to investigate the function of MYO1, an unconventional myosin gene in Tetrahymena thermophila . Phenotypic analysis of a transformed strain that lacked a functional MYO1 gene was conducted at both 20 degrees C and 35 degrees C . At either temperature the delta MYO1 strain had a smaller cytoplasm/nucleus ratio than wild type . At 20 degrees C, delta MYO1 populations had a longer doubling time than wild type, lower saturation density, and a reduced rate of food vacuole formation . However, at 35 degrees C, these characteristics were comparable to wild type . Although micronuclear division and cytokinesis appeared normal in delta MYO1 cells, failure of the macronucleus to elongate properly resulted in unequal segregation of macronuclear DNA in cells maintained at either 20 degrees C or 35 degrees C.

J Eukaryot Microbiol, 2000 Nov-Dec, 47(6), 532 - 7
A giant phosphoprotein localized at the spongiome region of Crithidia luciliae thermophila; Baqui MM et al.; A giant protein with an apparent molecular mass of 2,300-kDa was identified in the Triton X-100 soluble fraction of Crithidia luciliae thermophila . Polyclonal antibody raised against this protein reacted by immunoblot analysis with proteins of similar molecular mass in Crithidia fasciculata and Crithidia oncopelti . In addition, the antibody immunoprecipitates the protein either after in vivo phosphorylation with {32P}orthophosphoric acid or after metabolically labeling with {35S}methionine . Indirect immunofluorescence microscopy analysis performed either with fixed or with live parasites showed a single fluorescent spot at the level of the flagellar pocket region . Immunogold electron microscopy of thin sections of the parasite revealed that the antigen is localized at a restricted area of the spongiome, between the contractile vacuole and the flagellar pocket . Furthermore, Triton X-114 phase separation of whole cell membrane proteins, metabolically labeled with {35S}methionine, demonstrated that the giant protein remains in the aqueous phase . These results indicate that this phosphoprotein behaves as a peripheral membrane protein localized at the spongiome region, suggesting that it might be involved in the osmoregulatory process.

Lett Appl Microbiol, 2000 Dec, 31(6), 405 - 10
Potent fibrinolytic enzyme from a thermophilic Streptomyces megasporus strain SD5; Chitte RR et al.; As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues . A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent broth of thermophilic Streptomyces megasporus strain SD5 . The crude enzyme was concentrated using ammonium sulphate, dialysis and lyophilization . Approximately 0.11 mg ml(-1) crude enzyme with a specific activity of 4.2 U microg(-1) was obtained . Post-electrophoretic reactivity revealed a monomeric form of the enzyme with a molecular weight of 35 kDa . The optimum pH and temperature for production of the enzyme were 8 and 55 degrees C, respectively . The enzyme was resistant to a broad range of pH ranging from 6 to 9 and temperature ranging from 37 to 60 degrees C . The enzyme was a chymotrypsin-like serine peptidase and the activity of the enzyme was N-terminal-dependent . The in vitro clot lysis by the enzyme at 37 degrees C was encouraging.

J Appl Microbiol, 2000 Dec, 89(6), 1059 - 65
Characterization of phage receptors in Streptococcus thermophilus using purified cell walls obtained by a simple protocol; Quiberoni A et al.; A simple protocol was designed and applied to obtain Streptococcus thermophilus purified cell walls . To identify the structures involved in phage adsorption, the cell walls of two Strep . thermophilus strains were treated with sodium dodecyl sulphate and proteinase K . These treatments did not reduce the adsorption of phages CYM and 0BJ to the cell walls of Strep . thermophilus YSD10 and Strep . thermophilus BJ15, respectively . However, phage binding was reduced when the cell envelopes were treated with mutanolysin or trichloroacetic acid 5%, suggesting that the phage receptor component is part of the peptidoglycan or a polymer closely linked to it . The ability of several saccharides to inactivate both phages was also assayed . These phage inhibition experiments suggested that the phage CYM adsorbed to a component involving glucosamine and rhamnose, while glucosamine and ribose interfered with the adsorption of phage 0BJ.

Biochim Biophys Acta, 2000 Dec 1, 1494(3), 217 - 25
The ruv proteins of Thermotoga maritima: branch migration and resolution of Holliday junctions; Gonzalez S et al.; In homologous recombination in bacteria, the RuvAB Holliday junction-specific helicase catalyzes Holliday junction branch migration, and the RuvC Holliday junction resolvase catalyzes formation of spliced or patched structures . RuvAB and RuvC from the hyperthermophile Thermotoga maritima were expressed in Escherichia coli and purified to homogeneity . An inverted repeat sequence with unique termini was produced by PCR, restriction endonuclease cleavage, and head-to-tail ligation . A second inverted repeat sequence was derived by amplification of a second template containing a three-nucleotide insertion . Reassociation products from a mixture of these two sequences were homoduplex linear molecules and heteroduplex heat-stable Holliday junctions, which acted as substrates for both T . maritima RuvAB and RuvC . The T . maritima RuvAB helicase catalyzed energy-dependent Holliday junction branch migration at 70 degrees C, leading to heteroduplex linear duplex molecules with two three-nucleotide loops . Either ATP or ATP gamma S hydrolysis served as the energy source . T . maritima RuvC resolved Holliday junctions at 70 degrees C . Remarkably, the cleavage site was identical to the preferred cleavage site for E . coli RuvC {(A/T)TT(downward arrow)(G/C)} . The conservation of function and the ease of purification of wild-type and mutant thermophilic proteins argues for the use of T . maritima proteins for additional biochemical and structural studies.

J Appl Microbiol, 2000 Nov, 89(5), 768 - 77
Microbial succession during a composting process as evaluated by denaturing gradient gel electrophoresis analysis; Ishii K et al.; Microbial succession during a laboratory-scale composting process of garbage was analysed by denaturing gradient gel electrophoresis (DGGE) combined with measurement of physicochemical parameters such as temperature, pH, organic acids, total dissolved organic carbon and water-soluble humic substance . From the temperature changes, a rapid increase from 25 to 58 degrees C and then a gradual decrease, four phases were recognized in the process as follows; mesophilic (S), thermophilic (T), cooling (C) and maturing (M) . The polymerase chain reaction-amplified 16S rDNA fragments with universal (907R) and eubacterial (341F with GC clamp) primers were subjected to DGGE analysis . Consequently, the DGGE band pattern changed during the composting process . The direct sequences from DGGE bands were related to those of known genera in the DNA database . The microbial succession determined by DGGE was summarized as follows: in the S phase some fermenting bacteria, such as lactobacillus, were present with the existing organic acids; in the T phase thermophilic bacillus appeared and, after the C phase, bacterial populations were more complex than in previous phases and the phylogenetic positions of those populations were relatively distant from strains so far in the DNA database . Thus, the DGGE method is useful to reveal microbial succession during a composting process.

J Appl Microbiol, 2000 Nov, 89(5), 727 - 34
Antimicrobial resistance profiling and DNA Amplification Fingerprinting (DAF) of thermophilic Campylobacter spp . in human, poultry and porcine samples from the Cork region of Ireland; Lucey B et al.; Antimicrobial resistance (R) typing and DNA Amplification Fingerprinting (DAF) of a random collection of 84 Irish thermophilic Campylobacter isolates is described . The collection included human, veterinary (porcine) and poultry isolates cultured between 1996 and 1998 in the Cork region of Ireland . Biochemical and molecular methods were used to identify Campylobacter jejuni and Camp . coli . Many of these isolates were simultaneously resistant to several common antimicrobial agents . In particular, resistance to ampicillin, spectinomycin, sulphafurazole and tetracycline was common . A total of 74 DAF profiles was identified among the study collection, showing a high degree of diversity . Dendrogram analysis of the DNA patterns identified three main clusters at the 50% similarity level, which included two clusters of Camp . coli and a third containing a mixture of Camp . jejuni and Camp . coli.

J Microbiol Methods, 2001 Jan, 43(3), 197 - 212
Structural diversity of microorganisms in chemically perturbed soil assessed by molecular and cytochemical approaches; Kozdroj J et al.; Until recently, our understanding of microbial community development in soil ecosystems exposed to different inorganic and organic pollutants has been limited to culturable microorganisms because of the techniques available . The discovery that most soil microorganisms are non-culturable but potentially viable and metabolically active accelerated the application of different culture-independent methods for structural diversity assessments of the microbial community . This review examines the results of recent studies on the impact of heavy metals and organic pollutants on the diversity of the microflora obtained with methods based on analyses of signature biomarkers such as nucleic acids and fatty acids . The application of these techniques allowed researchers to pinpoint reduction of microbial diversity in contaminated soil, and significant shifts in the community structure, leading to the dominance of only a few populations (species) and the disappearance of others, some of which were never isolated by conventional methods (e.g . an increase in Acidobacterium or a decrease in terrestrial non-thermophilic Crenarchaeota) . Although the new techniques are not free from limitations, they allow the monitoring of the virtual impact of stressors on soil microorganisms and the direction of resuscitation of the microbial community during natural or induced bioremediation, especially when using combined approaches.

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 635 - 8
Activation of prostacyclin synthesis by mechanical stimulation in the ciliated protozoan Tetrahymena thermophila; Iwamoto M et al.; We detected a HPLC peak corresponding to 6-keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI(2)), in {1-(14)C}arachidonate metabolites from the ciliated protozoan Tetrahymena thermophila . Quantitative analysis of 6-keto-PGF(1alpha) by enzyme immunoassay revealed that the synthesis and release were rapidly activated by the mechanical stimulation of a short centrifugation . The activation was suppressed significantly by a cyclooxygenase inhibitor, ibuprofen, and was independent of the extracellular Ca(2+) . External addition of PGI(2) and its stable analogue, beraprost, caused a transient increase in the tumbling frequency of swimming . Other prostanoids, PGE(2) and PGF(2alpha), have no effect on the swimming . These results indicate that a free-living ciliate, T . thermophila, synthesizes and has a specific sensitivity to PGI(2) .

Enzyme Microb Technol, 2001 Jan 2, 28(1), 8 - 15
Capacity of thermomonospora alba XylA to impart thermostability in family F/10 chimeric xylanases; Ahsan MM et al.; To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and XylA of Thermomonospora alba ULJB1, into a single chimeric xylanase . The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s . The shuffled constructs, designated as FXYN-M3/4-XylA, FXYN-M9/10-XylA, and FXYN-M14/15-XylA were cloned and their nucleotide sequences were confirmed . Two chimera, FXYN-M3/4-XylA and FXYN-M14/15-XylA, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60 . The homogeneously pure chimeric proteins, FXYN-M3/4-XylA and FXYN-M14/15-XylA showed improved thermal and pH profiles compared to those of one of the parents, FXYN . This was apparently due to the influence of amino acids inherited from thermophilic XylA . Measured K(m) and kcat values were closer to those of the other parent, XylA . Interestingly, a significant level of heat tolerance up to 60 degrees C, was recorded for FXYN-M3/4-XylA in comparison to only 40 degrees C for FXYN-M14/15-XylA though their temperature optima did not correlates with their thermal stability . These results indicated that the amino acid residues of the larger T . alba XylA DNA fragment present in FXYN-M3/4-XylA were responsible for inducing its thermal stability.

Enzyme Microb Technol, 2000 Dec, 27(10), 789 - 792
Superoxide dismutase biosynthesis by two thermophilic bacteria; Gligic L et al.; Some high-molecular weight antioxidant defense system components of two thermophilic bacteria isolated from spa waters of Serbia (Yugoslavia) and identified as Bacillus stearothermophilus and Thermothrix sp . were studied . In addition to superoxide dismutase (SOD; EC 1.15.1.1), qualitative analyses demonstrated the presence of catalase (EC 1.11.1.6), peroxidases and oxidases in both bacterial strains . Cell-free extracts were subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE) and SOD activity in the eluates of the corresponding bands was examined in the presence of several specific inhibitors . A slight decrease of SOD activity observed in the presence of 0.3 M potassium cyanide and its complete insensitivity to hydrogen peroxide (5 mM) and sodium azide (20 mM) action suggest that the enzyme occurring in the two thermophiles represents Mn SOD . A high SOD activity recorded in cell-free extracts strongly recommends these two bacterial strains as potential producers of this important antioxidant defense system component at industrial scale.

J Bacteriol, 2001 Jan, 183(1), 155 - 61
Renaturation of Bacillus thermoglucosidasius HrcA repressor by DNA and thermostability of the HrcA-DNA complex in vitro; Watanabe K et al.; HrcA, a negative control repressor for chaperone expression from the obligate thermophile Bacillus thermoglucosidasius KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility . Renaturation trials revealed that the addition of DNA to purified B . thermoglucosidasius HrcA can result in solubilization of HrcA free from the denaturing agent urea . Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize B . thermoglucosidasius HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii) B . thermoglucosidasius HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii) B . thermoglucosidasius HrcA binding to the CIRCE element was stable at below 50 degrees C whereas the complex was rapidly denatured at 70 degrees C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes . These results will help to determine the nature of HrcA protein molecules.

J Bacteriol, 2001 Jan, 183(1), 71 - 6
Recombinant Thermus aquaticus RNA polymerase, a new tool for structure-based analysis of transcription; Minakhin L et al.; The three-dimensional structure of DNA-dependent RNA polymerase (RNAP) from thermophilic Thermus aquaticus has recently been determined at 3.3 A resolution . Currently, very little is known about T . aquaticus transcription and no genetic system to study T . aquaticus RNAP genes is available . To overcome these limitations, we cloned and overexpressed T . aquaticus RNAP genes in Escherichia coli . Overproduced T . aquaticus RNAP subunits assembled into functional RNAP in vitro and in vivo when coexpressed in E . coli . We used the recombinant T . aquaticus enzyme to demonstrate that transcription initiation, transcription termination, and transcription cleavage assays developed for E . coli RNAP can be adapted to study T . aquaticus transcription . However, T . aquaticus RNAP differs from the prototypical E . coli enzyme in several important ways: it terminates transcription less efficiently, has exceptionally high rate of intrinsic transcript cleavage, and is highly resistant to rifampin . Our results, together with the high-resolution structural information, should now allow a rational analysis of transcription mechanism by mutation.

Cell, 2000 Nov 22, 103(5), 793 - 803
Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase; Fukai S et al.; Valyl-tRNA synthetase (ValRS) strictly discriminates the cognate L-valine from the larger L-isoleucine and the isosteric L-threonine by the tRNA-dependent "double sieve" mechanism . In this study, we determined the 2.9 A crystal structure of a complex of Thermus thermophilus ValRS, tRNA(Val), and an analog of the Val-adenylate intermediate . The analog is bound in a pocket, where Pro(41) allows accommodation of the Val and Thr moieties but precludes the Ile moiety (the first sieve), on the aminoacylation domain . The editing domain, which hydrolyzes incorrectly synthesized Thr-tRNA(Val), is bound to the 3' adenosine of tRNA(Val) . A contiguous pocket was found to accommodate the Thr moiety, but not the Val moiety (the second sieve) . Furthermore, another Thr binding pocket for Thr-adenylate hydrolysis was suggested on the editing domain.

Biochemistry, 2000 Dec 19, 39(50), 15531 - 9
Psychrophilic elongation factor Tu from the antarctic Moraxella sp . Tac II 25: biochemical characterization and cloning of the encoding gene; Masullo M et al.; The elongation factor Tu was isolated from a psychrophilic eubacterial Antarctic Moraxella strain (MoEF-Tu) and its molecular and functional properties were determined . It catalyzed the synthesis of poly(Phe) and bound specifically guanine nucleotides with an affinity for GDP about 12-fold higher than that for GTP . The affinity toward guanine nucleotides was lower than that of other eubacterial EF-Tu . The intrinsic GTPase activity of MoEF-Tu was hardly detectable but was accelerated by 2 orders of magnitude in the presence of the antibiotic kirromycin (GTPase(k)) . Such a property resembled Escherichia coli EF-Tu (EcEF-Tu) even though the affinity of MoEF-Tu for the antibiotic was lower . MoEF-Tu showed a thermophilicity higher than that of EcEF-Tu; its temperature for half-denaturation was 44 degrees C . The MoEF-Tu encoding gene corresponding to E . coli tufA was cloned and sequenced . The translated protein had a calculated molecular weight of 43 288 and contained the GTP-binding sequence motifs . Concerning its primary structure, MoEF-Tu showed sequence identity with E . coli and Thermus thermophilus EF-Tu equal to 84% and 74%, respectively, while the identity with EF-1 alpha from the archaeon Sulfolobus solfataricus was equal to 32%.

Biochemistry, 2000 Dec 19, 39(50), 15500 - 12
Evidence for changes in the nucleotide conformation in the active site of H(+)-ATPase as determined by pulsed EPR spectroscopy; Schneider B et al.; The conformation of di- and triphosphate nucleosides in the active site of ATPsynthase (H(+)-ATPase) from thermophilic Bacillus PS3 (TF1) and their interaction with Mg(2+)/Mn(2+) cations have been investigated using EPR, ESEEM, and HYSCORE spectroscopies . For a ternary complex formed by a stoichiometric mixture of TF1, Mn(2+), and ADP, the ESEEM and HYSCORE data reveal a (31)P hyperfine interaction with Mn(2+) (|A((31)P)| approximately 5.20 MHz), significantly larger than that measured for the complex formed by Mn(2+) and ADP in solution (|A((31)P)| approximately 4.50 MHz) . The Q-band EPR spectrum of the Mn.TF1.ADP complex indicates that the Mn(2+) binds in a slightly distorted environment with |D| approximately 180 x 10(-4) cm(-1) and |E| approximately 50 x 10(-4) cm(-1) . The increased hyperfine coupling with (31)P in the presence of TF1 reflects the specific interaction between the central Mn(2+) and the ADP beta-phosphate, illustrating the role of the enzyme active site in positioning the phosphate chain of the substrate for efficient catalysis . Results with the ternary Mn.TF1.ATP and Mn.TF1.AMP-PNP complexes are interpreted in a similar way with two hyperfine couplings being resolved for each complex (|A((31)P(beta))| approximately 4.60 MHz and |A((31)P(gamma))| approximately 5.90 MHz with ATP, and |A((31)P(beta))| approximately 4.20 MHz and |A((31)P(gamma))| approximately 5.40 MHz with AMP-PNP) . In these complexes, the increased hyperfine coupling with (31)P(gamma) compared with (31)P(beta) reflects the smaller Mn.P distance with the gamma-phosphate compared with the beta-phosphate as found in the crystal structure of the analogous enzyme from mitochondria {3.53 vs 3.70 A (Abrahams, J . P., Leslie, A . G . W., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628)} and the different binding modes of the two phosphate groups . The ESEEM and HYSCORE data of a complex formed with Mn(2+), ATP, and the isolated beta subunit show that the (31)P hyperfine coupling is close to that measured in the absence of the protein, indicating a poorly structured nucleotide site in the isolated beta subunit in the presence of ATP . The inhibition data obtained for TF1 incubated in the presence of Mg(2+), ADP, Al(NO(3))(3), and NaF indicate the formation of the inhibited complex with the transition state analogue namely Mg.TF1.ADP.AlF(x) with the equilibrium dissociation constant K(D) = 350 microM and rate constant k = 0.02 min(-1) . The ESEEM and HYSCORE data obtained for an inhibited TF1 sample, Mn.TF1.ADP.AlF(x), confirm the formation of the transition state analogue with distinct spectroscopic footprints that can be assigned to Mn.(19)F and Mn.(27)Al hyperfine interactions . The (31)P(beta) hyperfine coupling that is measured in the inhibited complex with the transition state analogue (|A((31)P(beta))| approximately 5.10 MHz) is intermediate between those measured in the presence of ADP and ATP and suggests an increase in the bond between Mn and the P(beta) from ADP upon formation of the transition state.

