Appl Environ Microbiol, 1997 Jul, 63(7), 2647 - 53 Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation; Borneman J et al.; Although the Amazon Basin is well known for its diversity of flora and fauna, this report represents the first description of the microbial diversity in Amazonian soils involving a culture-independent approach . Among the 100 sequences of genes coding for small-subunit rRNA obtained by PCR amplification with universal small-subunit rRNA primers, 98 were bacterial and 2 were archaeal . No duplicate sequences were found, and none of the sequences had been previously described . Eighteen percent of the bacterial sequences could not be classified in any known bacterial kingdom . Two sequences may represent a unique branch between the vast majority of bacteria and the deeply branching, predominantly thermophilic bacteria . Five sequences formed a clade that may represent a novel group within the class Proteobacteria . In addition, rRNA intergenic spacer analysis was used to show significant microbial population differences between a mature forest soil and an adjacent pasture soil. Arch Microbiol, 1997 Jul, 168(1), 1 - 7 Pressure and temperature effects on growth and viability of the hyperthermophilic archaeon Thermococcus peptonophilus; Canganella F et al.; We studied the effects of high temperatures and elevated hydrostatic pressures on the physiological behavior and viability of the extremely thermophilic deep-sea archaeon Thermococcus peptonophilus . Maximal growth rates were observed at 30 and 45 MPa although no significant increases in cell yields were detected . Growth at 60 MPa was slower . The optimal growth temperature shifted from 85 degrees C at 30 MPa to 90-95 degrees C at 45 MPa . Cell viability during the stationary phase was also enhanced under high pressure . A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated at increasing growth temperatures . The viability of cells during starvation, at high temperature (90, 95 degrees C), and at low temperature (10 degrees C) was enhanced at 30 and 45 MPa as compared to atmospheric pressure . These results show that the extremely thermophilic archaeon T . peptonophilus is a barophile. Curr Microbiol, 1997 Jul, 35(1), 59 - 63 Identification of a cold shock gene in lactic acid bacteria and the effect of cold shock on cryotolerance; Kim WS et al.; When Lactic Acid Bacterial cultures were frozen at -20 degrees C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10 degrees C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp . lactis strains (25-37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp . cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2 . When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance . Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested . The PCR product from Lactococcus lactis ssp . lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's . Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested. FEBS Lett, 1997 Jun 30, 410(2-3), 141 - 4 Effect of polar side chains at position 172 on thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus; Akanuma S et al.; To understand the role of the amino acid residue at position 172 in the conformational stability, four mutant enzymes of Thermus thermophilus 3-isopropylmalate dehydrogenase in which Ala172 was replaced with Asp, Glu, Asn, and Gln were prepared by site-directed mutagenesis . Three mutants were more stable than the wild-type enzyme . No significant change in catalytic properties was found in the mutant enzymes . The molecular modeling studies suggested that the enhanced thermostability of the mutant enzymes resulted from the formation of extra electrostatic interactions and/or improvement of hydrophobic packing of the interior core. Virology, 1997 Jun 23, 233(1), 136 - 48 Characterization of the lysogeny DNA module from the temperate Streptococcus thermophilus bacteriophage phi Sfi21; Bruttin A et al.; Phage phi Sfi21, the only temperate Streptococcus thermophilus phage from our phage collection, showed extensive DNA homology with virulent phages from lytic group I . Southern blot hybridizations demonstrated that the phi Sfi21-specific DNA was clustered in an approximately 6.6-kb-long region, the putative lysogeny module . Sequence analysis and database research identified an integrase within this module; orf 203 with homology to an anonymous orf 258 from the temperate lactococcal phage BK5-T; orf 127 and orf 122 with weak homology to the N- and C-terminal parts, respectively, of the cl-like repressor from lactococcal phages Tuc2009 and BK5-T; orf 75 with homology to a repressor protein from lambdoid phage 434 and an anti-repressor ant with homology to phage P1 . The molecular arrangement of the predicted orfs in phage phi Sfi21 was very similar to that of the lactococcal phage BK5-T . The transition from phi Sfi21-specific DNA into DNA shared with virulent phages was abrupt and flanked at one side by notable DNA repeats . Sequence analysis identified a holin protein to the left of the lysogeny module . A site-specific deletion of 2.4 kb, which reproducibly transformed phi Sfi21 into a lytic phage, was localized in the lysogeny module . It was flanked at both sides by conspicuous DNA repeats . One repeat region reflected the DNA around the attP site, while the other reflected the putative genetic switch region between repressor and anti-repressor genes . S . thermophilus host Sfi1 transformed with a plasmid containing int and orf 203 showed resistance to superinfection by heterologous phages, but not by the homologous phi Sfi21 . Part of the int gene could be deleted without loss of this activity, while a deletion in orf 203 resulted in loss of the phage resistance . We speculate on the possibility of a bipartite immunity system for the control of lysogeny in phi Sfi21. Int J Food Microbiol, 1997 Jun 17, 37(1), 87 - 91 Characterization of monoclonal antibodies for the rapid detection of foodborne campylobacters; Lu P et al.; The specificity of 97 monoclonal antibodies (MAbs) to the Campylobacter jejuni Lior serogroup 6 reference strain was assessed using an indirect enzyme linked immunosorbent assay (ELISA) . Four MAbs, M316, M337, M357 and M637, reacted with whole cells of the C . jejuni, C . coli and C . lari reference strains of the 20 most common Lior serogroups and 25 recent C . jejuni and C . coli isolates, and did not react with most of the 42 other Campylobacter and non-Campylobacter spp . tested . Immunoblot analysis revealed that MAbs M337 and M357 reacted with a protein component with molecular mass of approximately 62 kiloDaltons (kDa) while M316 and M637 reacted with protein components of approximately 92 and 31 kDa, respectively . The detection limit of M357 in an indirect ELISA was 10(5) colony forming units . These four highly specific MAbs may be useful reagents of an immunoassay for the rapid detection of thermophilic campylobacters in foods and clinical samples. FEBS Lett, 1997 Jun 16, 409(3), 325 - 32 The complete inventory of the yeast Saccharomyces cerevisiae P-type transport ATPases; Catty P et al.; A total of sixteen open reading frames encoding for P-type ATPases have been identified in the complete genome sequence of Saccharomyces cerevisiae . Phylogenetic analysis distinguishes 6 distinct families . Topology predictions, identification of aminoacid sequence motifs and phenotype analysis of the available mutants suggest that these families correspond to ATPases transporting either H+ (2 members), Ca2+ (2 members), Na+ (3 members), heavy metals (2 members), possibly aminophospholipids (5 members including 4 new ones) or unknown substrates (2 new members) . It is proposed that the latter family which has homologs in Tetrahymena thermophila, Plasmodium falciparum and Caenorhabditis elegans constitutes a new group called P4-ATPases with characteristic topology and aminoacid signatures. Structure, 1997 Jun 15, 5(6), 825 - 36 The crystal structure of the nucleotide-free alpha 3 beta 3 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer; Shirakihara Y et al.; BACKGROUND: F1-ATPase, an oligomeric assembly with subunit stoichiometry alpha 3 beta 3 gamma delta epsilon, is the catalytic component of the ATP synthase complex, which plays a central role in energy transduction in bacteria, chloroplasts and mitochondria . The crystal structure of bovine mitochondrial F1-ATPase displays a marked asymmetry in the conformation and nucleotide content of the catalytic beta subunits . The alpha 3 beta 3 subcomplex of F1-ATPase has been assembled from subunits of the moderately thermophilic Bacillus PS3 made in Escherichia coli, and the subcomplex is active but does not show the catalytic cooperativity of intact F1-ATPase . The structure of this subcomplex should provide new information on the conformational variability of F1-ATPase and may provide insights into the unusual catalytic mechanism employed by this enzyme . RESULTS: The crystal structure of the nucleotide-free bacterial alpha 3 beta 3 subcomplex of F1-ATPase, determined at 3.2 A resolution, shows that the oligomer has exact threefold symmetry . The bacterial beta subunits adopt a conformation essentially identical to that of the nucleotide-free beta subunit in mitochondrial F1-ATPase; the alpha subunits have similar conformations in both structures . CONCLUSIONS: The structures of the bacterial F1-ATPase alpha and beta subunits are very similar to their counterparts in the mitochondrial enzyme, suggesting a common catalytic mechanism . The study presented here allows an analysis of the different conformations adopted by the alpha and beta subunits and may ultimately further our understanding of this mechanism. Carbohydr Res, 1997 Jun 11, 301(1-2), 41 - 50 Structural characterisation of the exocellular polysaccharide produced by Streptococcus thermophilus OR 901; Bubb WA et al.; The exocellular polysaccharide of Streptococcus thermophilus OR 901, isolated from partially deproteinised whey, is a heteropolymer of D-galactopyranose and L-rhamnopyranose residues in the molar ratio 5:2 . The structure was established by methylation analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by partial acid hydrolysis, using methylation analysis and 1D 1H NMR spectroscopy . The polysaccharide has a branched heptasaccharide repeating unit with the following structure: {sequence: see text} Biochim Biophys Acta, 1997 Jun 6, 1335(3), 283 - 9 Effects of magnesium