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 thermoph