Gene, 2000 Nov 27, 258(1-2), 9 - 14
Efficient insertional mutagenesis in Streptococcus thermophilus; Baccigalupi L et al.; Bacteria have always been considered ideal organisms for genetic analysis . While this is true for some model organisms, like Escherichia coli, Bacillus subtilis and, more recently, Lactococcus lactis, genetic analysis of other organisms is often prevented by lack of valuable tools, like vectors, transposons and methods for transformation, gene inactivation and random insertional mutagenesis . This is the case of the moderately thermophilic bacterium Streptococcus thermophilus, an organism that, in spite of its widespread use for food fermentations, is only poorly characterized . We report here an insertional mutagenesis system that allows efficient random mutagenesis, easy characterization of the interrupted genes and construction of stable null mutations . This may become a powerful S . thermophilus tool for both genetic analysis and construction of 'food-grade' mutants of this biotechnologically relevant microorganism.

Can J Microbiol, 2000 Nov, 46(11), 1029 - 35
Isolation and characterization of highly thermophilic xylanolytic Thermus thermophilus strains from hot composts; Lyon PF et al.; This is the first detailed report of xylanolytic activity in Thermus strains . Two highly thermophilic xylanolytic bacteria, very closely related to non-xylanolytic T . thermophilus strains, have been isolated from the hottest zones of compost piles . Strain X6 was investigated in more detail . The growth rate (optical density monitoring) on xylan was 0.404.h-1 at 75 degrees C . Maximal growth temperature was 81 degrees C . Xylanase activity was mainly cell-bound, but was solubilized into the medium by sonication . It was induced by xylan or xylose in the culture medium . The temperature and pH optima of the xylanases were determined to be around 100 degrees C and pH 6, respectively . Xylanase activity was fairly thermostable; only 39% of activity was lost after an incubation period of 48 h at 90 degrees C in the absence of substrate . Xylanolytic T . thermophilus strains could contribute to the degradation of hemicellulose during the thermogenic phase of industrial composting.

Can J Microbiol, 2000 Nov, 46(11), 1021 - 8
Effects of mesophilic and thermophilic composts on suppression of Fusarium root and stem rot of greenhouse cucumber; Kannangara T et al.; Three composts were tested for their ability to suppress root and stem rot caused by the soil borne fungal pathogen Fusarium oxysporum f . sp . radicis-cucumerinum (FORC) on cucumber . Two of the composts were prepared from separated dairy solids either by windrow (WDS) or vermicomposting (VMC) while the third, obtained from International Bio-Recovery (IBR), was prepared from vegetable refuse using aerobic digestion . Three sets of potting mixes were prepared by mixing the composts with sawdust at varying ratios, and seeded with cucumber cv . Corona . After 14 days of growth in the greenhouse, inoculum of FORC (20 mL of 5 x 10(6) micro-conidia per mL) was applied to each pot at three different times (14, 21, and 35 days) . In unamended inoculated pots, the pathogen caused stunted growth and reduced flowers . Amendment of WDS in the potting mix suppressed these symptoms, while VMC and IBR had no effect . All three composts reduced the FORC colony forming units (cfu) at the end of the experiment (10 weeks) . There was a large increase of fluorescent bacteria near the vicinity of roots particularly in WDS amended potting mixes . When water extracts of the composts were plated onto acidified potato dextrose agar (APDA), only IBR contained a potent thermostable inhibitor to FORC . This inhibitor was removed by activated charcoal but was not partitioned into petroleum ether at acid, basic, or neutral pH . Inhibition of FORC by IBR was not due to electrical conductivity or trace elements in the compost . Contrasting effectiveness of the WDS and VMC made from the same waste suggests that composting method can influence the disease suppression properties of the finished compost.

Int Microbiol, 1999 Dec, 2(4), 217 - 26
Isolation and typing methods for the epidemiologic investigation of thermotolerant campylobacters; Barros-Velazquez J et al.; Thermotolerant campylobacters, C . jejuni, C . coli, C . lari and C . upsaliensis, are spiral bacteria involved in human enteric disease . The prevalence of these emerging pathogens, mainly C . jejuni and to a lesser extent C . coli, as etiologic agents of enteric disease in industrialized countries has increased over the last decade . The isolation and culture of these microorganisms is tedious and time-consuming mainly due to their complex nutritional and environmental requirements . This review discusses the techniques and methods developed for the selective isolation of thermotolerant campylobacters from food, environmental and clinical samples . Additionally, both traditional and newer molecular biology techniques applied to this group of thermophilic organisms for typing and taxonomic purposes are summarized.

Syst Appl Microbiol, 2000 Oct, 23(3), 426 - 32
A thermophilic Bacillus isolated from an Eolian shallow hydrothermal vent, able to produce exopolysaccharides; Nicolaus B et al.; A thermophilic aerobic microorganism, able to produce two exocellular polysaccharides (EPS1 and EPS2), was isolated from a shallow hydrothermal vent at Vulcano island (Eolian Islands, Italy) . EPS1 and EPS2 were based on mannose and glucose although in a different ratio . EPS2 possessed a trisaccharide repeating unit with a manno-pyranoside configuration . New isolate phenotype was studied by physiological and morphological observations, including biochemical and antimicrobial susceptibility tests (134) . Previous analyses carried out on 87 field isolates and 8 thermophilic reference bacilli displayed low phenotypic similarity level (S(SM) = 65%) with Bacillus thermodenitrificans DSM 465 . Optimal growth occurs at 65 degrees C and pH 7.0 . Oxidase and catalase are negative . The guanine-plus-cytosine (G+C) content of DNA is 52.7% . Genotypic investigations demonstrated the diversity of the isolate with fifteen selected thermophilic Bacillus spp . when we compared the restriction patterns of the amplified 16S rDNA . The membrane lipids are based on fatty acids mainly belonging to the iso-family.

Eur J Biochem, 2000 Dec, 267(24), 7058 - 64
Temporal secretion of a multicellulolytic system in Myxobacter sp . AL-1 . Molecular cloning and heterologous expression of cel9 encoding a modular endocellulase clustered in an operon with cel48, an exocellobiohydrolase gene; Avitia CI et al.; The Gram-negative soil micro-organism Myxobacter sp . AL-1 possesses at least five extracellular cellulases, the production of which is regulated by the growth cycle . We cloned the complete gene for one of these cellulases, termed cel9, which encoded a 67-kDa modular family 9 endoglycohydrolase, which was produced during the stationary phase of growth and was strongly enhanced by avicel . The predicted product of cel9 matches the structural architecture of family 9 cellulases such as Thermonospora fusca endo/exocellulase E4 . Cel9 protein was synthesized in Escherichia coli from a multicopy plasmid and in Bacillus subtilis from the isopropyl thiogalactoside-inducible Pspac promoter and was purified from the culture medium . Thermal stability, optimum pH and temperature dependence of Cel9 were similar when expressed from either source, and were indistinguishable from related cellulases produced by thermophilic bacteria . Downstream from cel9 was found a partial ORF, designated cel48, the deduced product of which was highly similar to bacterial exocellobiohydrolases and processive endoglucanases belonging to family 48 of the glycosyl hydrolases . The cel9 and cel48 genes appear to be arranged as part of an operon.

RNA, 2000 Nov, 6(11), 1649 - 59
Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7; Robert F et al.; Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half . The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an alpha-helical domain with a beta-ribbon extension . To gain further insights into its interaction with rRNA, we cloned the S7 gene from Escherichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence . The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes . Deletion of the N-terminal 17 residues or deletion of the B hairpins (residues 72-89) severely decreased S7 affinity for the rRNA . Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal alpha-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding . Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the alpha-helices . Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain.

Biochem J, 2000 Dec 15, 352 Pt 3, 783 - 8
Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase; Konno H et al.; Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit . In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity . To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins . The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography . The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation . This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized . In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.

Am J Clin Nutr, 2000 Dec, 72(6), 1474 - 9
Chronic consumption of fresh but not heated yogurt improves breath-hydrogen status and short-chain fatty acid profiles: a controlled study in healthy men with or without lactose maldigestion; Rizkalla SW et al.; BACKGROUND: Ingestion of fermented dairy products induces changes in the equilibrium and metabolism of the intestinal microflora and may thus have beneficial effects on the host . OBJECTIVE: We compared the effects of chronic consumption of yogurt with (fresh) or without (heated) live bacterial cultures (Lactobacillus bulgaricus and Streptococcus thermophilus) on plasma glucose, insulin, triacylglycerols, cholesterol, fatty acids, and short-chain fatty acids . DESIGN: Two groups of 12 healthy men with or without lactose malabsorption were selected with use of a breath-hydrogen test after a 30-g lactose load . Subjects were randomly assigned in a crossover design to 500 g/d of either fresh or heated yogurt for 2 periods of 15 d each, separated by a 15-d washout interval . RESULTS: Chronic consumption of fresh or heated yogurt had no detrimental effects on plasma glucose, insulin, or fatty acid areas under the curve in response to acute ingestion of 500 g yogurt in healthy men with or without lactose malabsorption . There were also no detectable changes in fasting plasma glucose, insulin, fatty acid, triacylglycerol, or cholesterol concentrations . In contrast, plasma butyrate was higher (P: < 0.03) and plasma propionate tended to be higher (P: = 0.059) in subjects without lactose malabsorption after fresh yogurt consumption than after heated yogurt consumption . There were no significant changes in plasma acetate . In subjects with lactose malabsorption, 15 d of fresh yogurt consumption also increased propionate production compared with values at baseline (P: < 0.04) . In the same group, the production of breath hydrogen was lower after fresh yogurt consumption than after heated yogurt consumption (P: < 0.01) . CONCLUSIONS: In men with lactose malabsorption, chronic consumption of yogurt containing live bacterial cultures ameliorated the malabsorption, as evidenced by lower breath-hydrogen excretion, but increased propionate concentrations . In subjects without lactose malabsorption, such yogurt tended to increase propionate and increased butyrate.

Biotechnol Prog, 2000 Nov-Dec, 16(6), 1008 - 17
Acetic acid production from lactose by an anaerobic thermophilic coculture immobilized in a fibrous-bed bioreactor; Talabardon M et al.; An anaerobic thermophilic coculture consisting of a heterofermentative bacterium (Clostridium thermolacticum) and a homoacetogen (Moorella thermoautotrophica) was developed for acetic acid production from lactose and milk permeate . The fermentation kinetics with free cells in conventional fermentors and immobilized cells in a recycle batch fibrous-bed bioreactor were studied . The optimal conditions for the cocultured fermentation were found to be 58 degrees C and pH 6.4 . In the free-cell fermentation, C . thermolacticum converted lactose to acetate, ethanol, lactate, H(2) and CO(2), and the homoacetogen then converted lactate, H(2), and CO(2) to acetate . The overall acetate yield from lactose ranged from 0.46 to 0.65 g/g lactose fermented, depending on the fermentation conditions . In contrast, no ethanol was produced in the immobilized-cell fermentation, and the overall acetate yield from lactose increased to 0.8-0.96 g/g lactose fermented . The fibrous-bed bioreactor also gave a higher final acetate concentration (up to 25 . 5 g/L) and reactor productivity (0.18-0.54 g/L/h) as compared to those from the free-cell fermentation (final acetate concentration, 15 g/L; productivity, 0.06-0.08 g/L/h) . The superior performance of the fibrous-bed bioreactor was attributed to the high cell density (20 g/L) immobilized in the fibrous-bed and adaptation of C . thermolacticum cells to tolerate a higher acetate concentration . The effects of yeast extract and trypticase as nutrient supplements on the fermentation were also studied . For the free-cell fermentation, nutrient supplementation was necessary for the bacteria to grow in milk permeate . For the immobilized-cell fermentation, plain milk permeate gave a high acetate yield (0.96 g/g), although the reactor productivity was lower than those with nutrient supplementation . Balanced growth and fermentation activities between the two bacteria in the coculture are important to the quantitative conversion of lactose to acetic acid . Lactate and hydrogen produced by C . thermolacticum must be timely converted to acetic acid by the homoacetogen to avoid inhibition by these metabolites.

Biochemistry, 2000 Dec 5, 39(48), 14739 - 44
Crystallization and the crystal properties of the oxygen-evolving photosystem II from Synechococcus vulcanus; Shen JR et al.; A photosystem II (PSII) complex highly active in oxygen evolution was purified and crystallized from a thermophilic cyanobacterium, Synechococcus vulcanus . The PSII complex in the crystals contained the D1/D2 reaction center subunits, CP47 and CP43 (two chlorophyll-binding core antenna proteins of photosystem II), cytochrome b-559 alpha- and beta-subunits, several low molecular weight subunits, and three extrinsic proteins, that is, 33 and 12 kDa proteins and cytochrome c-550 . The PSII complex also retained a high rate of oxygen evolution . The apparent molecular mass of the PSII in the crystals was determined to be 580 kDa by gel filtration chromatography, indicating that the PSII crystallized is a dimer . The crystals diffracted to a maximum resolution of 3.5 A at a cryogenic temperature using X-rays from a synchrotron radiation source, SPring-8 . The crystals belonged to an orthorhombic system, and the space group was P2(1)2(1)2(1) with unit cell dimensions of a = 129.7 A, b = 226.5 A, and c = 307.8 A . Each asymmetric unit contained one PSII dimer, which gave rise to a specific volume (V(M)) of 3.6 A(3)/Da based on the calculated molecular mass of 310 kDa for a PSII monomer and an estimated solvent content of 66% . Multiple data sets of native crystals have been collected and processed to 4.0 A, indicating that our crystals are suitable for structure analysis at this resolution.

J Mol Biol, 2000 Dec 8, 304(4), 657 - 68
Thermostability and thermoactivity of citrate synthases from the thermophilic and hyperthermophilic archaea, Thermoplasma acidophilum and Pyrococcus furiosus; Arnott MA et al.; Citrate synthases from Thermoplasma acidophilum (optimal growth at 55 degrees C) and Pyrococcus furiosus (100 degrees C) are homo-dimeric enzymes that show a high degree of structural homology with each other, and thermostabilities commensurate with the environmental temperatures in which their host cells are found . A comparison of their atomic structures with citrate synthases from mesophilic and psychrophilic organisms has indicated the potential importance of inter-subunit contacts for thermostability, and here we report the construction and analysis of site-directed mutants of the two citrate synthases to investigate the contribution of these interactions . Three sets of mutants were made: (a) chimeric mutants where the large (inter-subunit contact) and small (catalytic) domains of the T . acidophilum and P . furiosus enzymes were swapped; (b) mutants of the P . furiosus citrate synthase where the inter-subunit ionic network is disrupted; and (c) P . furiosus citrate synthase mutants in which the C-terminal arms that wrap around their partner subunits have been deleted . All three sets of mutant enzymes were expressed as recombinant proteins in Escherichia coli and were found to be catalytically active . Kinetic parameters and the dependence of catalytic activity on temperature were determined, and the stability of each enzyme was analysed by irreversible thermal inactivation experiments . The chimeric mutants indicate that the thermostability of the whole enzyme is largely determined by the origin of the large, inter-subunit domain, whereas the dependence of catalytic activity on temperature is a function of the small domain . Disruption of the inter-subunit ionic network and prevention of the C-terminal interactions both generated enzymes that were substantially less thermostable . Taken together, these data demonstrate the crucial importance of the subunit contacts to the stability of these oligomeric enzymes . Additionally, they also provide a clear distinction between thermostability and thermoactivity, showing that stability is necessary for, but does not guarantee, catalytic activity at elevated temperatures .

Appl Environ Microbiol, 2000 Dec, 66(12), 5360 - 7
Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides; Deutsch SM et al.; The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening . To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used . Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry . Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp . lactis ITG LL14 and ITG LL51, L . delbrueckii subsp . bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein . The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry . Regardless of strain, L . helveticus was the most efficient in hydrolyzing beta-casein peptides . Interestingly, cell extracts of S . thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline . With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities . Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.

Appl Environ Microbiol, 2000 Dec, 66(12), 5306 - 11
Physiological study of Lactobacillus delbrueckii subsp . bulgaricus strains in a novel chemically defined medium; Chervaux C et al.; We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp . bulgaricus (L . bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h(-1) . MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus . The growth characteristics of L . bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved . For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose . Lactose concentrations below 0.4% were limiting for growth . We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842 . CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter . In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain . We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL . The MPL medium should thus be useful in conducting physiological studies of L . bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.

Appl Environ Microbiol, 2000 Dec, 66(12), 5128 - 33
Branched-chain amino acid biosynthesis is essential for optimal growth of Streptococcus thermophilus in milk; Garault P et al.; Lactic acid bacteria are nutritionally demanding bacteria which need, among other things, amino acids for optimal growth . We identified the branched-chain amino acid (BCAA) biosynthesis pathway as an essential pathway for optimal growth of Streptococcus thermophilus in milk . Through random insertional mutagenesis, we isolated and characterized two mutants for which growth in milk is affected as a consequence of ilvB and ilvC gene interruptions . This situation demonstrates that the BCAA biosynthesis pathway is active in S . thermophilus . BCAA biosynthesis is necessary but not sufficient for optimal growth of S . thermophilus and is subject to retro-inhibition processes . The specificity of the BCAA biosynthesis pathway in S . thermophilus lies in the independent transcription of the ilvC gene encoding a keto acid reductoisomerase acting on acetolactate at the junction of the BCAA and acetoin biosynthesis pathways . The possible advantages for S . thermophilus of keeping this biosynthesis pathway active could be linked either to adaptation of the organism to milk, which is different than that of other dairy bacteria, or to the role of the pathway in maintaining the internal pH.

Biochem Biophys Res Commun, 2000 Nov 30, 278(3), 621 - 6
Molecular characterization of the SerL paralogs of Tetrahymena thermophila; Doerder FP et al.; In the pond ciliate Tetrahymena thermophila, expression of genes encoding variant forms of the cell surface immobilization antigen (i-ag) is regulated by environmental conditions . Multiple isoforms of the L i-ags are found on the surface of cells grown at <20 degrees C as well as on the surface of rseC mutants which express SerL genes constitutively . Five cDNAs encoding variant L i-ags of rseC were sequenced and their expression studied . Two additional SerL genes from natural isolates were sequenced . Members of the SerL family encode polypeptides with 148, 316, or 371 amino acids, and the i-ags have two, five, or six imperfect repeats, respectively, flanked by putative ER translocation and GPI addition signals . Each repeat contains six periodic cysteines, in contrast to eight or ten in other i-ags of T . thermophila . At least three of the five genes constitutively expressed in rseC mutants are differentially expressed in cells expressing other i-ags . Northern analysis and RT-PCR indicate that expression of some members of the SerL family is regulated by both transcription and mRNA stability while another member is regulated primarily by mRNA stability .

Biochemistry (Mosc), 2000 Oct, 65(10), 1157 - 66
Interaction of T . thermophilus phenylalanyl-tRNA synthetase with the 3'-terminal nucleotide of tRNAPhe; Vasil'eva IA et al.; The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3;-terminal nucleotide of tRNAPhe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3;-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates . tRNAPhe derivatives containing a photoreactive 4-thiouridine (tRNAPhe-s4U-76) or 6-thioguanosine residue (tRNAPhe-s6G-76) in the 3;-end have been prepared using terminal tRNA nucleotidyl transferase . Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3;-terminal adenosine in productive interaction of tRNAPhe with the enzyme: tRNAPhe-s4U-76 cannot be aminoacylated; the replacement of A-76 with s6G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased Vmax value . Relative cross-linking of the s6G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNAPhe-s4U-76 . The dialdehyde derivative, tRNAPhe-Aox-76, with periodate-oxidized 3;-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNAPhe-s6G-76 . The results suggest specific binding of the 3;-terminal nucleotide of tRNAPhe by the catalytic subunit of PheRS in the absence of other substrates . Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNAPhe acceptor end with PheRS . The correct positioning of the 3;-terminal nucleotide of tRNAPhe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.