and temperature on the conformation and reassociation of Escherichia coli and Sulfolobus solfataricus ribosomes; Pedone F et al.; The structural response of the ribosomes of the extremely thermophilic archaeon Sulfolobus solfataricus was analysed and compared to that of the mesophilic (E . coli) ribosomes by assaying ethidium bromide (EB) binding to the native 70S particles as a function of magnesium concentration . We found that the thermophilic ribosomes bound more EB than their mesophilic counterparts; on the other hand, inhibition of EB binding by Mg2+ ions was more effective in the E . coli 70S particle . In Sulfolobus, the separated 30S and 50S subunits and the 70S particle bound the drug in a similar fashion, whereas the E . coli 70S had a reduced number of binding sites with respect to the subunits . Light scattering measurements as a function of Mg2+ concentration were carried out at various temperatures to study the interaction between the ribosomal subunits from the thermophilic and the mesophilic bacteria . As expected, the association of ribosomal subunits in E . coli was magnesium dependent and could be observed also at low temperature . By contrast, the interaction between Sulfolobus ribosomal subunits was obligatorily dependent upon both magnesium ions and a temperature of at least 80 degrees C, close to the physiological optimum for cell growth (87 degrees C). Protein Eng, 1997 Jun, 10(6), 665 - 72 Sequence and homology model of 3-isopropylmalate dehydrogenase from the psychrotrophic bacterium Vibrio sp . I5 suggest reasons for thermal instability; Wallon G et al.; The leuB gene from the psychrotrophic strain Vibrio sp . I5 has been cloned and sequenced . The gene codes for 3-isopropylmalate dehydrogenase, a 360-residue, dimeric enzyme involved in the biosynthesis of leucine . Three recently solved homologous isopropylmalate dehydrogenase (IPMDH) crystal structures from thermophilic and mesophilic organisms have been used to build a homology model for the psychrotrophic IPMDH and to deduce the possible structural reasons for its decreased thermostability . According to our model the psychrotrophic IPMDH contains fewer stabilizing interactions than its mesophilic and thermophilic counterparts . Elements that have been identified as destabilizing in the comparison of the psychrotrophic, mesophilic and thermophilic IPMDHs are a smaller number of salt-bridges, a reduction in aromatic-aromatic interactions, fewer proline residues and longer surface loops . In addition, there are a number of substitutions of otherwise strictly conserved residues that can be linked to thermostability. Mol Gen Genet, 1997 Jun, 255(2), 152 - 6 Primary structure, sequence analysis, and expression of the thermostable D-hydantoinase from Bacillus stearothermophilus SD1; Kim GJ et al.; The gene coding for the thermostable D-hydantoinase from the thermophilic bacterium Bacillus stearothermophilus SD1 was cloned and its nucleotide sequence was completely determined . The D-hydantoinase protein showed considerable amino acid sequence homology (20-28%) with other hydantoinases and functionally related allantoinases and dihydroorotases . Strikingly the sequence of the enzyme from B . stearothermophilus SD1 exhibited greater than 89% identity with hydantoinases from thermophilic bacteria . Despite the extremely high amino acid homology among the hydantoinases from thermophiles, the C-terminal regions of the enzymes were completely different in both sequence and predicted secondary structure, implying that the C-terminal region plays an important role in determining the biochemical properties of the enzymes . Alignment of the sequence of the D-hydantoinase from B . stearothermophilus SD1 with those of other functionally related enzymes revealed four conserved regions, and five histidines and an acidic residue were found to be conserved, suggesting a close evolutionary relationship between all these enzymes. Mol Cell Probes, 1997 Jun, 11(3), 177 - 85 Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene; van Doorn LJ et al.; Polymerase chain reaction (PCR) mediated DNA fingerprinting has resulted in the identification of a novel Campylobacter jejuni gene, encoding a GTPase protein . The gene, consisting of 383 amino acids contained semi-conserved GTP-binding sites (designated G-1 to G-4), that are characteristic for members of the GTPase protein superfamily . Remarkably, this gene from C . Jejuni appears to encode a member of a novel family of GTP-binding proteins, containing two separate putative GTP-binding domains, each comprising a series of semi-conserved GTP-binding motifs . Spacing between these motifs is highly conserved . Based on this novel gene, a general PCR strategy for the identification of C . jejuni, C . coli, C . lari and C . upsaliensis was developed . PCR primers were deduced from GTP-binding motifs G-1 and G-3 of the first GTP-binding domain . These GTP-binding sites flank a variable region of precisely 117 bp in the four Campylobacter spp . that allowed the development of species-specific probes . This PCR-hybridization assay offers a novel tool for rapid molecular detection and specific identification of the thermophilic Campylobacter spp. J Cell Sci, 1997 Jun, 110 ( Pt 12), 1345 - 50 When helicase and topoisomerase meet! Duguet M. Several examples of direct interactions between helicases and topoisomerases have recently been described . The data suggest a possible cooperation between these enzymes in major DNA events such as the progression of a replication fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, and finally recombination, repair, and genomic stability . A first example is the finding of a strong interaction between T antigen and topoisomerase I in mammalian cells, that may trigger unwinding of the parental DNA strands at the replication forks of Simian Virus 40 . A second example is the reverse gyrase from thermophilic prokaryotes, composed of a putative helicase domain, and a topoisomerase domain in the same polypeptide . This enzyme may be required to maintain genomic stability at high temperature . A third example is the finding of an interaction between type II topoisomerase and the helicase Sgs1 in yeast . This interaction possibly allows the faithful segregation of newly replicated chromosomes in eukaryotic cells . A fourth example is the interaction between the same helicase Sgs1 and topoisomerase III in yeast, that may control recombination level and genetic stability of repetitive sequences . Recently, in humans, mutations in genes similar to Sgs1 have been found to be responsible for Bloom's and Werner's syndromes . The cooperation between helicases and topoisomerases is likely to be extended to many aspects of DNA mechanisms including chromatin condensation/decondensation. Eur J Biochem, 1997 Jun 1, 246(2), 291 - 300 Ribosomal protein S15 from Thermus thermophilus--cloning, sequencing, overexpression of the gene and RNA-binding properties of the protein; Serganov A et al.; A 6-kb DNA fragment from an extreme thermophile, Thermus thermophilus, carrying the genes for cytochrome oxidase ba3 subunit I (cbaA) and the ribosomal protein S15 (rpsO) was cloned into Escherichia coli . The gene rpsO was sequenced . The deduced amino acid sequence exhibits 59% identity to the corresponding protein from E . coli . Expression of rpsO in E . coli requires the use of a fully repressed inducible promoter because S15 from T . thermophilus is toxic for E . coli cells . When purified without denaturation from either overproducing E . coli strain or from T . thermophilus ribosomes, the S15 protein is stable and binds a cloned T . thermophilus 16S rRNA fragment (nucleotides 559-753), with low identical dissociation constants (2.5 nM), thus demonstrating that the thermophilic protein folds correctly in a mesophilic bacterium . The rRNA fragment bound corresponds in position and structure to the 16S rRNA fragment of E . coli . A similar high affinity was also found for the binding of S15 from T . thermophilus or E . coli to the corresponding E . coli 16S rRNA fragment, whereas a slightly lower affinity was observed in binding experiments between E . coli S15 and T . thermophilus 16S rRNA fragment . These results suggest that S15 from T . thermophilus recognizes similar determinants in both rRNA fragments . Competition experiments support this conclusion. Lett Appl Microbiol, 1997 Jun, 24(6), 441 - 4 Degradation of 3-chlorobenzoate by thermophilic micro-organisms; Maloney SE et al.; Thermophilic (75 degrees C), anaerobic biodegradation of chlorobenzoates was investigated using different inocula from geothermal and non-geothermal environments . Microbial dehalogenation of 3-chlorobenzoate (0.5 mmol l-1 was achieved by two mixed cultures growing anaerobically at 75 degrees C . One culture consisted of a facultative anaerobe and two obligate anaerobes, one of which was a methanogen, isolated from terrestrial sediments from hot springs in New Zealand . The other culture, derived from a non-geothermal environment, consisted of a Clostridium spp . and a non-spore-forming obligate anaerobe . No degradation of either 2-chlorobenzoate or 4-chlorobenzoate was achieved by these thermophilic cultures over the same time period . This is the first reported biotransformation of this chlorinated aromatic at a temperature of 75 degrees C. J Appl Microbiol, 1997 Jun, 82(6), 695 - 704 Presence of additional peptidases in Streptococcus thermophilus CNRZ 302 compared to Lactococcus lactis; Rul F et al.; Streptococcus thermophilus is widely used in the dairy industry but little is known about its peptidase system . The aim of this study was to determine the biochemical and genetic characteristics of this system, and to compare it to the well known system of Lactococcus lactis . We separated the intracellular proteins of Strep . thermophilus CNRZ 302 and L . lactis NCDO 763 by ion-exchange chromatography and we detected the activity of the different types of peptidases . In both L . lactis and Strep . thermophilus strains, we showed 13 different peptidase activities with biochemical homologies between both species . Streptococcus thermophilus also possessed two peptidases which we did not find in L . lactis: an aminopeptidase and an oligopeptidase . We performed Southern blot experiments and among the eight peptidase genes tested, only the genes encoding the general aminopeptidases, pepC and pepN, were homologous between the L . lactis and Strep . thermophilus strains . Besides biochemical and genetic similarities, the peptidase systems of Strep . thermophilus and L . lactis thus differed by the presence of additional peptidases in Strep . thermophilus. Mol Biol Cell, 1997 Jun, 8(6), 1051 - 61 An unusual fibrillarin gene and protein: structure and functional implications; David E et al.; The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing . The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing . We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein . The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins . The remainder of the amino acid sequence of the molecule is more conserved . Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations . Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells . Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena . The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested. J Dairy Sci, 1997 Jun, 80(6), 1031 - 7 Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation and the effects of bile; Marteau P et al.; This study was conducted to validate a dynamic model of the stomach and small intestine to quantify the survival of lactic acid bacteria and to assess the influence of gastrointestinal secretions . The survival of a single strain of each of the following species, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Streptococcus thermophilus, was measured under physiological conditions (e.g., peristalsis, changes in pH, and changes in concentrations of enzymes and bile) and were compared with data obtained from humans . No significant differences were found between the in vitro and in vivo data, indicating that the model has a predictive value for the survival of these bacteria in humans . The survival of these strains of lactic acid bacteria in the gastrointestinal model was investigated under two different conditions in the small intestine: simulation of physiological secretion of bile and low bile secretion . Reductions in viability were significantly different between the bacterial species . The dose-response effect of bile on the survival of the tested bacteria was significant, demonstrating the bactericidal effect of bile salts . This study demonstrates the differences among bacterial species in their sensitivity to gastric and intestinal secretions. J Bacteriol, 1997 Jun, 179(12), 3997 - 4002 A genetic locus involved in iron utilization unique to some Campylobacter strains; Guerry P et al.; Two genes involved in iron utilization in Campylobacter coli VC167 T1 have been characterized . The cfrA gene encodes a protein with a predicted Mr of 77,653 which, after processing of the leader sequence, has a predicted Mr of 75,635 . This protein has significant sequence identity to siderophore receptors of several bacteria, and site-specific mutants defective in cfrA do not synthesize one of two major iron-repressible outer membrane proteins . An adjacent gene encodes a TonB-like protein; a mutant in this gene lost the ability to utilize hemin, ferrichrome, and enterochelin as iron sources . The cfrA and tonB genes of VC167 T1 hybridized to all strains of C . coli and most strains of C . jejuni examined but did not hybridize to several other strains of C . jejuni, suggesting that the thermophilic campylobacters can be separated into two categories based on the presence of these two iron utilization genes. J Bacteriol, 1997 Jun, 179(12), 3899 - 913 Compositional biases of bacterial genomes and evolutionary implications; Karlin S et al.; We compare and contrast genome-wide compositional biases and distributions of short oligonucleotides across 15 diverse prokaryotes that have substantial genomic sequence collections . These include seven complete genomes (Escherichia coli, Haemophilus influenzae, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis sp . strain PCC6803, Methanococcus jannaschii, and Pyrobaculum aerophilum) . A key observation concerns the constancy of the dinucleotide relative abundance profiles over multiple 50-kb disjoint contigs within the same genome . (The profile is rhoXY* = fXY*/fX*fY* for all XY, where fX* denotes the frequency of the nucleotide X and fY* denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complementary sequence.) On the basis of this constancy, we refer to the collection {rhoXY*} as the genome signature . We establish that the differences between {rhoXY*} vectors of 50-kb sample contigs of different genomes virtually always exceed the differences between those of the same genomes . Various di- and tetranucleotide biases are identified . In particular, we find that the dinucleotide CpG=CG is underrepresented in many thermophiles (e.g., M . jannaschii, Sulfolobus sp., and M . thermoautotrophicum) but overrepresented in halobacteria . TA is broadly underrepresented in prokaryotes and eukaryotes, but normal counts appear in Sulfolobus and P . aerophilum sequences . More than for any other bacterial genome, palindromic tetranucleotides are underrepresented in H . influenzae . The M . jannaschii sequence is unprecedented in its extreme underrepresentation of CTAG tetranucleotides and in the anomalous distribution of CTAG sites around the genome . Comparative analysis of numbers of long tetranucleotide microsatellites distinguishes H . influenzae . Dinucleotide relative abundance differences between bacterial sequences are compared . For example, in these assessments of differences, the cyanobacteria Synechocystis, Synechococcus, and Anabaena do not form a coherent group and are as far from each other as general gram-negative sequences are from general gram-positive sequences . The difference of M . jannaschii from low-G+C gram-positive proteobacteria is one-half of the difference from gram-negative proteobacteria . Interpretations and hypotheses center on the role of the genome signature in highlighting similarities and dissimilarities across different classes of prokaryotic species, possible mechanisms underlying the genome signature, the form and level of genome compositional flux, the use of the genome signature as a chronometer of molecular phylogeny, and implications with respect to the three putative eubacterial, archaeal, and eukaryote domains of life and to the origin and early evolution of eukaryotes. Appl Environ Microbiol, 1997 Jun, 63(6), 2252 - 7 The methanogenic archaeon Methanosarcina thermophila TM-1 possesses a high-affinity glycine betaine transporter involved in osmotic adaptation; Proctor LM et al.; Methanogenic Archaea are found in a wide range of environments and use several strategies to adjust to changes in extracellular solute concentrations . One methanogenic archaeon, Methanosarcina thermophila TM-1, can adapt to various osmotic conditions by synthesis of alpha-glutamate and a newly discovered compatible solute, Ne-acetyl-beta-lysine, or by accumulation of glycine betaine (betaine) and potassium ions from the environment . Since betaine transport has not been characterized for any of the methanogenic Archaea, we examined the uptake of this solute by M . thermophila TM-1 . When cells were grown in mineral salts media containing from 0.1 to 0.8 M NaC1, M . thermophila accumulated betaine in concentrations up to 140 times those of a concentration gradient within 10 min of exposure to the solute . The betaine uptake system consisted of a single, high-affinity transporter with an apparent K3 of 10 microM and an apparent maximum transport velocity of 1.15 nmol/min/mg of protein . The transporter appeared to be specific for betaine, since potential substrates, including glycine, sarcosine, dimethyl glycine, choline, and proline, did not significantly inhibit betaine uptake . M . thermophila TM-1 cells can also regulate the capacity for betaine accumulation, since the rate of betaine transport was reduced in cells pregrown in a high-osmolarity medium when 500 microM betaine was present . Betaine transport appears to be H+ and/or Na+ driven, since betaine transport was inhibited by several types of protonophores and sodium ionophores. J Bacteriol, 1997 Jun, 179(11), 3746 - 55 Composition and primary structure of the F1F0 ATP synthase from the obligately anaerobic bacterium Clostridium thermoaceticum; Das A et al.; The subunit composition and primary structure of the proton-translocating F1F0 ATP synthase have been determined in Clostridium thermoaceticum . The isolated enzyme has a subunit composition identical to that of the F1F0 ATP synthase purified from Clostridium thermoautotrophicum (A . Das, D . M . Ivey, and L . G . Ljungdahl, J . Bacteriol . 179:1714-1720, 1997), both having six different polypeptides . The molecular masses of the six subunits were 60, 50, 32, 17, 19, and 8 kDa, and they were identified as alpha, beta, gamma, delta, epsilon, and c, respectively, based on their reactivity with antibodies against the F1 ATPase purified from C . thermoautotrophicum and by comparing their N-terminal amino acid sequences with that deduced from the cloned genes of the C . thermoaceticum atp operon . The subunits a and b found in many bacterial ATP synthases could not be detected either in the purified ATP synthase or crude membranes of C . thermoaceticum . The C . thermoaceticum atp operon contained nine genes arranged in the order atpI (i), atpB (a), atpE (c), atpF (b), atpH (delta), atpA (alpha), atpG (gamma), atpD (beta), and atpC (epsilon) . The deduced protein sequences of the C . thermoaceticum ATP synthase subunits were comparable with those of the corresponding subunits from Escherichia coli, thermophilic Bacillus strain PS3, Rhodospirillum rubrum, spinach chloroplasts, and the cyanobacterium Synechococcus strain PCC 6716 . The analysis of total RNA by Northern hybridization experiments reveals the presence of transcripts (mRNA) of the genes i, a, and b subunits not found in the isolated enzyme . Analysis of the nucleotide sequence of the atp genes reveals overlap of the structural genes for the i and a subunits and the presence of secondary structures (in the b gene) which could influence the posttranscriptional regulation of the corresponding genes. J Bacteriol, 1997 Jun, 179(11), 3470 - 81 Structure and expression of a pyrimidine gene cluster from the extreme thermophile Thermus strain ZO5; Van de Casteele M et al.; On a 4.7-kbp HindIII clone of Thermus strain ZO5 DNA, complementing an aspartate carbamoyltransferase mutation in Escherichia coli, we identified