J Bacteriol, 2000 Dec, 182(24), 7014 - 20
pING family of conjugative plasmids from the extremely thermophilic archaeon Sulfolobus islandicus: insights into recombination and conjugation in Crenarchaeota; Stedman KM et al.; A novel family of conjugative plasmids from Sulfolobus comprising the active variants pING1, -4, and -6 and the functionally defective variants pING2 and -3, which require the help of an active variant for spreading, has been extensively characterized both functionally and molecularly . In view of the sparse similarity between bacterial and archaeal conjugation and the lack of a practical genetic system for Sulfolobus, we compared the functions and sequences of these variants and the previously described archaeal conjugative plasmid pNOB8 in order to identify open reading frames (ORFs) and DNA sequences that are involved in conjugative transfer and maintenance of these plasmids in Sulfolobus . The variants pING4 and -6 are reproducibly derived from pING1 in vivo by successive transpositions of an element from the Sulfolobus genome . The small defective but mobile variants pING2 and -3, which both lack a cluster of highly conserved ORFs probably involved in plasmid transfer, were shown to be formed in vivo by recombinative deletion of the larger part of the genomes of pING4 and pING6, respectively . The efficient occurrence of these recombination processes is further evidence for the striking plasticity of the Sulfolobus genome.

Appl Microbiol Biotechnol, 2000 Oct, 54(4), 521 - 7
Molecular characterization of xynX, a gene encoding a multidomain xylanase with a thermostabilizing domain from Clostridium thermocellum; Kim H et al.; A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined . The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa) . Sequence analysis of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain . A deletion mutant of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability . In terms of half-life at 70 degrees C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme, XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain . The optimum temperature of XynX-C was about 5-10 degrees C lower than that of XynX-TC.

Biochim Biophys Acta, 2000 Nov 30, 1543(1), 189 - 201
Distributions of structural features contributing to thermostability in mesophilic and thermophilic alpha/beta barrel glycosyl hydrolases; Panasik N et al.; Analysis of the structural basis for thermostability in proteins has come mainly from pairwise comparisons of mesophilic and thermophilic structures and has often yielded conflicting results . Interpretation of these results would be enhanced by knowing the normal range of features found for mesophilic proteins . In order to provide the average and distribution values of structural features among similar mesophilic proteins, we compared the amino acid composition, solvent accessible surface area, hydrogen bonds, number of ion pairs, and thermal factors of 22 structures of alpha/beta barrel glycosyl hydrolases . These distributions are then compared to values from seven alpha/beta barrel glycosyl hydrolases from thermophilic organisms . We find that the distribution of each structural feature is broad within the mesophilic proteins and illustrates the difficulty of making pairwise comparisons of mesophiles to thermophiles where differences for individual proteins may be within the normal range for the group . In comparing mesophiles to thermophiles as a group, we find that thermophilic structures have fewer glycines in a particular region of the structure and higher thermal factors at room temperature . These results suggest the basis for thermostability may be related to protein motion rather than to static features of protein structure.

Biochim Biophys Acta, 2000 Nov 30, 1543(1), 131 - 45
A primitive myoglobin from Tetrahymena pyriformis: its heme environment, autoxidizability, and genomic DNA structure; Korenaga S et al.; A myoglobin-like protein isolated from Tetrahymena pyriformis is composed of 121 amino acid residues . This is much smaller than sperm whale myoglobin by 32 residues, suggesting a distinct origin from the common globin gene . We have therefore examined this unique protein for its structural, spectral and stability properties . As a result, the rate of autoxidation of Tetrahymena oxymyoglobin (MbO(2)) was found to be almost comparable to that of sperm whale MbO(2) over a wide range of pH 4-12 in 0.1 M buffer at 25 degrees C . Moreover, both pH profiles exhibited the remarkable proton-assisted process, which can be performed in sperm whale myoglobin by the distal (E7) histidine as its catalytic residue . These kinetic observations are also in full accord with spectral examinations for the presence of a distal histidine in ciliated protozoa myoglobin . At the same time, we have isolated the globin genes both from T . pyriformis and Tetrahymena thermophila, and found that there is no intron in their genomic structures . This is in sharp contrast to previous reports on the homologous globin genes from Paramecium caudatum and Chlamydomonas eugametos . Rather, the Tetrahymena genes seemed to be related to the cyanobacterial globin gene from Nostoc commune . These contracted or truncated globins thus have a marked diversity in the cDNA, protein, and genomic structures.

Biochemistry, 2000 Nov 28, 39(47), 14481 - 6
Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica; Radfar R et al.; Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures . The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified . Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does . Neither binding site is located in the active site . The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding . Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules . The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98 . Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions . The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature . No significant increase was observed in E98D or E98S . The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding . The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme . These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.

Biochemistry, 2000 Nov 21, 39(46), 14356 - 62
Purification and initial characterization of RNA polymerase from Thermus thermophilus strain HB8; Xue Y et al.; Utilizing a novel and rapid two-column purification procedure, the DNA-dependent RNA polymerase (RNAP) from the thermophile, Thermus thermophilus HB8, was purified to electrophoretic homogeneity with a recovery of 65% (as determined by RNAP activity) in less than 2 days . The purified enzyme was characterized using DNA containing the lambdaP(R) promoter . KMnO(4) footprinting, abortive initiation assays, and the formation of the specific stalled elongation complex provide compelling evidence that T . thermophilus RNA polymerase can bind to DNA containing the lambdaP(R) promoter, form an open complex, and initiate transcription in a temperature-dependent manner . This evidence suggests that T . thermophilus RNAP possesses less intrinsic binding energy than E . coli RNAP . Instead, T . thermophilus relies on the high temperatures of its environment to provide the thermal energy required to stimulate open promoter complex formation, initiate transcription, and facilitate the conformational changes in RNA polymerase that result in nucleotide incorporation.

Carbohydr Res, 2000 Oct 20, 329(1), 65 - 73
Comparative study of new alpha-galactosidases in transglycosylation reactions; Spangenberg P et al.; We have studied the potential of several newly cloned alpha-galactosidases to catalyze the regioselective synthesis of disaccharides using 4-nitrophenylgalactoside as a donor . The kinetics of the reactions were followed by in situ NMR spectroscopy . The following thermophilic enzymes have been tested: Aga A and an isoenzyme Aga B obtained from the strain KVE39 and Aga 285 from the strain IT285 of Bacillus stearothermophilus; Aga T is an alpha-galactosidase from Thermus brockianus (strain IT360) . Two other non-thermophilic alpha-galactosidases have also been evaluated: Aga 1 (Streptococcus mutans, strain Ingbritt) and Raf A (Escherichia coli, strain D1021) . For all of the enzymes studied, high regioselectivity was observed leading to two (1 --> 6)-disaccharides: 4-nitrophenyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside and methyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, respectively.

Structure Fold Des, 2000 Nov 15, 8(11), 1203 - 14
Buried charged surface in proteins; Kajander T et al.; BACKGROUND: The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent . Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps . RESULTS: By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed . We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces . In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried . Furthermore, at all sizes few charged groups are fully exposed . As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure . In addition, free energy calculations of stability support the experimental results . CONCLUSIONS: Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment . Consistent with this view, thermophilic proteins often have less buried charge . Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.

Structure Fold Des, 2000 Oct 15, 8(10), R195 - 200
The bacterial ribosome at atomic resolution; Brimacombe R; X-ray crystallographic structures have just been published for the 30S ribosomal subunit of Thermus thermophilus at 3.4 A resolution and for the 50S subunit of Haloarcula marismortui at 2.4 A . These eagerly awaited structures will provide an enormous boost to research into the mechanisms involved in protein biosynthesis.

Gene, 2000 Oct 31, 257(2), 319 - 26
Sequence and expression of the SerJ immobilization antigen gene of Tetrahymena thermophila regulated by dominant epistasis; Doerder FP; In ciliates, variable surface protein genes encoding the immobilization antigen (-ag) are expressed under different environmental conditions, including temperature and salt stress . These i-ags are GPI-linked and coat the entire external surface of the cell, including the cilia . In Tetrahymena thermophila-ag in natural isolates is the result of dominant epistasis masking the expression of the H i-ag ordinarily expressed at 20-36 degrees C . This report describes the expression and sequence of the Ser-ag . J is present on the cell surface up to 38 degrees C; above 38 degrees C SerSeranked by an A-rich 5' UTR and a 3' UTR containing putative mRNA destabilization motifs . The encoded J polypeptide consists of 438 amino acids and is rich in alanine, cysteine, serine and threonine . The N- and resemble signal peptide and GPI-anchor addition sites, respectively . The majority of the molecule consists of four imperfect repeats with 10 periodic cysteines per repeat in the pattern CX(6)CX(2)CX(21)CX(4)CX(13-15)CX(2)CX(18)CX(3)CX(11)CX(9-10) . Although H i-ags encoded by paralogous SerH genes have 3.5 imperfect repeats with eight periodic cysteines per repeat, J nevertheless resembles H with respect to amino acid composition, codon usage, N- and C-termini, the arrangement of the cysteine periods, and regulation by mRNA stability . However, despite these similarities and epistasis, the evolutionary relationship between SerH and SerJ is unclear.

Biochim Biophys Acta, 2001 Nov 15, 1524(1), 27 - 37
Purification and characterization of alpha-galactosidase from a thermophilic fungus Thermomyces lanuginosus; Puchart V et al.; An extracellular alpha-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus . Molecular mass of the enzyme is 57 kDa . The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity . Isoelectric point of the major form was 5.2 . Enzyme is the most active against aryl alpha-D-galactosides but efficiently hydrolyzed alpha-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan . In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers . Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl alpha-galactopyranoside and galactosyl(1)mannotriose, followed by (1)H NMR spectroscopy, pointed out the alpha-anomer of D-galactose was the primary product of hydrolysis from which the beta-anomer was formed by mutarotation . Hence the enzyme is a retaining glycosyl hydrolase . In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl alpha-galactopyranoside as a glycosyl donor . Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.

Biochim Biophys Acta, 2001 Nov 15, 1524(1), 1 - 10
Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl; Martin DD et al.; Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates beta-glutamate and L-alpha-glutamate as osmolytes when grown in media with <1 M NaCl . When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N(epsilon)-acetyl-beta-lysine, is synthesized and becomes the dominant osmolyte . Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M . thermolithotrophicus to osmotic stress . There is a temporal hierarchy in the response of intracellular osmolytes . Changes in intracellular K(+) occur within the first few minutes of altering the external NaCl . Upon hypoosmotic shock, K(+) is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale . Upon hyperosmotic shock, M . thermolithotrophicus immediately internalizes K(+), far more than would be needed stoichiometrically to balance the new salt concentration . This is followed by a decrease to a new K(+) concentration (over 10-15 min), at which point synthesis and accumulation of primarily L-alpha-glutamate occur . Once growth of the M . thermolithotrophicus culture begins, typically 30-100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N(epsilon)-acetyl-beta-lysine) begins to represent a larger fraction of the intracellular pool . The observation that N(epsilon)-acetyl-beta-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N(epsilon)-acetyl-beta-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl . That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.

RNA, 2000 Oct, 6(10), 1432 - 44
Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch; Toyoda T et al.; Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides . The crystal structure of Thermus thermophilus RRF was determined at 2.6 A resolution . It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2) . Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349-2352), the interdomain angle differs by 33 degrees in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing . The hinge connects hydrophobic junctions of domains 1 and 2 . The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF . First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop . Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop . These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility . These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a "gooseneck" elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other . Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G . These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.

AIHAJ, 2000 Sep-Oct, 61(5), 727 - 32
Assessment of particulates and bioaerosols in eastern Canadian sawmills; Duchaine C et al.; The purpose of this study was to quantify and identify the airborne contamination in eastern Canadian sawmills . Seventeen sawmills were chosen to cover a wide range of size, geographic distribution, and wood species processed . Within each sawmill different work sites (debarking, sawing, sorting, or planing) were studied separately . Area sampling was performed for exposure assessment . Microbial contaminants were assessed with all-glass impingers 30 and six-stage Andersen microbial samplers; appropriate selective media and culture conditions for bacteria, thermophilic actinomycetes, molds, and yeasts were used . Inhalable dust, endotoxins, temperature, and humidity also were measured . Penicillium species were the most predominant molds with up to 40 different Penicillium species identified . Debarking was the working site most highly contaminated by molds, bacteria, and endotoxins (p=0.0001) . At this working site mold levels reached a maximum of 1.5 x 10(6) CFU/m3, whereas the median values for culturable bacteria and endotoxin were 21,620 CFU/m3 and 1,081 endotoxin units/m3, respectively . Planing sites were the most highly dust contaminated (median: 3.0 mg/m3) (p <0.05) . Sawmills of eastern Canada contain airborne biological contaminants that vary between working sites, and their microflora is different from that previously described in European sawmills.

Mol Microbiol, 2000 Oct, 38(2), 177 - 85
About the last common ancestor, the universal life-tree and lateral gene transfer: a reappraisal; Glansdorff N; An organismal tree rooted in the bacterial branch and derived from a hyperthermophilic last common ancestor (LCA) is still widely assumed to represent the path followed by evolution from the most primeval cells to the three domains recognized among contemporary organisms: Bacteria, Archaea and Eucarya . In the past few years, however, more and more discrepancies between this pattern and individual protein trees have been brought to light . There has been an overall tendency to attribute these incongruities to widespread lateral gene transfer . However, recent developments, a reappraisal of earlier evidence and considerations of our own lead us to a quite different view . It would appear (i) that the role of lateral gene transfer was overemphasized in recent discussions of molecular phylogenies; (ii) that the LCA was probably a non-thermophilic protoeukaryote from which both Archaea and Bacteria emerged by reductive evolution but not as sister groups, in keeping with a current evolutionary scheme for the biosynthesis of membrane lipids; and (iii) that thermophilic Archaea may have been the first branch to diverge from the ancestral line.

Lett Appl Microbiol, 2000 Nov, 31(5), 378 - 84
Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54; Malhotra R et al.; AIM: An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization . METHODS AND RESULTS: The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h . When the enzyme was partially purified using acetone (80%{v/v} saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded . The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively . The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C . Both alpha-amylase activity and production were Ca2+ independent . CONCLUSIONS: Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase . SIGNIFICANCE AND IMPACT OF THE STUDY: The calcium-independent and thermostable alpha-amylase of B . thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent . The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.

J Biol Chem, 2001 Mar 2, 276(9), 6537 - 44 Epub 2000 Nov 07.
Recombinant cytochrome rC557 obtained from Escherichia coli cells expressing a truncated Thermus thermophilus cycA gene . Heme inversion in an improperly matured protein; McRee DE et al.; Cytochrome rC(557) is an improperly matured, dimeric cytochrome c obtained from expression of the "signal peptide-lacking" Thermus thermophilus cycA gene in the cytoplasm of Escherichia coli . It is characterized by its Q(00) (or alpha-) optical absorption band at 557 nm in the reduced form (Keightley, J . A., Sanders, D., Todaro, T . R., Pastuszyn, A., and Fee, J . A . (1998) J . Biol . Chem . 273, 12006-12016) . We report results of a broad ranging, biochemical and spectral characterization of this protein that reveals the presence of a free vinyl group on the porphyrin and a disulfide bond between the protomers and supports His-Met ligation in both valence states of the iron . A 3-A resolution x-ray structure shows that, in comparison with the native protein, the heme moiety is rotated 180 degrees about its alpha,gamma-axis; cysteine 14 has formed a thioether bond with the 2-vinyl of pyrrole ring I instead of the 4-vinyl of pyrrole ring II, as occurs in the native protein; and a cysteine 11 from each protomer has formed an intermolecular disulfide bond . Numerous, minor perturbations exist within the structure of rC(557) in comparison with that of native protein, which result from heme inversion and protein-protein interactions across the dimer interface . The unusual spectral properties of rC(557) are rationalized in terms of this structure.

Microbiology, 2000 Nov, 146 ( Pt 11), 2793 - 802
The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide; Almiron-Roig E et al.; The cpsFGHIJKL genes from the cps cluster of Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of EPS were identified, cloned and nucleotide sequenced . The complete cps cluster is contained on an approximately 11.2 kb chromosomal region which contains 12 ORFs, including the previously cloned cpsABCDE genes . Functions were assigned to some of the predicted gene products on the basis of homology to known sequences as follows: cpsK encodes a protein thought to be involved in the polymerization and export of the polysaccharide; cpsE, cpsF, cpsG, cpsH, cpsI and cpsJ encode putative sugar transferases . Two insertion sequences, IS1193 and ISS1, were identified within and flanking the 3' end of the cps cluster respectively . Analysis of the expression of the cpsE gene in Escherichia coli demonstrated that it encodes a glucose-1-phosphate transferase; the enzyme which catalyses the first step in EPS biosynthesis in S . thermophilus NCFB 2393.

Biotechnol Bioeng, 2000 Dec 20, 70(6), 630 - 7
Modeling of olive oil degradation and oleic acid inhibition during chemostat and batch cultivation of Bacillus thermoleovorans IHI-91; Becker P et al.; Olive oil degradation by the thermophilic lipolytic strain Bacillus thermoleovorans IHI-91 in chemostat and batch culture was modeled to obtain a general understanding of the underlying principles and limitations of the process and to quantify its stoichiometry . Chemostat experiments with olive oil as the sole carbon source were successfully described using the Monod chemostat model extended by terms for maintenance requirements and wall growth . Maintenance requirements and biomass yield coefficients were in the range reported for mesophiles . For a chemostat experiment at D = 0.3 h(-1) the model was validated up to an olive oil feed concentration of about 3.0 g L(-1) above which an inhibitory effect occurred . Further analysis showed that the liberated oleic acid is the main cause for this inhibition . Using steady-state oleic acid concentrations measured in chemostat experiments with olive oil as substrate it was possible to derive a kinetic expression for oleic acid utilization, showing that a concentration of 430 mg L(-1) leads to a complete growth inhibition . Oleic acid accumulation observed during batch fermentations can be predicted using a model involving growth-associated lipase production and olive oil hydrolysis . Simulations confirmed that this accumulation is the cause for the sudden growth cessation occurring in batch fermentations with higher olive oil start concentrations . Further, an oscillatory behavior, as observed in some chemostat experiments, can also be predicted using the latter model . This work clearly demonstrates that thermophilic lipid degradation by Bacillus thermoleovorans IHI-91 is limited by long-chain fatty acid beta-oxidation rather than oil hydrolysis.

FEMS Microbiol Lett, 2000 Nov 15, 192(2), 169 - 73
High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate; Okubo Y et al.; We examined the effect of the pyridoxal 5'-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures . The decrease in the enzyme activity at incubation temperatures over 40 degrees C was consistent with the decrease in the amount of bound PLP . Unfolding of the enzyme at temperatures above 40 degrees C was suppressed in the presence of PLP . In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60 degrees C.

Appl Occup Environ Hyg, 2000 Nov, 15(11), 835 - 42
Indoor air quality in a middle school, Part II: Development of emission factors for particulate matter and bioaerosols; Scheff PA et al.; A middle school (grades 6 to 8) in a residential section of Springfield, Illinois, with no known air quality problems, was selected for a baseline indoor air quality survey . The study was designed to measure and evaluate air quality at the middle school with the objective of providing a benchmark for comparisons with measurements in schools with potential air quality problems . The focus of this article is on the development of emission factors for particulate matter and bioaerosols . The school was characterized as having no health complaints and good maintenance schedules . Four indoor locations including the cafeteria, a science classroom, an art classroom, the lobby outside the main office, and one outdoor location were sampled for various environmental comfort and pollutant parameters for one week in February 1997 . Integrated samples (eight-hour sampling time) for respirable and total particulate matter, and short-term measurements (two-minute samples, three times per day) for bioaerosols were collected on three consecutive days at each of the sampling sites . Continuous measurements of carbon dioxide were logged at all locations for five days . Continuous measurements of respirable particulate matter were also collected in the lobby area . A linear relationship between occupancy and corresponding carbon dioxide and particle concentrations was seen . A completely mixed space, one compartment mass balance model with estimated CO2 generation rates and actual CO2 and particulate matter concentrations was used to model ventilation and pollutant emission rates . Emission factors for occupancy were represented by the slope of emission rate versus occupancy scatter plots . The following particle and bioaerosol emission factors were derived from the indoor measurements: total particles: 1.28 mg/hr/person-hr; respirable particles: 0.154 g/hr/person-hr; total fungi: 167 CFU/hr/person-min; thermophilic fungi: 35.8 CFU/hr/person-min; mesophilic fungi: 119 CFU/hr/person-min; total bacteria: 227 CFU/hr/person-min; gram-negative bacteria: 69.5 CFU/hr/person-min; gram-positive bacteria: 191 CFU/hr/person-min; Aspergillus: 17.0 CFU/hr/person-min; Penicillium: 161 CFU/hr/person-min; and yeasts: 16.4 CFU/hr/person-min.

Nat Struct Biol, 2000 Nov, 7(11), 1046 - 50
MJ0109 is an enzyme that is both an inositol monophosphatase and the 'missing' archaeal fructose-1,6-bisphosphatase; Stec B et al.; In sequenced genomes, protein coding regions with unassigned function constitute between 10 and 50% of all open reading frames . Often key enzymes cannot be identified using sequence homology searches . For example, despite the fact that methanogens have an apparently functional gluconeogenesis pathway, standard tools have been unable to identify a fructose-1,6-bisphosphatase (FBPase) gene in the sequenced Methanoccocus jannaschii genome . Using a combination of functional and structural tools, we have shown that the protein product of the M . jannaschii gene MJ0109, which had been tentatively annotated as an inositol monophosphatase (IMPase), has both IMPase and FBPase activities . Moreover, several gene products annotated as IMPases from different thermophilic organisms also possess FBPase activity . Thus, we have found the FBPase that was 'missing' in thermophiles and shown that it also functions as an IMPase.

J Mol Biol, 2000 Nov 10, 303(5), 761 - 71
A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase; De Simone G et al.; EST2 is a novel thermophilic carboxylesterase, isolated and cloned from Alicyclobacillus (formerly Bacillus) acidocaldarius, which optimally hydrolyses esters with acyl chain lengths of six to eight carbon atoms at 70 degrees C . On the basis of the amino acid sequence homology, it has been classified as a member of the mammalian hormone-sensitive lipase (HSL) subfamily.The crystal structure of EST2, complexed with a sulphonyl derivative, has been determined at 2.6 A resolution by a multiple wavelength anomalous diffraction experiment on a seleno-methionine derivative . EST2 presents a canonical alpha/beta hydrolase core, shielded at the C-terminal side by a cap region built up of five helices . It contains the lipase-like catalytic triad, Ser155, His282 and Asp252, whereby the nucleophile is covalently modified . This allows an unambiguous view of the putative active site of EST2, detecting the oxyanion hole, in whose formation the amino acid sequence motif His81-Gly82-Gly83-Gly84 is involved, and the hydrophobic binding pocket for the acyl chain . The structural model here reported provides the first example of a transition state analogue of an esterase/lipase belonging to the HSL group, thus affording useful information for the design of medical inhibitors . Moreover, as the first X-ray structure of a thermophilic carboxylesterase, the comparison with its mesophilic homologue, the Brefeldin A esterase (BFAE) from Bacillus subtilis, allows the identification of putative determinants of thermal stability .

Extremophiles, 2000 Oct, 4(5), 315 - 20
Microbiology of acidic, geothermal springs of Montserrat: environmental rDNA analysis; Burton NP et al.; DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Caribbean island of Montserrat . 16S rRNA genes were amplified by PCR, cloned, sequenced, and examined to indicate some of the organisms that might be significant components of the in situ microbiota . A clone bank representing the lowest temperature pool that was sampled (33 degrees C) was dominated by genes corresponding to two types of acidophiles: Acidiphilium-like mesophilic heterotrophs and thermotolerant Acidithiobacillus caldus . Three clone types with origins in low- and moderate- (48 degrees C) temperature pools corresponded to bacteria that could be involved in metabolism of sulfur compounds: the aerobic A . caldus and putative anaerobic, moderately thermophilic, sulfur-reducing bacteria (from an undescribed genus and from the Desulfurella group) . A higher-temperature sample indicated the presence of a Ferroplasma-like organism, distinct from the other strains of these recently recognized acidophilic, iron-oxidizing members of the Euryarchaeota . Acidophilic Archaea from undescribed genera related to Sulfolobus and Acidianus were predicted to dominate the indigenous acidophilic archaeal population at the highest temperatures.

Extremophiles, 2000 Oct, 4(5), 305 - 13
A microbiological survey of Montserrat Island hydrothermal biotopes; Atkinson T et al.; In March 1996, a survey of hydrothermal sites on the island of Montserrat was carried out . Six sites (Galway's Soufriere . Gages Upper and Lower Soufrieres, Hot Water Pond, Hot River, and Tar River Soufriere) were mapped and sampled for chemical, ATP, and microbial analyses . The hydrothermal Soufriere sites on the slopes of the active Chances Peak volcano exhibited temperatures up to almost 100 degrees C and were generally either mildly acidic at pH 5-7 or strongly acidic at pH 1.5-3, but with some hot streams and pools of low redox potential at pH 7-8 . Hot Water Pond sites, comprising a series of heated pools near the western shoreline of the island . were neutral and saline, consistent with subsurface heating of entrained seawater . Biological activity shown by ATP analyses was greatest in near-neutral pH samples and generally decreased as acidity increased . A variety of heterotrophic and chemolithotrophic thermophilic organisms were isolated or observed in enrichment cultures . Most of the bacteria that were obtained in pure culture were familiar acidophiles and neutrophiles, but novel, iron-oxidizing species of Sulfobacillus were revealed . These species included the first mesophilic iron-oxidizing Sulfobacillus strains to be isolated and a strain with a higher maximum growth temperature (65 degrees C) than the previously described moderately thermophilic Sulfobacillus species.

Extremophiles, 2000 Oct, 4(5), 291 - 6
Sucrose transport by the alkaliphilic, thermophilic Bacillus sp . strain TA2.A1 is dependent on a sodium gradient; Peddie CJ et al.; An alkaliphilic Bacillus designated strain TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70 degrees C . Sodium chloride (>5mM) was an obligate requirement for the growth of strain TA2.A1 on sucrose, and growth on sucrose was inhibited by monensin, an ionophore that collapses the sodium gradient (ApNa+) across the cell membrane . Sucrose transport by strain TA2.A1 was sodium dependent and was inhibited by monensin . The Kt for sucrose transport was 33 microM and the Eadie-Hofstee plot was linear, suggesting one high-affinity uptake system for sucrose . The affinity for sodium was low (0.5 mM), and the Hill plot had a slope of 1.6, suggesting that sodium binding was noncooperative and that the sucrose transporter had more than one binding site for sodium . Based on these results, Bacillus strain TA2.A1 uses a sodium gradient for sucrose uptake, in addition to the sodium-dependent glutamate uptake system reported previously.

Extremophiles, 2000 Oct, 4(5), 279 - 84
The intracellular pH of the thermophilic bacterium Thermoanaerobacter wiegelii during growth and production of fermentation acids; Cook GM; The thermophilic glycolytic anaerobe Thermoanaerobacter wiegelii grows over the pH range 5.1-7.7, and no growth is observed below pH 5.0 or above 7.7 . When T . wiegelii was grown in pH-uncontrolled batch culture, glucose was fermented to ethanol, acetate, and lactate . Small amounts of lactic acid were produced once the external pH reached 6.0, and a fructose-1.6-diphosphate (FDP) activated lactate dehydrogenase (LDH) was detected in cell-free crude extracts . Maximal activation of LDH by FDP was observed at pH 6.2 . As the pH of the medium declined from 6.7 to 5.1 due to the production of acetate and lactate, the total protonmotive force (deltap) remained between - 110 and - 130mV, and the membrane potential (dekltapsi) decreased from -104 to -65mV . This decrease in deltapsi was paralleled by an increase in the chemical gradient of protons (ZdeltapH) from -31 to -62mV at pH 5.1 . Based on these results, T . wiegelii maintained a small deltapH (0.3-0.9 units, inside alkaline) as the medium pH declined and interconverted deltapsi to ZdeltapH to maintain the total deltap relatively constant . Intracellular potassium decreased from 150 mM at pH 6.70 to 50 mM at pH 5.1, and this represented a 33-mV decline in the transmembrane chemical potential of potassium . The ability to synthesize ATP remained constant as the external pH declined, and therefore metabolic energy per se was not a critical aspect of pH sensitivity.

Chemosphere, 2000 Aug, 41(3), 297 - 302
Aerobic thermophilic treatment of sewage sludge contaminated with 4-nonylphenol; Banat FA et al.; 4-Nonylphenol (4-NP) occurs in sewage sludge as a result of the breakdown of detergents which contains nonylphenol ethoxylates (NPEs) . 4-NP is of environmental concern because of its toxicity to biological systems . The present paper reports results of aerobic treatment under thermophilic conditions of sewage sludge artificially contaminated with 4-NP . Experiments were carried out using three parallel laboratory-scale batch reactors operating with blank, 50 and 100 mg/l of 4-NP concentration . For the two studied concentrations up to 66% 4-NP reduction was achieved at a specific air flow rate of 16 l/(l.h) and a thermophilic temperature of 60 degrees C, within 10 days of operation . The presence of 4-NP has minor effect on the rate of sludge oxidation and the nitrogen and phosphorous content in the sludge.

Appl Environ Microbiol, 2000 Nov, 66(11), 5066 - 72
Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes; Takai K et al.; We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities . A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed . A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed . Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved . The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant . Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.

Appl Environ Microbiol, 2000 Nov, 66(11), 4817 - 21
Advances in development of a genetic system for Thermoanaerobacterium spp.: expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome; Mai V et al.; Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described . We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485 . Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin . To develop further the genetic system for T . saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed . Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.

Appl Environ Microbiol, 2000 Nov, 66(11), 4772 - 8
Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization; Fernandez-Espla MD et al.; Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels . Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme) . The pure enzyme is a serine proteinase highly activated by Ca(2+) ions . Its activity was optimal at 37 degrees C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate . The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types . This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues . Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family . It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif . PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB) . The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.

Gene, 2000 Oct 3, 256(1-2), 215 - 21
Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway; Duffner FM et al.; Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway . Five enzymes used in the metabolism of phenol were cloned from B . thermoglucosidasius A7 into pUC18 . Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities . The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2 . The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens . Both components were neccessary for activity . The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp . CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping . The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp . strain CF600, encoded by dmpD . Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.

J Mol Biol, 2000 Nov 3, 303(4), 593 - 603
Structure of a mutant EF-G reveals domain III and possibly the fusidic acid binding site; Laurberg M et al.; The crystal structure of Thermus thermophilus elongation factor G (EF-G) carrying the point mutation His573Ala was determined at a resolution of 2.8 A . The mutant has a more closed structure than that previously reported for wild-type EF-G . This is obtained by a 10 degrees rigid rotation of domains III, IV and V with regard to domains I and II . This rotation results in a displacement of the tip of domain IV by approximately 9 A . The structure of domain III is now fully visible and reveals the double split beta-alpha-beta motif also observed for EF-G domain V and for several ribosomal proteins . A large number of fusidic acid resistant mutations found in domain III have now been possible to locate . Possible locations for the effector loop and a possible binding site for fusidic acid are discussed in relation to some of the fusidic acid resistant mutations .

Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1367 - 75
Structure of XynB, a highly thermostable beta-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 A resolution; McCarthy AA et al.; Microorganisms employ a large array of enzymes to break down the cellulose and hemicelluloses of plant biomass . These enzymes, especially those with high thermal stability, have many uses in biotechnology . We have solved the crystal structure of a beta-1, 4-xylanase, XynB, from the extremely thermophilic bacterium Dictyoglomus thermophilum, isolate Rt46B.1 . The protein crystallized from 1.6 M ammonium sulfate, 0.2 M HEPES pH 7.2 and 10% glycerol, with unit-cell parameters a = b = 91.3, c = 44.9 A and space group P4(3) . The structure was solved at high resolution (1.8 A) by X-ray crystallography, using the method of isomorphous replacement with a single mercury derivative, and refined to a final R factor of 18.3% (R(free) = 22.1%) . XynB has the single-domain fold typical of family 11 xylanases, comprising a jelly roll of two highly twisted beta-sheets that create a deep substrate-binding cleft . The two catalytic residues, Glu90 and Glu180, occupy this cleft . Compared with other family 11 xylanases, XynB has a greater proportion of polar surface and has a slightly extended C-terminus that, combined with the extension of beta-strand A5, gives additional hydrogen bonding and hydrophobic packing . These factors may account for the enhanced thermal stability of the enzyme.

J Bacteriol, 2000 Nov, 182(22), 6424 - 33
A DNA ligase from a hyperthermophilic archaeon with unique cofactor specificity; Nakatani M et al.; A gene encoding DNA ligase (lig(Tk)) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, has been cloned and sequenced, and its protein product has been characterized . lig(Tk) consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da . Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig(Tk) was an ATP-dependent DNA ligase . Phylogenetic analysis indicated that Lig(Tk) was closely related to the ATP-dependent DNA ligase from Methanobacterium thermoautotrophicum DeltaH, a moderate thermophilic archaeon, along with putative DNA ligases from Euryarchaeota and Crenarchaeota . We expressed lig(Tk) in Escherichia coli and purified the recombinant protein . Recombinant Lig(Tk) was monomeric, as is the case for other DNA ligases . The protein displayed DNA ligase activity in the presence of ATP and Mg(2+) . The optimum pH of Lig(Tk) was 8.0, the optimum concentration of Mg(2+), which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K(+) was 10 to 30 mM . Lig(Tk) did not display single-stranded DNA ligase activity . At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100 degrees C . Unexpectedly, Lig(Tk) displayed a relatively small, but significant, DNA ligase activity when NAD(+) was added as the cofactor . Treatment of NAD(+) with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD(+) solution . This unique cofactor specificity was also supported by the observation of adenylation of Lig(Tk) with NAD(+) . This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.

Biochemistry, 2000 Oct 31, 39(43), 13285 - 94
Enhancement of the enzymatic activity of ribonuclease HI from Thermus thermophilus HB8 with a suppressor mutation method; Hirano N et al.; A genetic method for isolating a mutant enzyme of ribonuclease HI (RNase HI) from Thermus thermophilus HB8 with enhanced activity at moderate temperatures was developed . T . thermophilus RNase HI has an ability to complement the RNase H-dependent temperature-sensitive (ts) growth phenotype of Escherichia coli MIC3001 . However, this complementation ability was greatly reduced by replacing Asp(134), which is one of the active site residues, with His, probably due to a reduction in the catalytic activity . Random mutagenesis of the gene encoding the resultant D134H enzyme, followed by screening for second-site revertants, allowed us to isolate three single mutations (Ala(12) --> Ser, Lys(75) --> Met, and Ala(77) --> Pro) that restore the normal complementation ability to the D134H enzyme . These mutations were individually or simultaneously introduced into the wild-type enzyme, and the kinetic parameters of the resultant mutant enzymes for the hydrolysis of a DNA-RNA-DNA/DNA substrate were determined at 30 degrees C . Each mutation increased the k(cat)/K(m) value of the wild-type enzyme by 2.1-4.8-fold . The effects of the mutations on the enzymatic activity were roughly cumulative, and the combination of these three mutations increased the k(cat)/K(m) value of the wild-type enzyme by 40-fold (5.5-fold in k(cat)) . Measurement of thermal stability of the mutant enzymes with circular dichroism spectroscopy in the presence of 1 M guanidine hydrochloride and 1 mM dithiothreitol showed that the T(m) value of the triple mutant enzyme, in which all three mutations were combined, was comparable to that of the wild-type enzyme (75.0 vs 77.4 degrees C) . These results demonstrate that the activity of a thermophilic enzyme can be improved without a cost of protein stability.

Biochemistry, 2000 Oct 31, 39(43), 13115 - 26
Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution; Benini S et al.; This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium . Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins . Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K . The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment . In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein . For the 0.97-A data, the phasing was based on a single data set . This is the most precise structure of a heme protein to date . The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus . This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure . The heme prosthetic group in B . pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement . The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion . The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges . A structural search of a representative set of protein structures reveals that B . pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes . Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein . In B . pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins . The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.

Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 11910 - 5
A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions; Michelitsch MD et al.; Glutamine/asparagine (Q/N)-rich domains have a high propensity to form self-propagating amyloid fibrils . This phenomenon underlies both prion-based inheritance in yeast and aggregation of a number of proteins involved in human neurodegenerative diseases . To examine the prevalence of this phenomenon, complete proteomic sequences of 31 organisms and several incomplete proteomic sequences were examined for Q/N-rich regions . We found that Q/N-rich regions are essentially absent from the thermophilic bacterial and archaeal proteomes . Moreover, the average Q/N content of the proteins in these organisms is markedly lower than in mesophilic bacteria and eukaryotes . Mesophilic bacterial proteomes contain a small number (0-4) of proteins with Q/N-rich regions . Remarkably, Q/N-rich domains are found in a much larger number of eukaryotic proteins (107-472 per proteome) with diverse biochemical functions . Analyses of these regions argue they have been evolutionarily selected perhaps as modular "polar zipper" protein-protein interaction domains . These data also provide a large pool of potential novel prion-forming proteins, two of which have recently been shown to behave as prions in yeast, thus suggesting that aggregation or prion-like regulation of protein function may be a normal regulatory process for many eukaryotic proteins with a wide variety of functions.

Protein Expr Purif, 2000 Nov, 20(2), 162 - 8
Purification of alpha-amylase isoenzymes from Scytalidium thermophilum on a fluidized bed of alginate beads followed by concanavalin A-agarose column chromatography; Roy I et al.; An alpha-amylase has been purified from the thermophilic fungus Scytalidium thermophilum . A ninefold purification was achieved in a single step using fluidized bed chromatography wherein alginate was used as the affinity matrix . There are at least two isoenzymes as shown by concanavalin A (Con A)-agarose column chromatography . The isoenzyme binding to Con A is stable for at least 3 h at 80 degrees C in the presence of calcium ions . The isoenzymes have similar molecular weights of around 45,000 Da as shown by SDS-PAGE analysis . The isoenzymes differ only slightly in their pH optima and temperature optima but the isoenzyme binding to Con A-agarose has slightly higher thermal stability .

Arch Latinoam Nutr, 2000 Mar, 50(1), 81 - 6
{Elaboration of an yogurt made of a milk and chickpea (Cicer arietinum) mixture}; Morales de Leon JC et al.; The objective of this work was to establish the experimental conditions for the production of yogurt extended with chickpea (Cicer arietinum), inoculated with St . thermophilus and L . bulgaricus and compare its chemical, microbiological and sensorial characteristics versus a yogurt made of skimmed milk . Results indicated that 70:30 and 80:20 (skimmed milk and chickpea extract) mixtures obtained by chemical score fulfilled with the proposed objectives . Yogurt made with 70:30 mixture added with modified starch (ULTRA SPERCE M and COL-FLO), did not remove syneresis present in these products and did not improve its sensory characteristics neither . Nevertheless, yogurt made with 80:20 mixture and modified starch (ULTRA SPERCE M) removed syneresis and present flavor and texture characteristics alike yogurt made of milk, this "extended" yogurt was accepted by the 80% of the judge and fulfill with yogurt specification established in the mexican regulations for this products.