a cluster of four potential open reading frames corresponding to genes pyrR, and pyrB, an unidentified open reading frame named bbc, and gene pyrC . The transcription initiation site was mapped at about 115 nucleotides upstream of the pyrR translation start codon . The cognate Thermus pyr promoter also functions in heterologous expression of Thermus pyr genes in E . coli . In Thermus strain ZO5, pyrB and pyrC gene expression is repressed three- to fourfold by uracil and increased twofold by arginine . Based on the occurrence of several transcription signals in the Thermus pyr promoter region and strong amino acid sequence identities (about 60%) between Thermus PyrR and the PyrR attenuation proteins of two Bacillus sp., we propose a regulatory mechanism involving transcriptional attenuation to control pyr gene expression in Thermus . In contrast to pyr attenuation in Bacillus spp., however, control of the Thermus pyr gene cluster would not involve an antiterminator structure but would involve a translating ribosome for preventing formation of the terminator RNA hairpin . The deduced amino acid sequence of Thermus strain ZO5 aspartate carbamoyltransferase (ATCase; encoded by pyrB) exhibits the highest similarities (about 50% identical amino acids) with ATCases from Pseudomonas sp . For Thermus strain ZO5 dihydroorotase (DHOase; encoded by pyrC), the highest similarity scores (about 40% identity) were obtained with DHOases from B . caldolyticus and Bacillus subtilis . The enzyme properties of ATCase expressed from truncated versions of the Thermus pyr gene cluster in E . coli suggest that Thermus ATCase is stabilized by DHOase and that the translation product of bbc plays a role in feedback inhibition of the ATCase-DHOase complex. Curr Microbiol, 1997 Jun, 34(6), 367 - 73 Cloning, sequence, and expression of the L-(+) lactate dehydrogenase of Streptococcus bovis; Wyckoff HA et al.; The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced . A genomic library of S . bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene . Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis . Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S . mutans ldh nucleic acid and amino acid sequences . Expression of the cloned S . bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E . coli FMJ39, by using an in-gel activity screen and by enzymatic assay . Increased LDH activity was observed in S . bovis JB1 containing the cloned ldh genes on a multicopy plasmid. J Biol Chem, 1997 May 30, 272(22), 14277 - 84 Thermophilin 13, a nontypical antilisterial poration complex bacteriocin, that functions without a receptor; Marciset O et al.; A novel broad host range antimicrobial substance, Thermophilin 13, has been isolated and purified from the growth medium of Streptococcus thermophilus . Thermophilin 13 is composed of the antibacterial peptide ThmA (Mr of 5776) and the enhancing factor ThmB (Mr of 3910); the latter peptide increased the activity of ThmA approximately 40 x . Both peptides are encoded by a single operon, and an equimolar ratio was optimal for Thermophilin 13 activity . Despite the antilisterial activity of Thermophilin 13, neither ThmA nor ThmB contain the YGNGV-C consensus sequence of Listeria-active peptides, and post-translational modifications comparable to that in the lantibiotics are also absent . Mass spectrometry did reveal the apparent oxidation of methionines in ThmA, which resulted in a peptide that could not be enhanced any longer by ThmB, whereas the intrinsic bactericidal activity was normal . Thermophilin 13 dissipated the membrane potential and the pH gradient in liposomes, and this activity was independent of membrane components from a sensitive strain (e.g . lipid or proteinaceous receptor) . Models of possible poration complexes formed are proposed on the basis of sequence comparisons, structure predictions, and the functional analysis of Thermophilin 13. J Mol Biol, 1997 May 23, 268(5), 922 - 33 Solution structure of ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus and its thermostability; Hatanaka H et al.; The three-dimensional structure of ferredoxin, purified from the thermophilic cyanobacterium Synechococcus elongatus, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance . In addition to the 946 distance constraints from nuclear Overhauser effect connectivities, we added 241 distance constraints derived from the crystal structure of Spirulina platensis ferredoxin to the 19 residues close to the {2Fe-2S} iron-sulfur center, where crosspeaks disappeared due to paramagnetic effects . The atomic root-mean-square difference of the ten converged structures from the mean structure was 0.61(+/-0.12) A for backbone atoms (N, C(alpha), C') . The main-chain structure was almost the same as the crystal structures of other mesophile ferredoxins, but comparison of the side-chain structures revealed an extension of the hydrophobic core, a unique hydrophobic patch on the surface of the large beta-sheet, and two unique charge networks in this thermostable ferredoxin structure, some of which might contribute to thermostability. J Clin Invest, 1997 May 15, 99(10), 2386 - 90 Interferon-gamma is necessary for the expression of hypersensitivity pneumonitis; Gudmundsson G et al.; Farmers lung disease is a common form of hypersensitivity pneumonitis (HP) and is characterized by inflammation and granuloma formation in the lung . Interferon-gamma is important for the expression of granulomatous diseases caused by infectious agents; however, the role this mediator in regulating expression of the granulomatous response to inhaled antigen is not known . To evaluate this, we compared the response to inhaled antigen of mice that do not express the gene coding for interferon-gamma (GKO) with that of their normal littermates (WT) . GKO and WT mice on a BALB/c background were exposed to 150 microg of the thermophilic bacteria Saccharopolyspora rectivirgula or saline alone, for three consecutive days a week, for 3 wk . After exposure to antigen, WT mice developed a marked granulomatous inflammation associated with an increase in lung weight and numbers of cells in bronchoalveolar lavage fluid (BAL) . Although GKO mice also exhibited an increase in lung weight and numbers of cells in BAL fluid, they developed minimal inflammation and no granulomas after a similar exposure to antigen . To further evaluate if the lack of a response to antigen in GKO mice was due to lack of IFN-gamma, we replaced this mediator via intraperitoneal injections . When given replacement IFN-gamma, the GKO mice developed granulomatous inflammation in the lung . These studies show that IFN-gamma is essential for the expression of hypersensitivity pneumonitis. Biochemistry, 1997 May 13, 36(19), 5902 - 11 Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl beta-D-cellobioside-inhibited forms, at 2.3 A resolution; Sulzenbacher G et al.; The mechanisms involved in the enzymatic degradation of cellulose are of great ecological and commercial importance . The breakdown of cellulose by fungal species is performed by a consortium of free enzymes, known as cellobiohydrolases and endoglucanases, which are found in many of the 57 glycosyl hydrolase families . The structure of the endoglucanase I (EG I), found in glycosyl hydrolase family 7, from the thermophilic fungus Fusarium oxysporum has been solved at 2.3 A resolution . In addition to the native enzyme, structures have also been determined with both the affinity label, 3,4-epoxybutyl beta-D-cellobioside, and the reaction product cellobiose . The affinity label is covalently bound, as expected, to the catalytic nucleophile, Glu197, with clear evidence for binding of both the R and S stereoisomers . Cellobiose is found bound to the -2 and -1 subsites of the enzyme . In marked contrast to the structure of EG I with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar {Sulzenbacher, G., et al . (1996) Biochemistry 35, 15280-15287}, the cellobiose complex shows no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the additional +1 subsite interactions and that the product is found, as expected, in its unstrained conformation. J Mol Biol, 1997 May 9, 268(3), 640 - 7 Recognition of tRNA(Gly) by three widely diverged glycyl-tRNA synthetases; Nameki N et al.; Glycyl-tRNA synthetase (GlyRS) is an unusual aminoacyl-tRNA synthetase because it varies in its quarternary structure between organisms; Escherichia coli GlyRS is an alpha2beta2 tetramer, whereas those of Thermus thermophilus and yeast are alpha2 dimers . In contrast, the tRNA(Gly) sequence is virtually identical in E . coli and T . thermophilus but very different in yeast . In this study, we examined the molecular recognition of tRNA(Gly) by three widely diverged GlyRSs using in vitro tRNA transcripts . Mutation studies showed that the discriminator base at position 73, the second base-pair, C2 x G71, in the acceptor stem, and the anticodon nucleotides, C35 and C36, contribute to the specific aminoacylation of all three GlyRSs, the discriminator base differing between prokaryotes (U73) and eukaryotes (A73) . However, we found differences between yeast and two bacteria around the second base-pair in the acceptor stem . The first base-pair, G1 x C72, is important for glycylation in E . coli and T . thermophilus, whereas the third base-pair, G3 x C70, is important for glycylation in yeast . These findings indicate that despite such large differences of the two prokaryotic GlyRSs, tRNA(Gly) identity has been essentially conserved in prokaryotes, and that there are also differences in the acceptor stem recognition between prokaryotes and yeast . The clear separation between prokaryotes and yeast is retained in the identity element location, whereas the apparent diversity of the two prokaryotic enzymes does not reflect on the tRNA recognition. J Biol Chem, 1997 May 9, 272(19), 12468 - 74 Chaperonin-mediated folding of green fluorescent protein; Makino Y et al.; Chaperonin-mediated folding of green fluorescent protein (GFP) was examined by real-time monitoring of recovery of fluorescence and by gel filtration high-performance liquid chromatography . Acid-denatured GFP can fold spontaneously upon dilution into the neutral buffer . When Escherichia coli GroEL/ES was present, folding of GFP was arrested . Folding was resumed by subsequent addition of 100 microM or 1 mM ATP, and native GFP was regenerated to 100% yield . When folding was resumed