Mol Cell Biol, 2000 Nov, 20(22), 8319 - 28
Developmentally regulated rpd3p homolog specific to the transcriptionally active macronucleus of vegetative Tetrahymena thermophila; Wiley EA et al.; A clear relationship exists between histone acetylation and transcriptional output, the balance of which is conferred by opposing histone acetyltransferases (HATs) and histone deacetylases (HDACs) . To explore the role of HDAC activity in determining the transcriptional competency of chromatin, we have exploited the biological features of Tetrahymena as a model . Each vegetative cell contains two nuclei: a somatic, transcriptionally active macronucleus containing hyperacetylated chromatin and a transcriptionally silent, germ line micronucleus containing hypoacetylated histones . Using a PCR-based strategy, a deacetylase gene (named THD1) encoding a homolog of the yeast HDAC Rpd3p was cloned . Thd1p deacetylates all four core histones in vitro . It resides exclusively in the macronucleus during vegetative growth and is asymmetrically distributed to developing new macronuclei early in their differentiation during the sexual pathway . Together, these data are most consistent with a potential role for Thd1p in transcriptional regulation and suggest that histone deacetylation may be important for the differentiation of micronuclei into macronuclei during development.

Biotechnol Bioeng, 2000 Dec 5, 70(5), 491 - 7
Toxicity effects of compressed and supercritical solvents on thermophilic microbial metabolism; Berberich JA et al.; Selection of biocompatible solvents is critical when designing bioprocessing applications for the in situ biphasic extraction of metabolic end-products . The prediction of the biocompatibility of supercritical and compressed solvents is more complicated than for liquid solvents, because their properties can change significantly with pressure and temperature . The activity of the anaerobic thermophilic bacterium, Clostridium thermocellum, was studied when the organism was incubated in the presence of compressed nitrogen, ethane, and propane at 333 K and multiple pressures . The metabolic activity of the organisms in contact with compressed solvents was analyzed using traditional indicators of solvent biocompatibility, such as log P, interfacial tension, and solvent density . The toxicity of the compressed solvents was compared with the phase and molecular toxicity effects measured in liquid alkanes at atmospheric pressure . Inactivation increased with time in the presence of the compressed solvents, but was constant in the presence of atmospheric liquid solvents . Knowledge of molecular and phase toxicity provides a framework for the interpretation of C . thermocellum metabolism in contact with atmospheric and compressed solvents .

J Struct Biol, 2000 Aug, 131(2), 83 - 9
Two different oligomeric states of the RuvB branch migration motor protein as revealed by electron microscopy; Miyata T et al.; In prokaryotes, the RuvA, B, and C proteins play major roles at the late stage of DNA homologous recombination, where RuvB complexed with RuvA acts as an ATP-dependent motor for branch migration . The oligomeric structures of negatively stained and frozen hydrated RuvB from Thermus thermophilus HB8 were investigated by electron microscopy . RuvB oligomers free of DNA formed a ring structure of about 14 nm in diameter . The averaged top view image clearly indicated a sevenfold symmetry, suggesting that it exists as a heptamer . The RuvB oligomers complexed with duplex DNA formed a smaller ring of about 13 nm in diameter . The averaged top view images represented a sixfold symmetry . This difference in oligomerization indicates that the oligomeric structure of RuvB may convert from a heptamer to a hexamer upon DNA binding . In addition, this finding provides the lesson that great care should be taken in investigating the subunit organizations of DNA binding proteins, because their oligomeric states are more sensitive to DNA interactions than expected .

J Dairy Res, 2000 Aug, 67(3), 381 - 92
DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction; De Urraza PJ et al.; DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR) . The method made it possible to identify 37 isolates from raw milk . industrial starters and yogurt . Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp . lactis, Lb . delbrueckii subsp bulgaricus and Str . thermophilus . BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.

Curr Microbiol, 2000 Mar, 40(3), 194 - 9
Biodegradability of food-associated extracellular polysaccharides; Ruijssenaars HJ et al.; Exopolysaccharides (EPSs) produced by lactic acid bacteria, which are common in fermented foods, are claimed to have various beneficial physiological effects on humans . Although the biodegradability of EPSs is important in relation to the bioactive properties, knowledge on this topic is limited . Therefore, the biodegradability of eight EPSs, six of which were produced by lactic acid bacteria, was compared with microorganisms from human feces or soil . EPS-degradation was determined from the decrease in polysaccharide-sugar concentration and by high-performance size exclusion chromatography (HPSEC) . Xanthan, clavan, and the EPSs produced by Streptococcus thermophilus SFi 39 and SFi 12 were readily degraded, in contrast to the EPSs produced by Lactococcus lactis ssp . cremoris B40, Lactobacillus sakei 0-1, S . thermophilus SFi20, and Lactobacillus helveticus Lh59 . Clearly, the susceptibility of exopolysaccharides to biological breakdown can differ greatly, implying that the physiological effects of these compounds may also vary a lot.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1829 - 32
Symbiobacterium thermophilum gen . nov., sp . nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth; Ohno M et al.; A Gram-negative and tryptophanase-positive thermophile, whose growth is dependent on co-culture with an associating Bacillus strain, had been reported and tentatively named Symbiobacterium thermophilum strain T(T) . Axenic culture of strain T(T) was recently established by dialysing cultures with the supporting bacterial strains or adding their culture broth . Phylogenetic analysis of strain T(T), based on the 16S rDNA sequence, was conducted for the validation of S . thermophilum . The sequence of strain T(T) was located at the outermost position in the high-G+C Gram-positive group distinctly isolated from any other branches hitherto known . Ten sequences identical to that of strain T(T), and one sequence closely related to it, were identified for the first time from soil and compost samples . The outer membrane of strain T(T) had a three-layered structure, outside the cytoplasmic membrane, which is similar to the S-layer in the cells of members of the Bacillaceae . Chemical analysis of the cells revealed that menaquinone-6 is a major component of the quinone system . According to these results, along with several previous observations (i.e . a G+C DNA content of 65 mol% and the identification of iso-C15:0 and iso-C17:0 acids as major cellular fatty acids), the new taxon Symbiobacterium thermophilum gen . nov., sp . nov . is proposed . The type strain is S . thermophilum strain T(T) (= IAM 14863T).

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1741 - 53
Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich archipelago, with the proposal of Bacillus fumarioli sp . nov; Logan NA et al.; Aerobic endospore-forming bacteria were isolated from soils taken from active fumaroles on Mount Rittmann and Mount Melbourne in northern Victoria Land, Antarctica, and from active and inactive fumaroles on Candlemas Island, South Sandwich archipelago . The Mt Rittmann and Mt Melbourne soils yielded a dominant, moderately thermophilic and acidophilic, aerobic endospore-former growing at pH 5.5 and 50 degrees C, and further strains of the same organism were isolated from a cold, dead fumarole at Clinker Gulch, Candlemas Island . Amplified rDNA restriction analysis, SDS-PAGE and routine phenotypic tests show that the Candlemas Island isolates are not distinguishable from the Mt Rittmann strains, although the two sites are 5600 km apart, and 16S rDNA sequence comparisons and DNA relatedness data support the proposal of a new species, Bacillus fumarioli, the type strain of which is LMG 17489T.

J Biol Chem, 2001 Jan 12, 276(2), 1345 - 52
The structure of the chloroplast F1-ATPase at 3.2 A resolution; Groth G et al.; The structure of the F(1)-ATPase from spinach chloroplasts was determined to 3.2 A resolution by molecular replacement based on the homologous structure of the bovine mitochondrial enzyme . The crystallized complex contains four different subunits in a stoichiometry of alpha(3)beta(3)gammaepsilon . Subunit delta was removed before crystallization to improve the diffraction of the crystals . The overall structure of the noncatalytic alpha-subunits and the catalytic beta-subunits is highly similar to those of the mitochondrial and thermophilic subunits . However, in the crystal structure of the chloroplast enzyme, all alpha- and beta-subunits adopt a closed conformation and appear to contain no bound adenine nucleotides . The superimposed crystallographic symmetry in the space group R32 impaired an exact tracing of the gamma- and epsilon-subunits in the complex . However, clear electron density was present at the core of the alpha(3)beta(3)-subcomplex, which probably represents the C-terminal domain of the gamma-subunit . The structure of the spinach chloroplast F(1) has a potential binding site for the phytotoxin, tentoxin, at the alphabeta-interface near betaAsp(83) and an insertion from betaGly(56)-Asn(60) in the N-terminal beta-barrel domain probably increases the thermal stability of the complex . The structure probably represents an inactive latent state of the ATPase, which is unique to chloroplast and cyanobacterial enzymes.

J Biol Chem, 2001 Jan 26, 276(4), 2432 - 9 Epub 2000 Oct 16.
Characterization of the intramolecular electron transfer pathway from 2-hydroxyphenazine to the heterodisulfide reductase from Methanosarcina thermophila; Murakami E et al.; Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens . HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM . HDR from Methanosarcina thermophila contains two b-hemes and two {Fe(4)S(4)} clusters . The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen) . This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM . Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm . Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2) . Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2) . We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 439 - 44
Thermophilic acidification of dairy wastewater; Yu HQ et al.; Acidification of simulated dairy wastewater was conducted in an upflow reactor at 55 degrees C . Results showed that the degree of acidification decreased with the increase in chemical oxygen demand (COD) loading rate, from 60.8% at 4 g l(-1) day(-1) to 27.1% at 24 g l(-1) day(-1) . Carbohydrate was readily degraded at all loading rates, but degradation of protein and lipid decreased with the increase in loading rate . Most carbohydrate degradation occurred at the reactor bottom, whereas protein was degraded mainly after the carbohydrate became depleted . The predominant acidification products were acetate, propionate, butyrate and ethanol, whereas formate, i-butyrate, valerate, i-valerate, caproate, lactate, methanol, propanol and butanol were present in lesser quantities . The increase in loading rate resulted in the increase of propionate and the decrease of acetate, but had little effect on ethanol and butyrate productions . Only 2.5-8.8% of influent COD was converted to hydrogen and methane . The biomass yield was 0.30-0.43 mg VSS mg(-1) COD.

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 390 - 6
Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila; Hartmann M et al.; We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hypersecretion of phospholipase A1 (PLA1) . Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA1-hypersecretory mutants were isolated . The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants . About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening . Of these, four selected mutants showed 1.8-2.2 more PLA1 activity in the cell-free supernatants compared to the wild-type strain CU 438.1 . Hypersecretion was only observable for PLA1; no increased activity for two other lysosomal enzymes could be detected . These hypersecretory mutants of T . thermophila can be very useful for increasing the yield of PLA1 in fermentation processes . This is particularly relevant because, in contrast to other phospholipases, PLA1 is not available on the commercial market for fine chemicals and little is known about the role of PLA1 in cell signaling and metabolism.

J Bacteriol, 2000 Nov, 182(21), 6036 - 41
Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine; Hopfner KP et al.; The processing of DNA double-strand breaks is a critical event in nucleic acid metabolism . This is evidenced by the severity of phenotypes associated with deficiencies in this process in multiple organisms . The core component involved in double-strand break repair in eukaryotic cells is the Mre11-Rad50 protein complex, which includes a third protein, p95, in humans and Xrs2 in yeasts . Homologues of Mre11 and Rad50 have been identified in all kingdoms of life, while the Nbs1 protein family is found only in eukaryotes . In eukaryotes the Mre11-Rad50 complex has nuclease activity that is modulated by the addition of ATP . We have isolated the Mre11 and Rad50 homologues from the thermophilic archaeon Pyrococcus furiosus and demonstrate that the two proteins exist in a large, heat-stable complex that possesses single-strand endonuclease activity and ATP-dependent double-strand-specific exonuclease activity . These findings verify the identification of the P . furiosus Rad50 and Mre11 homologues and demonstrate that functional homologues with similar biochemical properties exist in all kingdoms of life.

J Bacteriol, 2000 Nov, 182(21), 5982 - 9
Control of lactose transport, beta-galactosidase activity, and glycolysis by CcpA in Streptococcus thermophilus: evidence for carbon catabolite repression by a non-phosphoenolpyruvate-dependent phosphotransferase system sugar; van den Bogaard PT et al.; Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source . Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS . In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, beta-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate . A catabolite-responsive element (cre) was identified in the promoter of the lacSZ operon, indicating a possible role for regulation by CcpA . Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose . This CcpA-mediated repression of lacSZ transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system . Lactose transport during fermentation was increased significantly in strains carrying a disrupted ccpA gene . Moreover, a ccpA disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose . Transcriptional analysis of the ldh gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner . A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a ccpA disruption mutant . We propose that CcpA in S . thermophilus acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose . In contrast to other bacteria, S . thermophilus possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux.

Curr Opin Biotechnol, 2000 Oct, 11(5), 497 - 504
Advances in the genetics of thermophilic lactic acid bacteria; Delcour J et al.; Molecular genetics of thermophilic lactic acid bacteria has advanced in several directions: exploitation of the milk proteins and sugars; primary and secondary metabolism; stress response; and molecular ecology of bacteria and their phages . These have singularly contributed to open new avenues of scientific interest in the field: comparative phage genomics; horizontal gene transfer events in bacterial or phage populations; and genetics of external polysaccharide production.

FEMS Microbiol Lett, 2000 Oct 15, 191(2), 243 - 7
Genetic analysis of Carboxydothermus hydrogenoformans carbon monoxide dehydrogenase genes cooF and cooS; Gonzalez JM et al.; Carboxydothermus hydrogenoformans is an extremely thermophilic, Gram-positive bacterium growing on carbon monoxide (CO) as single carbon and energy source and producing only H(2) and CO(2) . Carbon monoxide dehydrogenase is a key enzyme for CO metabolism . The carbon monoxide dehydrogenase genes cooF and cooS from C . hydrogenoformans were cloned and sequenced . These genes showed the highest similarity to the cooF genes from the archaeon Archaeoglobus fulgidus and the cooS gene from the bacterium Rhodospirillum rubrum, respectively . The cooS gene was identified immediately downstream of cooF, however, the cooF and cooS genes from C . hydrogenoformans have substantially different codon usage, and the cooF gene Arg codon usage pattern, dominated by AGA and AGG, resembles the archaeal pattern . The data therefore suggest lateral transfer of these genes, possibly from different donor species.

Nucleic Acids Res, 2000 Oct 15, 28(20), 3999 - 4004
Elongation of repetitive DNA by DNA polymerase from a hyperthermophilic bacterium Thermus thermophilus; Ogata N et al.; Short repetitive DNA sequences are believed to be one of the primordial genetic elements that served as a source of complex large DNA found in the genome of modern organisms . However, the mechanism of its expansion (increase in repeat number) during the course of evolution is unclear . We demonstrate that the DNA polymerase of the hyperthermophilic bacterium Thermus thermophilus can elongate oligoDNA with several tandem repeats to very long DNA in vitro . For instance, 48mer repetitive oligoDNA (TACATGTA)(6), which has 25% GC content and a palindromic sequence, can be elongated up to approximately 10 000 bases by DNA polymerase at 74 degrees C without template DNA . OligoDNA having a different GC content or a quasi-palindromic sequence can also be elongated, but less efficiently . A spectroscopic thermal melting experiment with the oligoDNA showed that its hairpin-coil transition temperature was very close to the elongation reaction temperature (74 degrees C), but was much higher than the temperature at which duplex oligoDNA can exist stably . Taken together, we conclude that repetitive oligoDNA with a palindromic or quasi-palindromic sequence is elongated extensively by a hyperthermophilic DNA polymerase through hairpin-coil transitions . We propose that such an elongation mechanism might have been a driving force to expand primordial short DNA.

Nucleic Acids Res, 2000 Oct 15, 28(20), 3910 - 7
PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif; Bohlke K et al.; The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans . This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain . Five different mutations of the tyrosine at position 387 (Tyr387-->Phe, Tyr387-->Trp, Tyr387-->His, Tyr387-->Asn and Tyr387-->Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme . Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity . Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation . With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis.

J Biol Chem, 2000 Dec 29, 275(52), 40703 - 9
DNA binding and protein-protein interaction sites in MutS, a mismatched DNA recognition protein from Thermus thermophilus HB8; Tachiki H et al.; The mismatch repair system repairs mismatched base pairs, which are caused by either DNA replication errors, DNA damage, or genetic recombination . Mismatch repair begins with the recognition of mismatched base pairs in DNA by MutS . Protein denaturation and limited proteolysis experiments suggest that Thermus thermophilus MutS can be divided into three structural domains as follows: A (N-terminal domain), B (central domain), and C (C-terminal domain) (Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K., and Kuramitsu, S . (1998) Nucleic Acids Res . 26, 4153-4159) . To investigate the functions of each domain in detail, truncated genes corresponding to the domains were designed . The gene products were overproduced in Escherichia coli, purified, and assayed for various activities . The MutS-MutS protein interaction site was determined by size-exclusion chromatography to be located in the B domain . The B domain was also found to possess nonspecific double-stranded DNA-binding ability . The C domain, which contains a Walker's A-type nucleotide-binding motif, demonstrated ATPase activity and specific DNA recognition of mismatched base pairs . These ATPase and specific DNA binding activities were found to be dependent upon C domain dimerization.

J Mol Biol, 2000 Oct 20, 303(2), 329 - 44
Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual amino acid modification, conservation and adaptation; Grabarse W et al.; The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes the terminal step of methane formation in the energy metabolism of all methanogenic archaea . In this reaction methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide of coenzyme M and coenzyme B . The crystal structures of methyl-coenzyme M reductase from Methanosarcina barkeri (growth temperature optimum, 37 degrees C) and Methanopyrus kandleri (growth temperature optimum, 98 degrees C) were determined and compared with the known structure of MCR from Methanobacterium thermoautotrophicum (growth temperature optimum, 65 degrees C) . The active sites of MCR from M . barkeri and M . kandleri were almost identical to that of M . thermoautotrophicum and predominantly occupied by coenzyme M and coenzyme B . The electron density at 1.6 A resolution of the M . barkeri enzyme revealed that four of the five modified amino acid residues of MCR from M . thermoautotrophicum, namely a thiopeptide, an S-methylcysteine, a 1-N-methylhistidine and a 5-methylarginine were also present . Analysis of the environment of the unusual amino acid residues near the active site indicates that some of the modifications may be required for the enzyme to be catalytically effective . In M . thermoautotrophicum and M . kandleri high temperature adaptation is coupled with increasing intracellular concentrations of lyotropic salts . This was reflected in a higher fraction of glutamate residues at the protein surface of the thermophilic enzymes adapted to high intracellular salt concentrations .

J Mol Biol, 2000 Oct 20, 303(2), 125 - 30
Increasing the thermostability of staphylococcal nuclease: implications for the origin of protein thermostability; Chen J et al.; Seven hyper-stable multiple mutants have been constructed in staphylococcal nuclease by various combinations of eight different stabilizing single mutants . The stabilities of these multiple mutants determined by guanidine hydrochloride denaturation were 3.4 to 5.6 kcal/mol higher than that of the wild-type . Their thermal denaturation midpoint temperatures were 12.6 to 22.9 deg . C higher than that of the wild-type . These are among the greatest increases in protein stability and thermal denaturation midpoint temperature relative to the wild-type yet attained . There has been great interest in understanding how proteins found in thermophilic organisms are stabilized . One frequently cited theory is that the packing of hydrophobic side-chains is improved in the cores of proteins isolated from thermophiles when compared to proteins from mesophiles . The crystal structures of four single and five multiple stabilizing mutants of staphylococcal nuclease were solved to high resolution . No large overall structural change was found, with most changes localized around the sites of mutation . Rearrangements were observed in the packing of side-chains in the major hydrophobic core, although none of the mutations was in the core . It is surprising that detailed structural analysis showed that packing had improved, with the volume of the mutant protein's hydrophobic cores decreasing as protein stability increased . Further, the number of van der Waals interactions in the entire protein showed an experimentally significant increase correlated with increasing stability . These results indicate that optimization of packing follows as a natural consequence of increased protein thermostability and that good packing is not necessarily the proximate cause of high stability . Another popular theory is that thermostable proteins have more electrostatic and hydrogen bonding interactions and these are responsible for the high stabilities . The mutants here show that increased numbers of electrostatic and hydrogen bonding interactions are not obligatory for large increases in protein stability .

Enzyme Microb Technol, 2000 Nov 1, 27(8), 560 - 569
Avicel-adsorbable endoglucanase production by the thermophilic fungus Scytalidium thermophilum type culture Torula thermophila; Arifoglu N et al.; Scytalidium thermophilum type culture Torula thermophila was isolated from mushroom compost and the total cellulase, endoglucanase, Avicel-adsorbable endoglucanase activities, as well as the fungal biomass generation and cellulose utilisation were analyzed in shake flask cultures with Avicel (microcrystalline cellulose) as the carbon source . Results were compared with an industrial strain of Scytalidium thermophilum type culture Humicola insolens . The pH and temperature optima for endoglucanase activities during enzyme assays were also analyzed for both organisms and determined to be pH 6.0 and 65 degrees C for type culture Torula thermophila, and pH 6.5 and 60 degrees C for type culture Humicola insolens . Analysis of the effect of growth temperature showed that type culture T . thermophila can grow and produce cellulases in the range of 35 to 55 degrees C although 40 to 50 degrees C seemed to favor growth and cellulase production . Although 45 degrees C was found optimal for fungal growth, both the specific endoglucanase and Avicel-adsorbable endoglucanase activities (U/mg protein) as well as the percentage of Avicel-adsorbable endoglucanase activity reached maxima at 50 degrees C and were higher as compared to type culture H . insolens . Results indicate that type culture T . thermophila, with further optimisations, is of potential use in the industrial production of cellulases.

FEBS Lett, 2000 Sep 29, 482(1-2), 159 - 62
Observation of RecA protein monomer by small angle X-ray scattering with synchrotron radiation; Kato R et al.; RecA protein is capable of forming homo-oligomers in solution . The oligomeric and monomeric states of Thermus thermophilus RecA protein were studied by small angle X-ray scattering, a direct method used to measure the overall dimensions of a macromolecule . In the presence of 3 M urea or 0.2 M lithium perchlorate, RecA dissociates from higher oligomeric states to form a hexamer with a radius of gyration (R(g)) of 52 A . The value of R(g) decreased to 36 A at a higher lithium perchlorate concentration (1.0 M) . The zero angle intensity, I(0), was consistent with the identification of the former state as a hexamer and the latter as a monomer.

Biochimie, 2000 Aug, 82(8), 765 - 72
Proteolytic fragmentation of polypeptide release factor 1 of Thermus thermophilus and crystallization of the stable fragments; Tin OF et al.; Polypeptide release factor one from Thermus thermophilus, ttRF1, was purified and subjected to crystallization . Thin crystalline needles were obtained but their quality was not satisfactory for X-ray diffraction . Stable fragments of ttRF1 suitable for crystallization were screened by limited proteolysis . Three major fragments were produced by thermolysinolysis and analyzed by N-terminal sequencing and electrospray mass spectrometry . They were N-terminal fragments generated by proteolysis at amino acid positions 211, 231 and 292 . The corresponding recombinant polypeptides, ttRF1(211), ttRF1(231) and ttRF1(292), were overproduced and subjected to crystallization . Of these polypeptides, ttRF1(292) gave rise to crystals that belong to P3(1) (or P3(2)) space group with unit cell parameters a = b = 64 . 5 A, c = 86.6 A and diffract up to 7 A resolution.

Nat Struct Biol, 2000 Oct, 7(10), 827 - 8
The road to RNase P; Altman S; In 1989, Sidney Altman and Thomas R . Cech shared the Nobel Prize in Chemistry for their discovery of catalytic properties of RNA . Cech was studying the splicing of RNA in a unicellular organism called Tetrahymena thermophila . He found that the precursor RNA could splice in vitro in the absence of proteins . Altman studied ribonuclease P (RNase P), a ribonucleoprotein that is a key enzyme in the biosynthesis of tRNA . RNase P is an RNA processing endonuclease that specifically cleaves precursors of tRNA, releasing 5' precursor sequences and mature tRNAs . RNase P is involved in processing all species of tRNA and is present in all cells and organelles that carry out tRNA synthesis . What follows is a personal recollection by Altman of how he came to study this remarkable enzyme.

Nature, 2000 Sep 21, 407(6802), 327 - 39
Structure of the 30S ribosomal subunit; Wimberly BT et al.; Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria . Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution . The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly . It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics . The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

Gig Sanit, 1998 Nov-Dec, (6), 24 - 7
{Effect of thermal power stations on the sanitary and biological conditions of water reservoirs}; Solovykh GN et al.; Discharge of thermal waters from power stations can result in the development of thermophilic microorganisms in the water reservoirs and increased water pollution . Increased water temperature changes relationships between lysozyme-active and antilysozyme-active bacteria . The quality of water gets worse.

Rev Argent Microbiol, 2000 Jul-Sep, 32(3), 153 - 6
{Thermophilic endospores in the environment of a sugar mill in Jujuy}; Carrillo L; Twenty six samples from green and scorched sugarcane stems and leaves, sugarmill air dust and raw sugar were analyzed . Thirty nine thermophilic bacilli strains were isolated . Physiological and morphological examinations were carried out according to Bergey's Manual . The strains were identified as B . licheniformis (66.7%), B . coagulans (17.9%), B . stearothermophilus (10.3%) y B . subtilis (5.1%).

Cell, 2000 Sep 1, 102(5), 615 - 23
Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution; Schluenzen F et al.; The small ribosomal subunit performs the decoding of genetic information during translation . The structure of that from Thermus thermophilus shows that the decoding center, which positions mRNA and three tRNAs, is constructed entirely of RNA . The entrance to the mRNA channel will encircle the message when a latch-like contact closes and contributes to processivity and fidelity . Extended RNA helical elements that run longitudinally through the body transmit structural changes, correlating events at the particle's far end with the cycle of mRNA translocation at the decoding region . 96% of the nucleotides were traced and the main fold of all proteins was determined . The latter are either peripheral or appear to serve as linkers . Some may assist the directionality of translocation.

Can J Microbiol, 2000 Sep, 46(9), 817 - 25
Physical degradation of wheat straw by the in-vessel and windrow methods of mushroom compost production; Lyons GA et al.; Mushroom compost manufacturers in Ireland are moving away from the traditional outdoor phase I windrow method, favouring in-vessel production . Composters and growers have reported better quality compost with faster spawn run and higher yields produced by this process . In the present study, physical examination of samples highlighted differences when comparing the windrow and in-vessel methods of compost production . Observations using scanning electron microscopy suggest that the cuticle of wheat straw from in-vessel production is damaged during phase I, peeling away from the surface in fragments, and exposing the epidermis . Changes in silicon levels on the straw surface acted as a marker for cuticle damage when comparing both composting systems . Cuticle damage may be important during composting and afterwards, as substrate colonisation is faster, and consequently spawn run is shorter . The phase I compost microbial community is altered by the in-vessel technique, producing a predominantly thermophilic bacterial flora in contrast to the mesophilic and thermophilic bacteria and fungi found in windrow phase I compost . These differences may be significant in mushroom compost production.

FEMS Microbiol Lett, 2000 Oct 1, 191(1), 103 - 7
The alternative sigma factor sigma(28) of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant; Studholme DJ et al.; Sigma factor sigma(28) (sigma(F), FliA, SigD) directs RNA polymerase to transcribe the genes required for flagellar biosynthesis and chemotaxis in many bacteria, including Bacillus subtilis, Legionella pneumophila, Salmonella typhimurium, Escherichia coli, Yersinia enterolytica, Treponema maltophilum and Pseudomonas aeruginosa . Remarkably the fliA gene from the extreme thermophile Aquifex aeolicus restored motility to the E . coli mutant at relatively low temperature, albeit partially . This clearly demonstrates that A . aeolicus sigma(28) is able to direct RNA polymerase to E . coli sigma(28)-dependent promoters and take part in the complex interactions required to support transcription of the flagellar apparatus in vivo . The ability of A . aeolicus sigma(28) to function with mesophilic components shows that critical functional interactions made by these sigma factors are well conserved, and are not dependent upon high temperature . We over-produced and purified the sigma(28) protein and demonstrated binding to E . coli core RNA polymerase in vitro . In common with SigD from B . subtilis, but unlike most sigma factors, A . aeolicus sigma(28) showed DNA binding activity in vitro but there was no evidence of sequence specificity . We note that A . aeolicus sigma(28) is a good candidate for structural studies.

FEMS Microbiol Lett, 2000 Oct 1, 191(1), 79 - 85
Purification, characterisation, cloning and sequencing of the gene encoding oligopeptidase PepO from Streptococcus thermophilus A; Chavagnat F et al.; The oligopeptidase PepO from Streptococcus thermophilus A was purified to protein homogeneity by a five-step chromatography procedure . It was estimated to be a serine metallopeptidase of 70 kDa, with maximal activity at pH 6.5 and 41 degrees C . PepO has endopeptidase activity on oligopeptides composed of between five and 30 amino acids . PepO was demonstrated to be active and stable at the pH, temperature and salt concentrations found in Swiss-type cheese during ripening . Using a battery of PCR techniques, the pepO gene was amplified, subcloned and sequenced, revealing an open reading frame of 1893 nucleotides . The amino acid sequence analysis of the pepO gene-translation product shows homology with PepO enzymes from other lactic acid bacteria and contains the signature sequence of the metallopeptidase family.

J Bacteriol, 2000 Oct, 182(20), 5911 - 5
Organization and expression of a Thermus thermophilus arginine cluster: presence of unidentified open reading frames and absence of a Shine-Dalgarno sequence; Sanchez R et al.; A group of genes regulated by arginine was found clustered in the order argF-ORF1-argC-argJ-ORF4 between other, as yet uncharacterized, open reading frames (ORFs) . Transcription starts were identified immediately upstream from argF and ORF4 . Arginine repressed transcription that was initiated at argF but induced transcription of ORF4 . The functions of ORF1 and ORF4 are unknown, but analysis of the sequence of ORF4 suggests that it is a membrane protein, possibly involved in transport of arginine or a related metabolite . Mobility shift and DNase I footprinting have revealed specific binding of pure Escherichia coli ArgR to the promoter region of Thermus thermophilus argF . These results suggest that argF transcription is controlled by a repressor homologous to those characterized in enteric bacteria and bacilli . Thermus argF mRNA is devoid of Shine-Dalgarno (SD) sequences . However, downstream from the ATG start codon of argF and many other Thermus genes (with or without an SD box), sequences were found to be complementary to nucleotides 1392 to 1409 of Thermus 16S rRNA, suggesting that an mRNA-rRNA base pairing in this region is important for correct translation initiation.

Biochim Biophys Acta, 2000 Jul 26, 1475(3), 191 - 206
Pyruvate carboxylase from Mycobacterium smegmatis: stabilization, rapid purification, molecular and biochemical characterization and regulation of the cellular level; Mukhopadhyay B et al.; This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium . The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages . We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene . We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month . The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg(2+), Co(2+), and Mn(2+), by aspartate, but not by glutamate and alpha-ketoglutarate . Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged . These observations would not fit the idea that the M . smegmatis enzyme fulfills a straightforward anaplerotic function; in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme . Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol . The PYCs of M . smegmatis and Mycobacterium tuberculosis were highly homologous to each other . In M . smegmatis, M . tuberculosis and M . lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in an identical operon-like arrangement . Thus, M . smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria . Also, the ease of purification and the extraordinary stability could make the M . smegmatis enzyme a model for studying the structure-function relationships of PYCs in general . It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya.

J Dairy Sci, 2000 Sep, 83(9), 1952 - 6
Influence of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus on Mozzarella cheese and cheese whey; Petersen BL et al.; We investigated the effect of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus starter bacteria on Mozzarella cheese functionality and whey viscosity . Mozzarella cheeses were manufactured with Lactobacillus helveticus LH100 paired with one of four S . thermophilus strains: MR-1C, a bacterium that produces a capsular exopolysaccharide; MTC360, a strain that secretes a ropy exopolysaccharide; TAO61, a nonexopolysaccharide-producing commercial cheese starter; and DM10, a nonencapsulated, exopolysaccharide-negative mutant of strain MR-1C . As expected, cheese moisture levels were significantly higher in Mozzarella cheeses made with exopolysaccharide-positive versus exopolysaccharide-negative streptococci, and melt properties were better in the higher moisture cheeses . Whey viscosity measurements showed that unconcentrated and ultrafiltered, fivefold concentrated whey from cheeses made with S . thermophilus MTC360 were significantly more viscous than whey from cheeses made with MR-1C, TAO61, or DM10 . No significant differences were noted between the viscosity of unconcentrated or concentrated whey from cheeses made with S . thermophilus MR-1C versus the industrial cheese starter TAO61 . These data indicate that encapsulated, but not ropy, exopolysaccharide-producing S . thermophilus strains can be utilized to increase the moisture level of cheese and to improve the melt properties of Mozzarella cheese without adversely affecting whey viscosity.

J Dairy Sci, 2000 Sep, 83(9), 1905 - 11
Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and nonprobiotic microflora in Argentinian Fresco cheese; Vinderola CG et al.; We evaluated the suitability of Argentinian Fresco cheese as a food carrier of probiotic cultures . We used cultures of Bifidobacterium bifidum (two strains), Bifidobacterium longum (two strains), Bifidobacterium sp . (one strain), Lactobacillus acidophilus (two strains), and Lactobacillus casei (two strains) in different combinations, as probiotic adjuncts . Probiotic, lactic starter (Lactococcus lactis and Streptococcus thermophilus), and contaminant (coliforms, yeasts, and molds) organisms were counted at 0, 30, and 60 d of refrigerated storage . Furthermore, the acid resistance of probiotic and starter bacteria was determined from hydrochloric solutions (pH 2 and 3) of Fresco cheese . The results showed that nine different combinations of bifidobacteria and L . acidophilus had a satisfactory viability (count decreases in 60 d <1 log order) in the cheese . Both combinations of bifidobacteria and L . casei cultures assayed also showed a satisfactory survival (counts decreased <1 log order for bifidobacteria but no decrease was detected for L . casei) . On the other hand, the three combinations of bifidobacteria, L . acidophilus, and L . casei tested adapted well to the Fresco cheese environment . When a cheese homogenate at pH 3 was used to partially simulate the acidic conditions in the stomach, the probiotic cultures had an excellent ability to remain viable up to 3 h . At pH 2, the cell viability was more affected; B . bifidum was the most resistant organism . This study showed that the Argentinian Fresco cheese could be used as an adequate carrier of probiotic bacteria.

Nucleic Acids Res, 2000 Oct 1, 28(19), 3811 - 6
Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants; Jackson JD et al.; Saccharomyces cerevisiae contains three genes that encode members of the histone H2A gene family . The last of these to be discovered, HTZ1 (also known as HTA3), encodes a member of the highly conserved H2A.Z class of histones . Little is known about how its in vivo function compares with that of the better studied genes (HTA1 and HTA2) encoding the two major H2As . We show here that, while the HTZ1 gene encoding H2A.Z is not essential in budding yeast, its disruption results in slow growth and formamide sensitivity . Using plasmid shuffle experiments, we show that the major H2A genes cannot provide the function of HTZ1 and the HTZ1 gene cannot provide the essential function of the genes encoding the major H2As . We also demonstrate for the first time that H2A.Z genes are functionally conserved by showing that the gene encoding the H2A.Z variant of the ciliated protozoan TETRAHYMENA: thermophila is able to rescue the phenotypes associated with disruption of the yeast HTZ1 gene . Thus, the functions of H2A.Z are distinct from those of the major H2As and are highly conserved.

Virology, 2000 Sep 30, 275(2), 294 - 305
Comparative genomics of the late gene cluster from Lactobacillus phages; Desiere F et al.; Three prophage sequences were identified in the Lactobacillus johnsoni strain NCC533 . Prophage Lj965 predicted a gene map very similar to those of pac-site Streptococcus thermophilus phages over its DNA packaging and head and tail morphogenesis modules . Sequence similarity linked the putative DNA packaging and head morphogenesis genes at the protein level . Prophage Lj965/S . thermophilus phage Sfi11/Lactococcus lactis phage TP901-1 on one hand and Lactobacillus delbrueckii phage LL-H/Lactobacillus plantarum phage phig1e/Listeria monocytogenes phage A118 on the other hand defined two sublines of structural gene clusters in pac-site Siphoviridae from low-GC Gram-positive bacteria . Bacillus subtilis phage SPP1 linked both sublines . The putative major head and tail proteins from Lj965 shared weak sequence similarity with phages from Gram-negative bacteria . A clearly independent line of structural genes in Siphoviridae from low-GC Gram-positive bacteria is defined by temperate cos-site phages including Lactobacillus gasseri phage adh, which also shared sequence similarity with phage D3 infecting a Gram-negative bacterium . A phylogenetic tree analysis demonstrated that the ClpP-like protein identified in four cos-site Siphoviridae from Lactobacillus, Lactococcus, Streptococcus, and Pseudomonas showed graded sequence relationships . The tree suggested that the ClpP-like proteins from the phages were not acquired by horizontal gene transfer from their corresponding bacterial hosts .

Virology, 2000 Sep 30, 275(2), 267 - 77
Broad-range bacteriophage resistance in Streptococcus thermophilus by insertional mutagenesis; Lucchini S et al.; Streptococcus thermophilus is a lactic acid bacterium used in industrial milk fermentation . To obtain phage-resistant starters, S . thermophilus strain Sfi1 was submitted to mutagenesis with the thermolabile insertional vector pG(+)host9:ISS1 followed by a challenge with the lytic S . thermophilus phage Sfi19 . Vector insertions into four distinct sites led to a phage-resistance phenotype . Three mutants were characterized further . They were protected against the homologous challenging phage and 14 heterologous phages . All three mutants adsorbed phages . No intracellular phage DNA synthesis was observed in mutants R7 and R71, while mutant R24 showed a delayed and diminished phage DNA synthesis compared to the parental Sfi1 strain . In mutant R7 a short deletion occurred next to the insertion site which removed the upstream sequences and the 15 initial codons from orf 394, encoding a likely transmembrane protein . Analogy with other phage systems suggests an involvement of this protein in the phage DNA injection process . In mutant R24 the vector was inserted into orf 269 predicting an oxido-reductase . When the vector sequence was removed via homologous recombination across the duplicated insertion elements, mutant R24 returned to the phage susceptibility of the parental strain . This observation suggested that inactivation of orf 269 was not crucial for the resistance phenotype . A gene encoding a likely restriction subunit of a type I restriction-modification system was located directly downstream of the insertion site in mutant R24 . hsdM and hsdS genes encoding the modification and specificity subunits of a type I R-M system and biological evidence for an active R-M system were detected in strain Sfi1, suggesting involvement of a type I R-M system in the resistance phenotype of R24 .