by 10 microM ATP (1.4 mol/mol GroEL subunit), about 60% of GFP recovered native structure, and one-half of them (30%) was found to be still bound to GroEL/ES, indicating the occurrence of folding in the central cavity of the GroEL ring underneath GroES (cis-folding) . Because the overall rates of GroEL/ES-, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded that cis-folding proceeded as fast as spontaneous folding . The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the folding mixture . Holo-chaperonin from Thermus thermophilus, which was purified as a cpn60/10 complex, exhibited the similar cis-folding . Consistently, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed. Biochemistry (Mosc), 1997 May, 62(5), 537 - 42 RNA-binding properties of an unusual ribosomal protein TL5 from Thermus thermophilus; Meshcheryakov VA et al.; The gene encoding the 5S rRNA-binding ribosomal protein TL5 from Thermus thermophilus, an extremely thermophilic species, was expressed in E . coli . A method for isolation of TL5 from the overproducing strain was developed . Samples of TL5 protein isolated from ribosomes and the overproducing strain displayed identical RNA-binding properties . Circular dichroic spectroscopy was used to calculate the secondary structure of the protein . TL5 was shown to form a stable complex with the 3'-terminal fragment of 5S rRNA, which is similar to the fragment of E . coli RNA that binds to L25 protein . The data suggest that TL5 from T . thermophilus and L25 from E . coli bind to similar sites on the 5S rRNA molecule. Biochimie, 1997 May, 79(5), 287 - 92 Cloning and overexpression of polypeptide release factor 1 of Thermus thermophilus; Ito K et al.; A prfA gene encoding polypeptide release factor RF1 was cloned from Thermus thermophilus . T thermophilus RF1 shares 68% homology with Escherichia coli RF1, and its overproduction reduced readthrough translation of UAG, not of UGA, in the lacZ gene . Rapid purification of T thermophilus RF1 was achieved by T7-RNA polymerase driven overexpression of T thermophilus RF1 protein with a C-terminal histidine tag. Pathology, 1997 May, 29(2), 209 - 16 Disc susceptibility testing for thermomphilic campylobacters; Huysmans MB et al.; The minimum inhibitory concentrations (MICs) and zone diameters around NCCLS strength discs of 100 clinical isolates of thermophilic Campylobacter species, including 79 strains of Campylobacter jejuni subsp . jejuni, 19 of C . coli and two of C . lari, plus three type strains of these species, were determined for erythromycin, clindamycin, nalidixic acid, norfloxacin, ciprofloxacin, ampicillin, piperacillin, cephalothin, ceftriaxone, chloramphenicol, gentamicin and tetracycline . Using error-rate bounded analysis and adjustment of MIC breakpoints to fit natural populations, tentative interpretive zone diameter criteria were set for each of the antimicrobials . Application of these criteria showed that resistance to quinolones was not detected in species other than C . lari . Two strains of C . jejuni subsp . jejuni were susceptible to cephalothin . The type strain of C . lari was susceptible to erythromycin and resistant to clindamycin . Full resistance to erythromycin, chloramphenicol or gentamicin was not found in any strain, while nine strains were resistant to tetracycline . This disc method should provide a simple approach to resistance detection for surveillance or routine testing of invasive isolates. Lett Appl Microbiol, 1997 May, 24(5), 347 - 50 Survival of Escherichia coli O157:H7 in yoghurt during preparation and storage at 4 degrees C; Massa S et al.; Cow's milk was inoculated with ca 10(3) and 10(7) cfu ml-1 Escherichia coli O157:H7 . After fermentation at 42 degrees C for 0-5 h, the yoghurt was stored at 4 degrees C . Two kinds of yoghurt were used: traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E . coli O157:H7 decreased from 3.52 to 2.72 log10 cfu ml-1 and from 7.08 to 5.32 log10 cfu ml-1 in TY, and from 3.49 to 2.73 log10 cfu ml-1 and from 7.38 to 5.41 log10 cfu ml-1 in BY . The pH values of yoghurt dropped from 6.6 to 4.5 and 4.4 in TY (for low and high pathogen inocula, respectively), and from 6.6 to 4.6 and 4.5 in BY (for low and high pathogen inocula, respectively). Biochem J, 1997 May 1, 323 ( Pt 3), 833 - 40 Effects of temperature and SDS on the structure of beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus; D'auria S et al.; The effects of temperature and SDS on the three-dimensional organization and secondary structure of beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus were investigated by CD, IR spectroscopy and differential scanning calorimetry . CD spectra in the near UV region showed that the detergent caused a remarkable change in the protein tertiary structure, and far-UV CD analysis revealed only a slight effect on secondary structure . Infrared spectroscopy showed that low concentrations of the detergent (up to 0.02%) induced slight changes in the enzyme secondary structure, whereas high concentrations caused the alpha-helix content to increase at high temperatures and prevented protein aggregation. Microbiology, 1997 May, 143 ( Pt 5), 1587 - 94 A 16 kDa protein family overexpressed by Streptococcus thermophilus PB18 in acid environments; Gonzalez-Marquez H et al.; The one- and two-dimensional protein patterns of Streptococcus thermophilus PB18 in the exponential and stationary phases of growth were analysed . One-dimensional SDS-PAGE showed that a 16 kDa protein was overexpressed in stationary phase as well as 2 h after an acid shock, and that it was not expressed when the bacteria reached the stationary phase in medium with limiting lactose concentrations (5 or 10 g l-1), in which the pH (5.5) was not as acid as in control cultures (pH 4.7, lactose 20 g l-1) . The results support the idea that this protein is expressed in response to the acidic environment and not in response to the growth phase . Two-dimensional PAGE showed that nine proteins were expressed only during the exponential phase and ten others only during the stationary phase . The 16 kDa band seen in one-dimensional SDS-PAGE corresponded to a 16 kDa protein family observed on two-dimensional SDS-PAGE/IEF gels, whose expression was increased 8.5-fold when the extracellular pH reached a critical value below 5.0 . The N-terminal sequences of proteins from two spots on the two-dimensional gels (members of the 16 kDa family) were determined and found to be identical . The physiological role of this protein family has not yet been elucidated. J Bacteriol, 1997 May, 179(10), 3298 - 303 Rates of spontaneous mutation in an archaeon from geothermal environments; Jacobs KL et al.; To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid . Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively . To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments . The measured rates of forward mutation at the pyrE and pyrF loci in S . acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli . The normal rate of spontaneous mutation in E . coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA . Our results provide indirect evidence that S . acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells. Proteins, 1997 May, 28(1), 117 - 30 Structural basis for thermostability and identification of potential active site residues for adenylate kinases from the archaeal genus Methanococcus; Haney P et al.; Sequence comparisons of highly related archaeal adenylate kinases (AKs) from the mesophilic Methanococcus voltae, the moderate thermophile Methanococcus thermolithotrophicus, and two extreme thermophiles Methanococcus igneus and Methanococcus jannaschii, allow identification of interactions responsible for the large variation in temperatures for optimal catalytic activity and thermostabilities observed for these proteins . The tertiary structures of the methanococcal AKs have been predicted by using homology modeling to further investigate the potential role of specific interactions on thermal stability and activity . The alignments for the methanococcal AKs have been generated by using an energy-based sequence-structure threading procedure against high-resolution crystal structures of eukaryotic, eubacterial, and mitochondrial adenylate and uridylate (UK) kinases . From these alignments, full atomic model structures have been produced using the program MODELLER . The final structures allow identification of potential active site interactions and place a polyproline region near the active site, both of which are unique to the archaeal AKs . Based on these model structures, the additional polar residues present in the thermophiles could contribute four additional salt bridges and a higher negative surface charge . Since only one of these possible salt bridges is interior, they do not appear significantly to the thermal stability . Instead, our model structures indicate that a larger and more hydrophobic core, due to a specific increase in aliphatic amino acid content and aliphatic side chain volume, in the thermophilic AKs is responsible for increased thermal stability. Protein Sci, 1997 May, 6(5), 1074 - 83 Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase; Peretz M et al.; The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2{14C}iodoacetic acid, via S-alkylation . The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label . In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT . NADP protected the enzyme from both modification and inactivation by the biradical . RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue . A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline . This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC . The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203 . Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability . The status of these cysteine residues was verified by site directed mutagenesis. J Bacteriol, 1997 May, 179(9), 3068 - 72 Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria; Piard JC et al.; The M6 protein from Streptococcus pyogenes is the best-characterized member of a family of cell envelope-associated proteins . Based on the observation that the C-terminal sorting signals of these proteins can drive cell wall anchoring of heterologous unanchored proteins, we have cloned and expressed the emm6 structural gene for the M6 protein in various lactic acid bacteria (LAB) . The emm6 gene was successfully expressed from lactococcal promoters in several Lactococcus lactis strains, an animal-colonizing Lactobacillus fermentum strain, Lactobacillus sake, and Streptococcus salivarius subsp . thermophilus . The M6 protein was efficiently anchored to the cell wall in all strains tested . In lactobacilli, essentially all detectable M6 protein was cell wall associated . These results suggest the feasibility of using the C-terminal anchor moiety of M6 for protein surface display in LAB. Genetics, 1997 May, 146(1), 135 - 47 Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment; Cassidy-Hanley D et al.; Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development . Both macronuclear and micronuclear transformants were recovered . Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the macronucleus . Evidence is given for transient retention of the introduced plasmid . Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome. Arch Microbiol, 1997 May, 167(5), 275 - 9 Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6; Yoon KS et al.; Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography . The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an alphabetagammadelta-structure . The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H . thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen . NAD, NADP, and ferredoxins from Chlorella spp . and Clostridium pasteurianum were ineffective as the electron acceptor . The temperature optimum for pyruvate oxidation was approximately 80 degrees C . The pH optimum was 7.6-7.8 . The apparent Km values for pyruvate and coenzyme A at 70 degrees C were 3.45 mM and 54 microM, respectively . The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t50%) at 70 degrees C was approximately 8 h. Biochem Biophys Res Commun, 1997 Apr 28, 233(3), 834 - 7 Analysis of the mismatch and insertion/deletion binding properties of Thermus thermophilus, HB8, MutS; Whitehouse A et al.; The methyl-directed long patch repair pathway in Escherichia coli is involved in increasing the fidelity of replication specific repair of DNA polymerase incorporation errors . This pathway is mediated by three gene products, MutS, MutL, and MutH, which are conserved in higher eukaryotes . Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC) . A MutS homologue has recently been identified in the extremely thermophilic bacterium, Thermus thermophilus . Here we describe analysis of the binding properties of this protein, which has indicated it can identify all specific base mismatches as well as one, two and three base pair insertion/deletion mutations . We therefore believe this protein may be generally useful for applications involving mismatch detection. J Mol Biol, 1997 Apr 18, 267(5), 1104 - 12 Assessing the reliability of RNA folding using statistical mechanics; Huynen M et al.; We have analyzed the base-pairing probability distributions of 16 S and 16 S-like, and 23 S and 23 S-like ribosomal RNAs of Archaea, Bacteria, chloroplasts, mitochondria and Eukarya, as predicted by the partition function approach for RNA folding introduced by McCaskill . A quantitative analysis of the reliability of RNA folding is done by comparing the base-pairing probability distributions with the structures predicted by comparative sequence analysis (comparative structures) . We distinguish two factors that show a relationship to the reliability of RNA minimum free energy structure . The first factor is the dominance of one particular base-pair or the absence of base-pairing for a given base within the base-pairing probability distribution (BPPD) . We characterize the BPPD per base, including the probability of not base-pairing, by its Shannon entropy (S) . The S value indicates the uncertainty about the base-pairing of a base: low S values result from BPPDs that are strongly dominated by a single base-pair or by the absence of base-pairing . We show that bases with low S values have a relatively high probability that their minimum free energy (MFE) structure corresponds to the comparative structure . The BPPDs of prokaryotes that live at high temperatures (thermophilic Archaea and Bacteria) have, calculated at 37 degrees C, lower S values than the BPPDs of prokaryotes that live at lower temperatures (mesophilic and psychrophilic Archaea and Bacteria) . This reflects an adaptation of the ribosomal RNAs to the environmental temperature . A second factor that is important to consider with regard to the reliability of MFE structure folding is a variable degree of applicability of the thermodynamic model of RNA folding for different groups of RNAs . Here we show that among the bases that show low S values, the Archaea and Bacteria have similar, high probabilities (0.96 and 0.94 in 16 S and 0.93 and 0.91 in 23 S, respectively) that the MFE structure corresponds to the comparative structure . These probabilities are lower in the chloroplasts (16 S 0.91, 23 S 0.79), mitochondria (16 S-like 0.89, 23 S-like 0.69) and Eukarya (18 S 0.81, 28 S 0.86). Biochem Biophys Res Commun, 1997 Apr 17, 233(2), 568 - 71 Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic red algae with a strong specificity for CO2 fixation; Uemura K et al.; Strongly carboxylase-specific ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was found in Galdieria partita and Cyanidium caldarium (Cyanidiophyceae) . The relative specificity, VcKo/VoKc, of Galdieria and Cyanidium RuBisCO was 238 and 222, respectively; 2.4 to 2.5-fold that of higher plant RuBisCOs . The apparent Km of RuBisCO from the thermophilic red algae for CO2 was 6 to 7 microM and the smallest of the values reported so far for other RuBisCOs . The pre-rhodophyte Porphiridium purpureum, which lives at moderate temperatures, had RuBisCO with the relative specificity value of 144 . A large difference (5.2 kcal x mol(-1)) in the activation energies between the carboxylase and oxygenase activities in Galdieria RuBisCO was a cause of the strong specificity for the carboxylase activity. FEBS Lett, 1997 Apr 7, 406(1-2), 142 - 6 Cloning and expression of superoxide dismutase from Aquifex pyrophilus, a hyperthermophilic bacterium; Lim JH et al.; A superoxide dismutase (SOD) gene of Aquifex pyrophilus, a marine hyperthermophilic bacterium, was cloned, sequenced, expressed in Escherichia coli, and its gene product characterized . This is the first SOD from a hyperthermophilic bacterium that has been cloned . It is an iron-containing homo-oligomeric protein with a monomeric molecular mass of 24.2 kDa . The DNA-derived amino acid sequence is more similar to those of known Mn- and Fe-SODs from thermophilic archaea than of Cu, Zn-SODs . The metal binding residues found in all SOD sequences from different species are also conserved in A . pyrophilus SOD . The protein is biochemically active only as an oligomer and is resistant to thermal denaturation. J Biotechnol, 1997 Apr 4, 54(1), 77 - 80 Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus; Stoyan T et al.; The L-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced . The open reading frame has 1101 bp and codes for a protein of 39.9 kDa . The deduced amino acid sequence of the LeuDH from B . cereus shares 70-80% identity with LeuDH's from the thermophilic strains B . stearothermophilus and Thermoactinomyces intermedius . The active protein was overexpressed in Escherichia coli to yield approximately 30% of the total soluble protein. J Mol Biol, 1997 Apr 4, 267(3), 749 - 61 Structure-function relationships within the peptide deformylase family . Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion; Meinnel T et al.; Thermus thermophilus peptide deformylase was characterized . Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities . In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand . The study of site-directed mutants of the E . coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme . Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases . Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp . could be identified . The characterization of the product of the open reading frame from B . stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E . coli enzyme . Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved . This motif is also located in the active site according to the three-dimensional structure of the E . coli enzyme . Site-directed variants of E . coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme . Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion . Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E . coli enzyme . This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure. Biochimie, 1997 Apr, 79(4), 195 - 203 Organization of the Thermus thermophilus nusA/infB operon and overexpression of the infB gene in Escherichia coli; Vornlocher HP et al.; The structural gene for translation initiation factor IF2 from Thermus thermophilus was identified on the basis of the N-terminal amino acid sequence of intact T thermophilus IF2 and an internal 25 kDa IF2 fragment . A total of 5135 bp was cloned and sequenced, comprising the open reading frames for p15A, NusA, p10A, IF2, p10B and SecD, which may form an operon . There are pronounced similarities between the operon arrangement and primary sequence of the T thermophilus genes and proteins, respectively, and their counterparts from other organisms . The T thermophilus infB gene was expressed to a high level in E coli . Four hundred milligrams of homogenous T thermophilus IF2 were prepared from 60 g of overproducing cells. Bioorg Khim, 1997 Apr, 23(4), 257 - 61 {Recombinant Thermus thermophilus His6-DNA polymerase with reverse transcriptase activity}; Smirnov IuV et al.; The Tth DNA polymerase genes from the thermophilic bacterium Thermus thermophilus (strans HB-8 and Tt-111) were cloned and expressed in Escherichia coli cells . The target protein contained a polyhistidine tag at the N-terminus that allowed its single-stage isolation by affinity chromatography on Ni-NTA-agarose . The isolated enzyme displayed both high DNA polymerase and reverse transcriptase activities. Cell Biol Int, 1997 Apr, 21(4), 213 - 6 A rapid bioassay to detect mycotoxins using a melanin precursor overproducer mutant of the ciliate Tetrahymena thermophila; Martin Gonzalez A et al.; Four different mycotoxins (patulin, T-2 toxin, diacetoxyscirpenol and roquefortine) were used to study growth inhibitory effects on a melanin precursor overproducer mutant of the ciliate Tetrahymena thermophila . This strain is especially sensitive to diacetoxyscirpenol and T-2 toxin . The secretion capacity of melanin precursors into the culture medium by this mutant and its biosensor capacity are very useful characteristics to elaborate a rapid bioassay to detect some specific mycotoxins. Biochem J, 1997 Apr 1, 323 ( Pt 1), 259 - 64 Stability of a thermophilic TIM-barrel enzyme: indole-3-glycerol phosphate synthase from the thermophilic archaeon Sulfolobus solfataricus; Andreotti G et al.; The stability and activity of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus were studied as a function of pH and temperature . In this paper we focus on three points: (1) the long-term stability of the protein to irreversible denaturation at high temperature; (2) the short-term stability of the protein to reversible temperature-driven unfolding; and (3) the dependence of its activity on temperature . Results can be summarized as follows: (a) the same first-order kinetic constant (0.020+/-0.003 min-1) was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 degrees C; (b) short-term stability experiments revealed different behaviour in two different pH ranges (6.5-8.0, 8.5-9.5), suggesting that the melting temperature is higher at alkaline than at neutral pH; (c) the dependence of activity on temperature was investigated at pH 7.0 and 9.0, and a discontinuity was observed in the Arrhenius plot of kcat values at pH 9.0 . We also investigated the stability in the presence of guanidinium chloride at 20 degrees C either at pH 7.0 or at pH 9.0, and we present data that indicate that the unfolding mechanism closely approaches a two-state model at pH 7.0 and a more complex mechanism at pH 9.0 . Satisfactory fitting of the equilibrium unfolding transition obtained by fluorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms . At 20 degrees C the folded conformation is more stable than the unfolded conformation by (14 . 7+/-1.2) kJ/mol at pH 7.0 and by (25.5+/-1.8) kJ/mol at pH 9.0. Arch Microbiol, 1997 Apr, 167(4), 233 - 8 Biochemical and phylogenetic characterization of two novel deep-sea Thermococcus isolates with potentially biotechnological applications; Canganella F et al.; The partial 16S rDNA gene sequences of two thermophilic archaeal strains, TY and TYS, previously isolated from the Guaymas Basin hydrothermal vent site were determined . Lipid analyses and a comparative analysis performed with 16S rDNA sequences of similar thermophilic species showed that the strains isolated from deep-sea vents were not identical to the other species belonging to the genus Thermococcus . On the basis of the results of the phylogenetic analyses, lipid analyses, and previously reported physiological data, we believe that strains TY and TYS are significantly different from the previously described Thermococcus species . According to specific physiological and molecular features, we propose the use of these isolates as potential tools for the development of biotechnological applications in the field of starch processing and DNA technology. Microbiology, 1997 Apr, 143 ( Pt 4), 1335 - 43 Analysis of the genetic polymorphism between three Streptococcus thermophilus strains by comparing their physical and genetic organization; Roussel Y et al.; The physical maps of Streptococcus thermophilus CNRZ368 and NST2280 strains were constructed by analysing PFGE patterns obtained with the low-frequency-cutting enzymes SmaI, BssHII and SfiI . Their chromosomes are 1864 and 1840 kb circular molecules, respectively . Comparison of their physical maps with that of the reference A054 strain revealed a relatively conserved organization of the restriction sites . Three variable regions were detected with the map of CNRZ368 whereas 15 were found with the map of NST2280 . To construct the genetic maps, probes corresponding to 10 single-copy genes, the rrn genes and the insertion sequences IS1191, IS981 and ISS1 were hybridized to Southern blots of chromosomal DNA digested with the different mapping enzymes . Comparison of the genetic maps of the three strains showed a conserved location of the mapped single-copy genes . However, six rrn loci were present in the chromosome of A054 and CNRZ368 whereas five were present in the NST2280 chromosome . A polymorphism was also found in the copy number of the insertion sequences between the three strains. Mol Microbiol, 1997 Apr, 24(1), 61 - 72 Surface proteins and a novel transcription factor regulate the expression of the S-layer gene in Thermus thermophilus HB8; Fernandez-Herrero LA et al.; We have identified proteins that control the expression of slpA, the gene encoding the crystalline surface layer of Thermus thermophilus HB8 . We cloned three genes from T . thermophilus that specifically repressed the expression of the slpA promoter in Escherichia coli . The proteins encoded by two of them (Rep6 and Rep29) bound in vitro to the slpA promoter, while that from the third (Rep54) bound specifically to the 5'-untranslated region (5'UTR) of the slpA mRNA . Rep6 protein was identified as a C-fragment from a Thermus cytoplasmic basic protein of 28 kDa, whose coding gene, slrA (for S-layer regulator), was characterized . Surprisingly, Rep29 was identified as a C-fragment of SlpM, an S-layer-like protein that is overexpressed in slpA mutants . Insertional inactivation of slrA and slpM demonstrated their in vivo function in the control of slpA transcription: SlrA acts as a repressor, and SlpM as an activator . Even more surprising was the identification of Rep54, the 5'UTR mRNA-binding protein, as a C-terminal fragment of the SlpA protein . This result, in addition to further in vivo evidence presented here, supports the existence of a translational autoregulation in slpA expression . The physiological meaning of overlapping transcriptional and translational controls of S-layer expression, and its relationships with other systems, are discussed. Eur J Biochem, 1997 Apr 1, 245(1), 129 - 36 Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase II is a thermostable M(r) 540,000 homohexameric alpha-glucosidase with both exo-alpha-1,4-glucosidase and oligo-1,6-glucosidase activities; Suzuki Y et al.; alpha-Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477; growth over 30-66 degrees C) was purified to a homogeneous state . Its M(r) was estimated as 90000 by SDS/PAGE . However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions . The enzyme was not glycosylated . Its isoelectric point was estimated as 5.7 . The N-terminal sequence of 20 residues was determined asAla1-Ile-Gln-Pro-Glu-Gln-Asp-Asp-Lys-Thr-Gln-Glu-Asp-Gly- Tyr-Ile-Asp-Ile-Gly-Asn20 . The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo-alpha-1,4-glucosidase and the monomeric oligo-1,6-glucosidase from the same microorganism . The alpha-glucosidase II had no antigenic group shared with the latter two enzymes . Analysis of substrate specificity showed that the alpha-glucosidase II has dual activity towards oligo-1,6-glucosidases and exo-alpha-1,4-glucosidases, but its preference is for non-reducing terminal alpha-1,4 glucosidic bonds in substrates . Kinetic studies proved that both activities are attributed to the same catalytic site . The enzyme was most active at 81 degrees C and pH 7.0 . Its half-life at pH 6.8 was 10 min at 81 degrees C, and 5 h at 55 degrees C in 6.4 M urea, 26% ethanol or 2.5% SDS . We suggest that the alpha-glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo-alpha-1,4-glucosidase and the oligo-1,6-glucosidase present in the same strain. Int J Food Microbiol, 1997 Apr 1, 35(2), 137 - 45 Incidence and detection of thermotolerant and thermophilic fungi from maize with particular reference to Thermoascus species; Wareing PW; A number of thermotolerant and thermophilic fungi were isolated from shipments of food-aid grain, and from large bag stacks of maize stored in sub-Saharan Africa . Thermotolerant fungi included Aspergillus candidus, A . fumigatus, A . flavus and Paecilomyces varioti; thermophilic fungi included Thermomyces lanuginosus, Rhizomucor pusillus, Thermoascus aurantiacus and T . crustaceous . Temperature profiles for Thermoascus spp . indicated that isolates of T . aurantiacus grew up to 60 degrees C, and T . crustaceous to 55 degrees C, whereas Paecilomyces could not grow above 50 degrees C . Thermoascus species isolated from grains conformed to published morphological descriptions . Problems associated with the detection and interpretation of fungal spoilage in relation to heat-damaged grain are discussed. Int J Syst Bacteriol, 1997 Apr, 47(2), 541 - 7 Thermoterrabacterium ferrireducens gen . nov., sp . nov., a thermophilic anaerobic dissimilatory Fe(III)-reducing bacterium from a continental hot spring; Slobodkin A et al.; A strain of a thermophilic, anaerobic, dissimilatory, Fe(III)-reducing bacterium, Thermoterrabacterium ferrireducens gen . nov., sp . nov . (type strain JW/AS-Y7T; DSM 11255), was isolated from hot springs in Yellowstone National Park and New Zealand . The gram-positive-staining cells occurred singly or in pairs as straight to slightly curved rods, 0.3 to 0.4 by 1.6 to 2.7 microns, with rounded ends and exhibited a tumbling motility . Spores were not observed . The temperature range for growth was 50 to 74 degrees C with an optimum at 65 degrees C . The pH range for growth at 65 degrees C was from 5.5 to 7.6, with an optimum at 6.0 to 6.2 . The organism coupled the oxidation of glycerol to reduction of amorphous Fe(III) oxide or Fe(III) citrate as an electron acceptor . In the presence as well as in the absence of Fe(III) and in the presence of CO2, glycerol was metabolized by incomplete oxidation to acetate as the only organic metabolic product; no H2 was produced during growth . The organism utilized glycerol, lactate, 1,2-propanediol, glycerate, pyruvate, glucose, fructose, mannose, and yeast extract as substrates . In the presence of Fe(III) the bacterium utilized molecular hydrogen . The organism reduced 9,10-anthraquinone-2,6-disulfonic acid, fumarate (to succinate), and thiosulfate (to elemental sulfur) but did not reduce MnO2, nitrate, sulfate, sulfite, or elemental sulfur . The G + C content of the DNA was 41 mol% (as determined by high-performance liquid chromatography) . The 16S ribosomal DNA sequence analysis placed the isolated strain as a member of a new genus within the gram-type-positive Bacillus-Clostridium subphylum. Int J Syst Bacteriol, 1997 Apr, 47(2), 505 - 