Structure Fold Des, 2000 Aug 15, 8(8), 875 - 82
Another piece of the ribosome: solution structure of S16 and its location in the 30S subunit; Allard P et al.; BACKGROUND: X-ray crystallography has recently yielded much-improved electron-density maps of the bacterial ribosome and its two subunits and many structural details of bacterial ribosome subunits are now being resolved . One approach to complement the structures and elucidate the details of rRNA and protein packing is to determine structures of individual protein components and model these into existing intermediate resolution electron density . RESULTS: We have determined the solution structure of the ribosomal protein S16 from Thermus thermophilus . S16 is a mixed alpha/beta protein with a novel folding scaffold based on a five-stranded antiparallel/parallel beta sheet . Three large loops, which are partially disordered, extend from the sheet and two alpha helices are packed against its concave surface . Calculations of surface electrostatic potentials show a large continuous area of positive electrostatic potential and smaller areas of negative potential . S16 was modeled into a 5.5 A electron-density map of the T . thermophilus 30S ribosomal subunit . CONCLUSIONS: The location and orientation of S16 in a narrow crevice formed by helix 21 and several other unassigned rRNA helices is consistent with electron density corresponding to the shape of S16, hydroxyl radical protection data, and the electrostatic surface potential of S16 . Two protein neighbors to S16 are S4 and S20, which facilitate binding of S16 to the 30S subunit . Overall, this work exemplifies the benefits of combining high-resolution nuclear magnetic resonance (NMR) structures of individual components with low-resolution X-ray maps to elucidate structures of large complexes.

Nahrung, 2000 Aug, 44(4), 257 - 60
Effect of spray drying temperature of yoghurt on the survival of starter cultures, moisture content and sensoric properties of yoghurt powder; Bielecka M et al.; A synergistic set of the strains Lactobacillus delbrueckii subsp . bulgaricus 151 and Streptococcus thermophilus MK-10 was preserved with the method of spray drying . Studies were performed on the effect of outlet air temperature in range of 60-80 degrees C upon the survival of yoghurt cultures, as well as the moisture content and sensoric properties of yoghurt powder . Survival of yoghurt cultures was the highest at 60 and 65 degrees C, but excessive moisture (10.2%) of yoghurt powder had a negative effect on its texture . Moisture content of the powder was low at 80 degrees C (4.4%), unfortunately sensoric faults appeared at this temperature, and survival of bacteria cultures decreased considerably . Temperatures within the range 70-75 degrees C ensured satisfactory survival of yoghurt cultures (L . delbrueckii subsp . bulgaricus--13.7-15.8%; S . thermophilus--51.6-54.7%), maintained proportion of the strains (L:S--1:3), satisfactory moisutre content (5.1-6.3%), and good sensoric properties of yoghurt powder.

J Biochem Biophys Methods, 2000 Sep 11, 45(2), 157 - 68
Direct characterisation by electrospray ionisation mass spectroscopy of mercuro-polypeptide complexes after deprotection of acetamidomethyl groups from protected cysteine residues of synthetic polypeptides; Boysen RI et al.; In this paper, we describe a rapid procedure to characterise the products generated in the presence of mercuric salts following removal of the acetamidomethyl (Acm)-protecting group from cysteine residues of synthetic polypeptides prepared by solid-phase peptide synthesis (SPPS) methods . In particular, electrospray ionisation mass spectrometry (ESI-MS) procedures have been employed to characterise the mercuro-polypeptide products related to the ribosomal L36 protein isolated from the bacterium Thermus thermophilus . The results demonstrate that very stable mercuro-polypeptide complexes can form under standard conditions of deprotection involving Hg(2+) salts in the presence of a reductant such as beta-mercaptoethanol . Metal ion exchange effects involving other divalent metal ions, such as Co(2+) or Zn(2+), can also be monitored by similar procedures, thus permitting the relative affinity and selectivity for metal ion-polypeptide interactions to be qualitatively assessed . Since the Thermus thermophilus ribosomal L36 protein contains a putative zinc finger binding CCCH motif, these procedures enable the formation of metal-ion complexes of synthetic polypeptides related to this structural motif to be directly examined.

J Bacteriol, 2000 Oct, 182(19), 5309 - 16
Site-specific mutational analysis of a novel cysteine motif proposed to ligate the 4Fe-4S cluster in the iron-sulfur flavoprotein of the thermophilic methanoarchaeon Methanosarcina thermophila; Leartsakulpanich U et al.; Isf (iron-sulfur flavoprotein) from Methanosarcina thermophila has been produced in Escherichia coli as a dimer containing two 4Fe-4S clusters and two FMN (flavin mononucleotide) cofactors . The deduced sequence of Isf contains six cysteines (Cys 16, Cys 47, Cys 50, Cys 53, Cys 59, and Cys 180), four of which (Cys 47, Cys 50, Cys 53, and Cys 59) comprise a motif with high identity to a motif (CX(2)CX(2)CX(4-7)C) present in all homologous Isf sequences available in the databases . The spacing of the motif is highly compact and atypical of motifs coordinating known 4Fe-4S clusters; therefore, all six cysteines in Isf from M . thermophila were altered to either alanine or serine to obtain corroborating biochemical evidence that the motif coordinates the 4Fe-4S cluster and to further characterize properties of the cluster dependent on ligation . All except the C16S variant were produced in inclusion bodies and were void of iron-sulfur clusters and FMN . Reconstitution of the iron-sulfur cluster and FMN was attempted for each variant . The UV-visible spectra of all reconstituted variants indicated the presence of iron-sulfur clusters and FMN . The reduced C16A/S variants showed the same electron paramagnetic resonance (EPR) spectra as wild-type Isf, whereas the reduced C180A/S variants showed EPR spectra identical to those of one of the two 4Fe-4S species present in the wild-type Isf spectrum . Conversely, EPR spectra of the oxidized C50A and C59A variants showed g values characteristic of a 3Fe-4S cluster . The spectra of the C47A and C53A variants indicated a 4Fe-4S cluster with g values and linewidths different from those for the wild type . The combined results of this study support a role for the novel CX(2)CX(2)CX(4-7)C motif in ligating the 4Fe-4S clusters in Isf and Isf homologues.

Arch Microbiol, 2000 Jul-Aug, 174(1-2), 97 - 103
The transposable element IS4712 prevents S-layer gene (sbsA) expression in Bacillus stearothermophilus and also affects the synthesis of altered surface layer proteins; Scholz H et al.; Cell surface (S)-layer protein synthesis in Bacillus stearothermophilus PV72/p6 is blocked when cells are grown at elevated temperature . From a culture exhibiting the S-layer-negative phenotype, the S-layer deficient mutant T5 (SbsA-) was isolated . Genetic analysis of the S-layer-encoding gene (sbsA) of mutant T5 revealed an insertion element (IS4712) integrated into the upstream regulatory region of the S-layer gene, thereby blocking sbsA transcription . The insertion element consists of 1371 base pairs which are flanked by two perfect inverted terminal repeats . Sequence similarity to other transposases of the IS4 family was detected . DNA-DNA hybridizations demonstrated that multiple homologues of IS4712 were also present within the genomes of several other thermophilic bacillus isolates . Attempts to isolate SbsA+ revertants failed . Instead, cells with altered surface proteins were detected . The synthesis of the altered S-layer proteins was correlated with the presence of IS4712 along with the occurrence of deletions in the sbsA coding region . Furthermore imprecise excision of IS4712 was detected . This work demonstrated that B . stearothermophilus is able to express at least four different S-layer proteins and that blocking of sbsA transcription by the insertion element IS4712 is associated with the expression of altered surface proteins.

Arch Microbiol, 2000 Jul-Aug, 174(1-2), 74 - 80
Nucleotide sequence, expression and transcriptional analysis of the Bifidobacterium longum MB 219 lacZ gene; Rossi M et al.; The gene encoding beta-galactosidase was isolated by functional complementation of Escherichia coli from Bifidobacterium longum MB219, which exhibited the highest activity among ten Bifidobacterium strains tested of the species B . longum, B . breve, B . adolescentis, B . indicum, B . animalis and B . cuniculi . The nucleotide sequence of the 5.0-kb fragment conferring the positive beta-galactosidase phenotype to E . coli revealed the presence of a lacZ-type gene encoding a 1023-amino-acid protein that was preceded by a ribosome binding site . A sequence showing 72% identity with the proline tRNA of Bacillus subtilis and a gene probably encoding the DNA-3-methyladenine glycosydase I were located downstream from the lacZ gene, after a gap of 30-50 unsequenced base pairs . By primer-extension analysis, the transcription start site of the lacZ gene was mapped 65 nt upstream from the start codon, and it enabled identification of the -10 region of the putative promoter . The nucleotide sequence of lacZ and its deduced amino acid sequence were compared with those of beta-galactosidase genes and enzymes from other microorganisms . High similarity was demonstrated between the B . longum beta-galactosidase and its counterparts in Lactobacillus delbruckii subsp . bulgaricus, Streptococcus salivarius subsp . thermophilus, E . coli, Clostridium acetobutylicum, Leuconostoc lactis, Klebsiella pneumoniae and Kluyveromyces marxianus var . lactis, all belonging to the LacZ family . The B . longum MB219 lacZ gene was cloned in Bifidobacterium and its expression was observed in strains with otherwise low levels of endogenous activity . The expression increased by factors of 1.5-50 and enabled those strains that do not grow on lactose to use this sugar as sole carbon source.

Nucleic Acids Res, 2000 Sep 15, 28(18), 3570 - 80
PSI-BLAST searches using hidden markov models of structural repeats: prediction of an unusual sliding DNA clamp and of beta-propellers in UV-damaged DNA-binding protein; Neuwald AF et al.; We have designed hidden Markov models (HMMs) of structurally conserved repeats that, based on pairwise comparisons, are unconserved at the sequence level . To model secondary structure features these HMMs assign higher probabilities of transition to insert or delete states within sequence regions predicted to form loops . HMMs were optimized using a sampling procedure based on the degree of statistical uncertainty associated with parameter estimates . A PSI-BLAST search initialized using a checkpoint-recovered profile derived from simulated sequences emitted by such a HMM can reveal distant structural relationships with, in certain instances, substantially greater sensitivity than a normal PSI-BLAST search . This is illustrated using two examples involving DNA- and RNA-associated proteins with structurally conserved repeats . In the first example a putative sliding DNA clamp protein was detected in the thermophilic bacterium Thermotoga maritima . This protein appears to have arisen by way of a duplicated beta-clamp gene that then acquired features of a PCNA-like clamp, perhaps to perform a PCNA-related function in association with one or more of the many archaeal-like proteins present in this organism . In the second example, beta-propeller domains were predicted in the large subunit of UV-damaged DNA-binding protein and in related proteins, including the large subunit of cleavage-polyadenylation specificity factor, the yeast Rse1p and human SAP130 pre-mRNA splicing factors and the fission yeast Rik1p gene silencing protein.

Appl Biochem Biotechnol, 2000 Jun, 87(3), 165 - 75
Amiodarone interactions with membrane lipids and with growth of Bacillus stearothermophilus used as a model; Rosa SM et al.; The thermophilic eubacterium Bacillus stearothermophilus was used as a model to study the effects of amiodarone (2-butyl-3-{3',5'diido-4'alpha-diethylaminoethoxybenzoyl}-be nzofuran) in lipid organization and in bacterial growth . Effects on the structural order of lipids were assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and of the propionic acid derivative 3-{p-(6-phenyl)-1,3,5-hexatrienyl} phenylpropionic acid (DPH-PA), probing the outer regions of the bilayer . Amiodarone fluidizes bacterial polar lipid bilayers for temperatures below the phase transition midpoint, and orders the fluid phase of the bacterial polar lipids, as evaluated by DPH and DPH-PA . The ordering and disordering effects, which are concentration dependent, are more extensive when detected by DPH relative to DPH-PA . Growth studies performed in parallel revealed that amiodarone inhibits bacterial growth as a function of concentration . Amiodarone concentrations in the range from 1 to 2.5 microM increased the lag time, decreased the specific growth rate, and decreased the final cell density . Furthermore, 3 microM amiodarone completely inhibited growth . These in vivo effects of amiodarone can be related to its ability to perturb the phospholipid bilayer structure, whose integrity is essential for cell function, viability, and growth.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 13 - 9
Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei; Te'o VS et al.; The catalytic domain of the xynB (xylanase) gene from the thermophilic bacterium Dictyoglomus thermophilum was reconstructed by PCR to match the codon preference of Trichoderma reesei . The 0.6-kb DNA fragment encoding the enzyme was first amplified by primer extension with a mixture of eight overlapping oligonucleotides, followed by PCR with outside primers containing restriction enzyme sites for directional cloning into Escherichia coli and T . reesei vectors . The synthetic gene was expressed in both organisms, producing a clearing halo around transformant colonies in plate assay utilizing an overlay of oat spelts xylan . Effective transcription of xyn B in T . reesei was obtained after changing 20 codons.

FEMS Microbiol Rev, 2000 Oct, 24(4), 335 - 66
Prokaryotic carbonic anhydrases; Smith KS et al.; Carbonic anhydrases catalyze the reversible hydration of CO(2) {CO(2)+H(2)Oright harpoon over left harpoon HCO(3)(-)+H(+)} . Since the discovery of this zinc (Zn) metalloenzyme in erythrocytes over 65 years ago, carbonic anhydrase has not only been found in virtually all mammalian tissues but is also abundant in plants and green unicellular algae . The enzyme is important to many eukaryotic physiological processes such as respiration, CO(2) transport and photosynthesis . Although ubiquitous in highly evolved organisms from the Eukarya domain, the enzyme has received scant attention in prokaryotes from the Bacteria and Archaea domains and has been purified from only five species since it was first identified in Neisseria sicca in 1963 . Recent work has shown that carbonic anhydrase is widespread in metabolically diverse species from both the Archaea and Bacteria domains indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized . A remarkable feature of carbonic anhydrase is the existence of three distinct classes (designated alpha, beta and gamma) that have no significant sequence identity and were invented independently . Thus, the carbonic anhydrase classes are excellent examples of convergent evolution of catalytic function . Genes encoding enzymes from all three classes have been identified in the prokaryotes with the beta and gamma classes predominating . All of the mammalian isozymes (including the 10 human isozymes) belong to the alpha class; however, only nine alpha class carbonic anhydrase genes have thus far been found in the Bacteria domain and none in the Archaea domain . The beta class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants as well as enzymes from phylogenetically diverse species from the Archaea and Bacteria domains . The only gamma class carbonic anhydrase that has thus far been isolated and characterized is from the methanoarchaeon Methanosarcina thermophila . Interestingly, many prokaryotes contain carbonic anhydrase genes from more than one class; some even contain genes from all three known classes . In addition, some prokaryotes contain multiple genes encoding carbonic anhydrases from the same class . The presence of multiple carbonic anhydrase genes within a species underscores the importance of this enzyme in prokaryotic physiology; however, the role(s) of this enzyme is still largely unknown . Even though most of the information known about the function(s) of carbonic anhydrase primarily relates to its role in cyanobacterial CO(2) fixation, the prokaryotic enzyme has also been shown to function in cyanate degradation and the survival of intracellular pathogens within their host . Investigations into prokaryotic carbonic anhydrase have already led to the identification of a new class (gamma) and future research will undoubtedly reveal novel functions for carbonic anhydrase in prokaryotes.

Biochim Biophys Acta, 2000 Sep 7, 1493(1-2), 82 - 90
Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus; Klein W et al.; The Bacillus subtilis transition state regulator AbrB(su) is a DNA-binding protein that acts on several genes either as activator, repressor, or preventer . However, among genes under its control, neither common binding sites could be identified nor could the structural features of this broad and specific interaction be elucidated . Attempts to elucidate these interesting features by crystallizing AbrB(su) have failed so far . Therefore, to solve this problem, we focused in this work on identifying an AbrB(su) homologue from Bacillus stearothermophilus . Using a novel method, the entire abrB(st) gene of B . stearothermophilus was cloned and sequenced . The gene encodes a 95 amino acid protein that shows 77% identity and 85% similarity to the mesophilic B . subtilis protein . A calmodulin binding peptide-tagged fusion of the thermophilic gene was constructed for overexpression and efficient affinity column purification of the AbrB(st) protein . The purified protein showed, after removal of the tag, an oligomerization behavior through hexamer formation that is essential for its DNA binding activity.

Enzyme Microb Technol, 2000 Oct 1, 27(7), 492 - 501
Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp . BR449 into Escherichia coli; Kim S et al.; A moderate thermophile, Bacillus sp . BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C . In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli . Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K . Spacings and orientation of the coding regions as well as their gene expression in E . coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed . Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases . The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains . When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts . Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.

Microbiol Mol Biol Rev, 2000 Sep, 64(3), 461 - 88
Thermophilic fungi: their physiology and enzymes; Maheshwari R et al.; Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C . As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C . Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea . However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests . This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi . Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi . Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general . Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used . Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass . The properties of their enzymes show differences not only among species but also among strains of the same species . Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi . Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects . Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis . By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi . Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.

J Biol Chem, 2000 Dec 1, 275(48), 37902 - 6
Second stalk of ATP synthase . Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis; Suzuki T et al.; ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits . The second stalk is expected to keep the stator subunits from spinning along with the rotor . We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc') . Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity . A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase . This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis . Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).

EMBO J, 2000 Sep 1, 19(17), 4745 - 58
Crystal structure of a eukaryote/archaeon-like protyl-tRNA synthetase and its complex with tRNAPro(CGG); Yaremchuk A et al.; Prolyl-tRNA synthetase (ProRS) is a class IIa synthetase that, according to sequence analysis, occurs in different organisms with one of two quite distinct structural architectures: prokaryote-like and eukaryote/archaeon-like . The primary sequence of ProRS from the hypothermophilic eubacterium Thermus thermophilus (ProRSTT) shows that this enzyme is surprisingly eukaryote/archaeon-like . We describe its crystal structure at 2.43 angstom resolution, which reveals a feature that is unique among class II synthetases . This is an additional zinc-containing domain after the expected class IIa anticodon-binding domain and whose C-terminal extremity, which ends in an absolutely conserved tyrosine, folds back into the active site . We also present an improved structure of ProRSTT complexed with tRNAPro(CGG) at 2.85 angstom resolution . This structure represents an initial docking state of the tRNA in which the anticodon stem-loop is engaged, particularly via the tRNAPro-specific bases G35 and G36, but the 3' end does not enter the active site . Considerable structural changes in tRNA and/or synthetase, which are probably induced by small substrates, are required to achieve the conformation active for aminoacylation.

Bioorg Med Chem Lett, 2000 Aug 21, 10(16), 1795 - 8
Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine; Shohda K et al.; A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed . Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy . A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes . The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair . On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair . It is concluded that s2Um has higher selectivity toward A over G than unmodified U.

J Biol Chem, 2000 Dec 1, 275(48), 37876 - 86
Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein; Howorka S et al.; The S-layer protein SbsB of the thermophilic, Gram-positive organism Bacillus stearothermophilus PV72/p2 forms a crystalline, porous array constituting the outermost component of the cell envelope . SbsB has a molecular mass of 98 kDa, and the corresponding S-layer exhibits an oblique lattice symmetry . To investigate the molecular structure and assembly of SbsB, we replaced 75 residues (mainly serine, threonine, and alanine), located throughout the primary sequence, with cysteine, which is not found in the wild-type protein . As determined by electron microscopy, 72 out of 75 mutants formed regularly-structured self-assembly products identical to wild-type, thereby proving that the replacement of most of the selected amino acids by cysteine does not dramatically alter the structure of the protein . The three defective mutants, which showed a greatly reduced ability to self-assemble, were, however, successfully incorporated into S-layers of wild-type protein . Monomeric SbsB mutants and SbsB mutants assembled into S-layers were subjected to a surface accessibility screen by targeted chemical modification with a 5-kDa hydrophilic cysteine-reactive polyethylene glycol conjugate . In the monomeric form of SbsB, 34 of the examined residues were not surface accessible, while 23 were classified as very accessible, and 18 were of intermediate surface accessibility . By contrast, in the assembled S-layers, 57 of the mutated residues were not accessible, six were very accessible, and 12 of intermediate accessibility . Together with other structural information, the results suggest a model for SbsB in which functional domains are segregated along the length of the polypeptide chain.

Biophys J, 2000 Sep, 79(3), 1629 - 36
Structural equilibrium fluctuations in mesophilic and thermophilic alpha-amylase; Fitter J et al.; By comparing a mesophilic alpha-amylase with its thermophilic homolog, we investigated the relationship between thermal stability and internal equilibrium fluctuations . Fourier transform infrared spectroscopy monitoring hydrogen/deuterium (H/D) exchange kinetics and incoherent neutron scattering measuring picosecond dynamics were used to study dynamic features of the folded state at room temperature . Fairly similar rates of slowly exchanging amide protons indicate about the same free energy of stabilization DeltaG(stab) for both enzymes at room temperature . With respect to motions on shorter time scales, the thermophilic enzyme is characterized by an unexpected higher structural flexibility as compared to the mesophilic counterpart . In particular, the picosecond dynamics revealed a higher degree of conformational freedom for the thermophilic alpha-amylase . The mechanism proposed for increasing thermal stability in the present case is characterized by entropic stabilization and by flattening of the curvature of DeltaG(stab) as a function of temperature.

Appl Microbiol Biotechnol, 2000 Aug, 54(2), 173 - 9
Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans; Kademi A et al.; The thermostable esterase from the moderate thermophile Bacillus circulans was purified to homogeneity using a four-step procedure . Esterase activity was associated with a protein of molecular mass 95 kDa, composed of three identical subunits of 30 kDa . The esterase activity was thermostable with a maximum activity at 55 degrees C using initial rate assay . The half-inactivation temperature was 71 degrees C after a 1-h treatment, which compared favorably to that of other enzymes . Activity at temperatures of 30-37 degrees C was high (about half of maximum), making this new enzyme very attractive for applications in this moderate temperature range . The esterase also showed high activity at a rather alkaline pH (higher than 10) . The specificity pattern showed a marked specificity for mid-chain-length fatty acids (3-8 carbon atoms), which classified the enzyme as a carboxylesterase.

Front Biosci, 2000 Sep 01, 5, D813 - 20
Adaptations of the archaeal cell membrane to heat stress; Albers SV et al.; In extreme environments varying from hot to cold, acidic to alkaline, and highly saline, mainly Archaea are found . Thermophilic and extremely acidophilic Archaea have a membrane that contains membrane spanning tetraether lipids . These tetra-ether membranes have a limited permeability for protons even at the high temperatures of growth and this property makes it possible for thermophilic archaea to maintain a viable proton motive force under the extreme conditions . -Ether lipids cannot be degraded easily and are highly stable which is also a requirement for life under extreme conditions . Psychrophilic and mesophilic Bacteria, and all Archaea adjust the lipid composition of their membranes so that the proton permeability of their membranes remains within a narrow range . This phenomenon is termed 'homeoproton permeability adaptation' . Thermophilic Bacteria are the only prokaryotes that are unable to control the proton permeability of their membranes . These organisms have to rely on the less permeable sodium ions in energy transducing processes in their membrane.

Appl Environ Microbiol, 2000 Sep, 66(9), 3951 - 9
Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater; LaPara TM et al.; The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied . These reactors were operated at both mesophilic (28 to 32 degrees C) and thermophilic (50 to 58 degrees C) temperatures . Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities . A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness . Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened . While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected . Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the beta subdivision of the division Proteobacteria (beta-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions . The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (alpha-proteobacteria, beta-proteobacteria, gamma-proteobacteria and delta-proteobacteria subdivisions) . The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes . This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity-a function of both the total number of species present (richness) and their relative distribution (evenness).

Appl Environ Microbiol, 2000 Sep, 66(9), 3798 - 806
Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge hydrothermal vent; Reysenbach AL et al.; The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23 degrees 22' N, 44 degrees 57' W) . The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20 degrees C . Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned . Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened . The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders . A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples . Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained . The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the epsilon subclass of the Proteobacteria (epsilon-Proteobacteria) (40%), and the genus Desulfurobacterium (25%) . Most of the clones (28%) were confined to a monophyletic clade within the epsilon-Proteobacteria with no known close relatives . The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S(0), SO(4), thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents . The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.

Protist, 2000 Aug, 151(2), 171 - 80
The cDNA sequences of three tetrins, the structural proteins of the Tetrahymena oral filaments, show that they are novel cytoskeletal proteins; Brimmer A et al.; The oral filaments of the ciliate Tetrahymena consist of the tetrins, insoluble polypeptides with molecular masses of around 85 kD . We characterised the tetrins of T . thermophila by two-dimensional gels and derived a large number of peptide sequences by in gel digestion . Using RT-PCR techniques and RACE-PCR, the complete cDNA sequences of tetrins A, B and C were established . Although tetrins differ strikingly in protein sequence they show a common structural principle . A N-terminal domain of 60 to 100 residues contains most of the proline residues of the tetrins and is probably globular . It is followed by a long alpha-helical domain of 620 to 640 residues which either lacks prolines or in tetrin A contains a single proline residue . Although this long domain has coiled coil forming ability, the individual heptad repeats are not extensive . Tetrins are novel cytoskeletal proteins unique to ciliates . Since the three tetrin sequences account for all 900 amino acid residues obtained by microsequencing of peptides, an additional major tetrin seems excluded . A minor component D is related to tetrin B by peptide sequences . The isoelectric variants, particularly obvious for tetrin A, most likely reflect post-translational modifications . These could arise by phosphorylation of serines and threonines in the proline rich N-terminal domain.

Protein Eng, 2000 Aug, 13(8), 527 - 33
The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method; Hori T et al.; A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation . In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (approximately 90 degrees C) was found to be localized at a region which includes a beta-turn and a loop located between the two domains of the enzyme . A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this beta-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T:(m) by approximately 10 degrees C) and no T-jump-induced structural change in this region was detected by our method . These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this beta-turn region.

Ann Occup Hyg, 2000 Sep, 44(6), 467 - 73
Exposure to microbes, endotoxins and total dust in cigarette and cigar manufacturing: an evaluation of health hazards; Reiman M et al.; The concentrations of airborne microbes, endotoxins and total dust were measured in one cigar and two cigarette factories in order to evaluate the risk of respiratory symptoms . The role of humidifiers as a source of microbes was investigated . Air samples for the analyses were collected near workers' breathing zones during different phases of production . Gram-negative bacteria, mesophilic fungi, thermotolerant fungi and thermophilic actinomycetes, but not Aspergillus glaucus fungi, were found in higher concentrations in the cigar factory than in the cigarette factories . High microbe concentrations (10(4)-10(5)cfu m(-3)) occurred throughout the production line in the cigar factory . The highest dust and endotoxin concentrations were found in the wick-making department in the cigar factory (3.3mg dust per m(3) and 38ng endotoxin per m(3)) and during the weighing or handling of raw tobacco in the cigarette factories (4.5 mg dust per m(3) and 106ng endotoxin per m(3)) . The spray humidifiers in the cigar factory were a more important source of microbes than was raw tobacco . In the cigarette factories, steam humidifiers were used; the humidified air was free of microbes . The microbe concentrations in the tobacco factories were lower than in environments known to have caused allergic alveolitis.

Acta Biochim Pol, 2000, 47(1), 209 - 14
Cloning, expression, and crystallization of Cpn60 proteins from Thermococcus litoralis; Osipiuk J et al.; Two genes of the extreme thermophilic archaeon Thermococcus litoralis homologous to those that code for Cpn60 chaperonins were cloned and expressed in Escherichia coli . Each of the Cpn60 subunits as well as the entire Cpn60 complex crystallize in a variety of morphological forms . The best crystals diffract to 3.6 A resolution at room temperature and belong to the space group 1422 with unit cell parameters a = b = 193.5 A, c = 204.2 A.

Antonie Van Leeuwenhoek, 2000 May, 77(4), 321 - 7
Thiosulfate reduction and alanine production in glucose fermentation by members of the genus Coprothermobacter; Etchebehere C et al.; Coprothermobacter platensis is an anaerobic, proteolytic, thermophilic bacterium, which is phylogenetically related to the genera Fervidobacterium and Thermotoga . The organism was found to reduce thiosulfate to sulfide during growth on carbohydrates and proteinaceous substrates . Growth on glucose was inhibited by hydrogen, but this inhibition was overcome by thiosulfate reduction, stirring, increasing the headspace volume and coculturing with a hydrogen-consuming methanogen . Alanine was detected during glucose fermentation, its formation was influenced by the hydrogen concentration in the gas phase suggesting an electron sink mechanism, as was previously reported for the phylogenetically related Thermotogales and the archaeal hyperthermophile Pyrococcus furiosus.

J Biol Chem, 2000 Nov 17, 275(46), 35746 - 50
Movement of the helical domain of the epsilon subunit is required for the activation of thermophilic F1-ATPase; Kato-Yamada Y et al.; The inhibitory effect of epsilon subunit in F(1)-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship . For this purpose, we designed a mutant for epsilon subunit similar to the one constructed by Schulenberg and Capaldi (Schulenberg, B., and Capaldi, R . A . (1999) J . Biol . Chem . 274, 28351-28355) . We introduced two cysteine residues at the interface of N-terminal beta-sandwich domain (S48C) and C-terminal alpha-helical domain (N125C) of epsilon subunit . The alpha(3)beta(3)gammaepsilon complex containing the reduced form of this mutant epsilon subunit showed suppressed ATPase activity and gradual activation during the measurement . This activation pattern was similar to the complex with the wild type epsilon subunit . The conformation of the mutant epsilon subunit must be fixed and similar to the reported three-dimensional structure of the isolated epsilon subunit, when the intramolecular disulfide bridge was formed on this subunit by oxidation . This oxidized mutant epsilon subunit could form the alpha(3)beta(3)gammaepsilon complex but did not show any inhibitory effect . The complex was converted to the activated state, and the cross-link in the mutant epsilon subunit in the complex was efficiently formed in the presence of ATP-Mg, whereas no cross-link was observed without ATP-Mg, suggesting the conformation of the oxidized mutant epsilon subunit must be similar to that in the activated state complex . A non-hydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate, could stimulate the formation of the cross-link on the epsilon subunit . Furthermore, the cross-link formation was stimulated by nucleotides even when this mutant epsilon subunit was assembled with a mutant alpha(3)beta(3)gamma complex lacking non-catalytic sites . These results indicate that binding of ATP to the catalytic sites induces a conformational change in the epsilon subunit and triggers transition of the complex from the suppressed state to the activated state.

J Biol Chem, 2000 Oct 27, 275(43), 33765 - 70
The role of histidines in the acetate kinase from Methanosarcina thermophila; Ingram-Smith C et al.; The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis . Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine . The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified . Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation . Comparison of the M . thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved . Diethylpyrocarbonate inactivation of the eight histidine --> alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation . Kinetic analyses of the eight variants showed that no other histidines are important for activity . Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis . Possible functions of His-180 are discussed.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1201 - 3
Identification of a potential metal cation-pi binding site in the structure of a thermophilic Bacillus stearothermophilus triosephosphate isomerase mutant; Wouters J et al.; A potential metal cation-pi interaction between a sodium cation (Na(+)) and the indole ring of a tryptophan residue was detected in the crystallographic structure (2 A) of the thermophilic Bacillus stearothermophilus triosephosphate isomerase H12N/K13G mutant (bTIMmut) . The cation-pi binding site is located near the surface of the protein, the alkali metal ion facing the benzo ring of Trp9 . The presence of Phe21 and Glu17 close to Trp9 could indicate an additional role for those residues in the stability of the sodium-indole interaction . The sodium cation lies in a position that is occupied by CE and NZ of Lys13 in the wild-type structure.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1173 - 5
Crystallization and preliminary X-ray diffraction analysis of a cytochrome P450 (CYP119) from Sulfolobus solfataricus; Park SY et al.; CYP119 is a cytochrome P450 with a molecular weight of 43 kDa which has been isolated from the thermophilic archaeon Sulfolobus solfataricus . This enzyme is extremely stable to high temperature and high pressure . The first crystallization and preliminary crystallographic study of CYP119 is reported here . Crystals of CYP119 were obtained by the sitting-drop vapour-diffusion method using a precipitant solution containing 20%(w/v) PEG 4000 and 0.2 M sodium thiocyanate at pH 6.4 . Using synchrotron radiation, the CYP119 crystal diffracted to 1.84 A resolution . It belongs to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 86.17 (0.07), c = 221.11 (0.04) A, in which the numbers in parentheses describe the standard deviations . Assuming two molecules of the CYP119 per asymmetric unit, the calculated molar volume (V(m)) is 2.38 A(3) Da(-1) . Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem irons . The complete crystallographically defined structure is currently in progress using MIR (multiple isomorphous replacement) and MAD (multiwavelength anomalous diffraction) techniques.

J Biol Chem, 2000 Oct 27, 275(43), 33504 - 11
High salt-induced conversion of Escherichia coli GroEL into a fully functional thermophilic chaperonin; Kusmierczyk AR et al.; The GroE chaperonin system can adapt to and function at various environmental folding conditions . To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate . Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5, 5')-8-anilino-1-naphthalene sulfonic acid to the chaperonin . The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins . Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 degrees C . At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins.

Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1531 - 3
Effect of Streptococcus thermophilus on the trp-P-1 level in the blood; Terahara M et al.; We examined the 3-amino-1,4-dimethyl-5H-Pyrido-{4,3-b}indole (Trp-P-1) concentration in the blood after administering Trp-P-1 (0.25 mg/ml) with or without Streptococcus thermophilus 1131 cells (10 mg/ml) to rats . The Trp-P-1 concentration in the blood from the portal vein was significantly lower in the rats that had been administered with Trp-P-1 with the strain 1131 cells than without them . However, there was no difference in the Trp-P-1 concentration in the blood taken from the abdominal aorta of these rats.

Proteins, 2000 Oct 1, 41(1), 75 - 85
Model-free analysis of a thermophilic Fe(7)S(8) protein compared with a mesophilic Fe(4)S(4) protein; Bertini I et al.; 15N T(1), T(2) and (1)H-(15)N NOE were measured for the thermophilic Fe(7)S(8) protein from Bacillus schlegelii and for the Fe(4)S(4) HiPIP protein from Chromatium vinosum, which is a mesophilic protein . The investigation was performed at 276, 300, and 330 K at 11.7 T for the former, whereas only the 298 K data at 14.1 T for the latter were acquired . The data were analyzed with the model-free protocol after correcting the measured parameters for the effect of paramagnetism, because both proteins are paramagnetic . Both thermophilic and mesophilic proteins are quite rigid, with an average value of the generalized order parameter S2at room temperature of 0.92 and 0.94 for Fe(7)S(8) and Fe(4)S(4) proteins, respectively . The analyzed nitrogens for the Fe(7)S(8) protein showed a significant decrease in S2with increasing temperature, and at the highest temperature >70% of the residues had an internal correlation time . This research shows that subnanosecond rigidity is not related to thermostability and provides an estimate of the effect of increasing temperature on this time scale.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1061 - 3
Crystallization and structure determination of the catalytic trimer of Methanococcus jannaschii aspartate transcarbamoylase; Vitali J et al.; Aspartate transcarbamoylase (ATCase) catalyzes the first step in the pyrimidine biosynthetic pathway, the reaction between carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and phosphate . The structural analysis of the ATCase catalytic trimer from Methanococcus jannaschii, a unicellular thermophilic archaeabacterium, has been undertaken in order to gain insight into the structural features that are responsible for the thermostability of the enzyme . As a first step, the catalytic trimer was crystallized in space group R32, with unit-cell parameters a = b = 265.3, c = 195.5 A and two trimers in the asymmetric unit . Its structure was determined using molecular replacement and Patterson methods . In general, structures containing multiple copies of molecules in the asymmetric unit are difficult to determine . In this case, the two trimers in the asymmetric unit are parallel to each other and use of the Patterson function greatly simplified the structure solution.

Biotechnol Bioeng, 2000 Oct 5, 70(1), 9 - 16
Engineering direct fructose production in processed potato tubers by expressing a bifunctional alpha-amylase/glucose isomerase gene complex; Beaujean A et al.; Manipulation of starch biosynthesis/degradation and formation of novel molecules in storage organs of plants through genetic engineering is an attractive but technically challenging goal . We report here, for the first time, that starch was degraded and glucose and fructose were produced directly when crushed potato tubers expressing a starch degrading bifunctional gene were heated for 45 minutes at 65 degrees C . To achieve this, we have constructed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus) . The chimeric gene was placed under the control of the granule-bound-starch synthase promoter . This enzymatic complex produced in transgenic tubers was only active at high temperature (65 degrees C) . More than 100 independent transgenic potato plants were regenerated . Molecular analyses confirmed the stable integration of the chimeric gene into the potato genome . The biochemical analyses performed on young and old tubers after high-temperature treatment (65 degrees C) revealed an increase in the formation rate of fructose and glucose by a factor of 16.4 and 5 . 7, respectively, in the transgenic tubers as compared to untransformed control tubers . No adverse discernible effect on plant development and metabolism including tuber formation and starch accumulation was observed in the transgenic plants before heat treatment . Our results demonstrate that it is possible to replace starch degradation using microbial enzymes via a system where the enzymes are produced directly in the plants, but active only at high temperature, thus offering novel and viable strategies for starch-processing industries .

J Biol Chem, 2000 Nov 17, 275(46), 35820 - 4
Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome; Martemyanov KA et al.; Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome . Their functional cycles depend on GTP binding and its hydrolysis . The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome . In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation . By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced . These findings indicate an essential contribution of domain III to activation of GTP hydrolysis . These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.

J Biol Chem, 2000 Oct 20, 275(42), 32383 - 6
Structural and genomic correlates of hyperthermostability; Cambillau C et al.; While most organisms grow at temperatures ranging between 20 and 50 degrees C, many archaea and a few bacteria have been found capable of withstanding temperatures close to 100 degrees C, or beyond, such as Pyrococcus or Aquifex . Here we report the results of two independent large scale unbiased approaches to identify global protein properties correlating with an extreme thermophile lifestyle . First, we performed a comparative proteome analyses using 30 complete genome sequences from the three kingdoms . A large difference between the proportions of charged versus polar (noncharged) amino acids was found to be a signature of all hyperthermophilic organisms . Second, we analyzed the water accessible surfaces of 189 protein structures belonging to mesophiles or hyperthermophiles . We found that the surfaces of hyperthermophilic proteins exhibited the shift already observed at the genomic level, i.e . a proportion of solvent accessible charged residues strongly increased at the expense of polar residues . The biophysical requirements for the presence of charged residues at the protein surface, allowing protein stabilization through ion bonds, is therefore clearly imprinted and detectable in all genome sequences available to date.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1601 - 9
Thermacetogenium phaeum gen . nov., sp . nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium; Hattori S et al.; A novel anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium, strain PB(T), was isolated from a thermophilic (55 degrees C) anaerobic methanogenic reactor which had been treating kraft-pulp waste water . The bacterium oxidized acetate in co-culture with a thermophilic hydrogenotrophic methanogen . Strain PB(T), a gram-positive, spore-forming, rod-shaped bacterium grew optimally at 58 degrees C and pH 6.8 . The bacterium grew acetogenically on several alcohols, methoxylated aromatics, pyruvate, glycine, cysteine, formate and hydrogen/CO2 . Strain PB(T) also oxidized acetate with reduction of sulfate or thiosulfate as the electron acceptor . The bacterium contained MK-7 as the major quinone . The G+C content of the DNA was 53.5 mol% . Comparative 16S rDNA analysis indicated that strain PB(T) belongs to the Bacillus-Clostridium subphylum . However, it was distant from any known genera or micro-organism . The closest known relative was Thermoterrabacterium ferrireducens with 87.4% similarity . The name Thermacetogenium phaeum gen . nov., sp . nov . is proposed . The type strain is strain PBT (= DSM 12270T).

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