9 Deferribacter thermophilus gen . nov., sp . nov., a novel thermophilic manganese- and iron-reducing bacterium isolated from a petroleum reservoir; Greene AC et al.; A thermophilic anaerobic bacterium, designated strain BMAT (T = type strain), was isolated from the production water of Beatrice oil field in the North Sea (United Kingdom) . The cells were straight to bent rods (1 to 5 by 0.3 to 0.5 microns) which stained gram negative . Strain BMAT obtained energy from the reduction of manganese (IV), iron(III), and nitrate in the presence of yeast extract, peptone, Casamino Acids, tryptone, hydrogen, malate, acetate, citrate, pyruvate, lactate, succinate, and valerate . The isolate grew optimally at 60 degrees C (temperature range for growth, 50 to 65 degrees C) and in the presence of 2% (wt/vol) NaCl (NaCl range for growth, 0 to 5% {wt/vol}) . The DNA base composition was 34 mol% G + C . Phylogenetic analyses of the 16S rRNA gene indicated that strain BMAT is a member of the domain Bacteria . The closest known bacterium is the moderate thermophile Flexistipes sinusarabici (similarity value, 88%) . Strain BMAT possesses phenotypic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, we propose that this isolate should be described as a member of a novel species of a new genus, Deferribacter thermophilus gen . nov., sp . nov. Int J Syst Bacteriol, 1997 Apr, 47(2), 408 - 13 Porphyrobacter tepidarius sp . nov., a moderately thermophilic aerobic photosynthetic bacterium isolated from a hot spring; Hanada S et al.; A new thermophilic bacterium, strain OT3T (T = type strain), was isolated from a brackish hot spring . Strain OT3T is an obligate aerobe that synthesizes bacteriochlorophyll a and has a photosynthetic apparatus . This isolate is a thermophilic bacterium with an optimal growth temperature of 40 to 48 degrees C . The cells are nonmotile, ovoid to short rods . An analysis of 16S rRNA sequences revealed that the new strain forms a coherent cluster with members of the alpha-4 group of the alpha subclass of the Proteobacteria, which contains the genera Erythrobacter, Erythromicrobium, and Porphyrobacter . The closest relative is Porphyrobacter neustonensis, with a 16S rRNA sequence similarity of 96.8% . The in vivo absorption spectrum has maxima at 460, 494, 596, 800, and 870 nm . The main carotenoids are OH-beta-carotene sulfate derivatives, nostoxanthin, and bacteriorubixanthinal . Growth occurs with glucose, acetate, glutamate, butyrate, Casamino Acids, and yeast extract as sole energy sources . The pigment composition and nutritional profile of the new isolate are similar to the pigment composition and nutritional profile of P . neustonensis . Although there are marked differences in cell morphology between the new isolate and the budding bacterium P . neustonensis, the results of phenotypic and genetic comparisons suggest that the new isolate is closely related to P . neustonensis . Consequently, we assign the new isolate to the genus Porphyrobacter and propose the name Porphyrobacter tepidarius sp . nov . for it; the type strain of P . tepidarius is strain OT3 (= DSM 10595). J Bacteriol, 1997 Apr, 179(8), 2529 - 33 Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32; Fenster KM et al.; An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32 . The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa . Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp . lactis DSM7290, L . helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp . cremoris AM2 . A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity . Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively . PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1) . PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected. Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3104 - 9 Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase; Dean AM et al.; Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) first evolved from an NAD-dependent precursor about 3.5 billion years ago . Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis . Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and the distantly related Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase . Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes . Reconstructed ancestral sequences indicate that these replacements are ancestral . Hence the adaptive history of molecular evolution is amenable to experimental investigation. Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2823 - 7 A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains; Bhattacharyya A et al.; The ribonucleoprotein (RNP) enzyme telomerase is required for replication of eukaryotic chromosomal termini . The RNA moiety of telomerase is essential for enzyme function and provides the template for telomeric DNA synthesis . However, the roles of its nontemplate domains have not been explored . Here we demonstrate that a novel interspecies telomerase RNA swap in vivo creates a functional but aberrant telomerase . Telomerase RNA from the ciliate Glaucoma chattoni was expressed in Tetrahymena thermophila cells . The telomerase RNAs from these two species have almost superimposable secondary structures . The template region base sequence is identical in the two RNAs, but elsewhere their sequences differ by 49% . This hybrid telomerase RNP was enzymatically active but added only short stretches of telomeric repeat tracts in vivo and in vitro . This new enzyme also had a strong, aberrant DNA cleavage activity in vitro . Thus, molecular interactions in the RNP involving nontemplate RNA domains affect specific aspects of telomerase enzyme function, raising the possibility that they may regulate telomerase activity. J Bacteriol, 1997 Apr, 179(7), 2418 - 20 Protein-tyrosine phosphorylation in the Archaea; Smith SC et al.; Sulfolobus sulfataricus ATCC 35091, Haloferax volcanii, and Methanosarcina thermophila TM-1, representing the Euryarchaeota and Crenarchaeota subdomains of the Archaea, contain proteins which are phosphorylated on tyrosine . These data raise fundamental questions as to the origin and evolution of tyrosine phosphorylation, a protein modification that is of pivotal importance in the regulation of the physiology of eukaryotic cells. Curr Microbiol, 1997 Apr, 34(4), 216 - 9 Isolation and characterization of transcription signal sequences from Streptococcus thermophilus; Solaiman DK et al.; Streptococcus thermophilus (ST) chromosomal DNA fragments generated by partial Sau3A digestion were cloned into the unique BamHI site upstream from the promoterless chloramphenicol acetyltransferase (cat) gene of the Escherichia coli (EC)promoter-probe vector pKK520-3 . Recombinant plasmids containing ST sequences with transcription-activation activity were isolated from chloramphenicol-resistant (CmR) EC transformants . A promoterless Streptomyces antibioticus melanin biosynthesis operon (melC) was inserted immediately downstream from the ST sequence to identify DNA with strong promoter activity . Several ST transcription-activation sequences, termed STPs, were isolated and subcloned, and their nucleic acid sequences determined . The -10 and -35 consensus sequences were identified in these putative ST promoters . Detailed analysis of STP3306 sequence data revealed two partial open reading frames (ORFs) with high degrees of homology to prokaryotic GTP-binding protein and DNA repair enzyme, thus providing valuable information for further study on DNA maintenance in this important lactic acid bacterium. J Biol Chem, 1997 Mar 28, 272(13), 8215 - 21 Catalytic activity of the alpha3beta3gamma complex of F1-ATPase without noncatalytic nucleotide binding site; Matsui T et al.; A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides . It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex . However, the initial rate decayed rapidly to an inactivated form . Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site . Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP . Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1) . In the presence of ATP, the wild-type complex dissociated the inhibitory {3H}ADP preloaded onto a catalytic site whereas the mutant complex did not . Lauryl dimethylamineoxide promoted release of preloaded inhibitory {3H}ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex . Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex . These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM. Biochem Biophys Res Commun, 1997 Mar 27, 232(3), 766 - 70 Characterization of an extremely thermophilic and oxygen-stable membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus; Nishihara H et al.; The membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus was characterized as highly oxygen-tolerant, extremely thermophilic and thermostable in its membrane-bound form . The optimum temperatures for H2 oxidation and H2 evolution were 90 and 80 degrees C, respectively . The enzyme retained 90% of its activity after heating at 70 degrees C for 50 min under air and retained full activity at 90 degrees C for 80 min under hydrogen . The optimum pH values were 5.5 (H2 evolution) and 9.4 (H2 oxidation), and the activity ratio (H2 evolution/H2 oxidation) was high (50.3) at pH 5.5 . The hydrogen evolution from reduced methyl viologen continued at high temperatures and acidic pH with the continuous addition of sodium dithionite. Biochemistry, 1997 Mar 25, 36(12), 3719 - 27 ADP-fluoroaluminate complexes are formed cooperatively at two catalytic sites of wild-type and mutant alpha3beta3gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3; Dou C et al.; Addition of Al3+ and F- to the alpha3beta3gamma subcomplex of the TF1-ATPase containing MgADP in one catalytic site causes slow, complete inactivation as the ADP-fluoroaluminate complex is formed . This conflicts with the "bisite" stochastic model suggested earlier (Issartel, J . P., Dupuis, A., Lunardi, J . & Vignais, P . V . (1991) Biochemistry 30, 4726-4733} on the finding that complete inactivation of the bovine mitochondrial F1-ATPase by Al3+, F-, Mg2+, and excess ADP occurs as ADP-fluoroaluminate complexes form in two catalytic sites . When Al3+ and F- were added to alpha3beta3gamma containing MgADP in two catalytic sites, inactivation accelerated 8-fold, indicating catalytic to catalytic site cooperativity . When added to alpha3beta3gamma containing MgADP bound to one or two catalytic sites prior to addition of Al3+ and F-, phosphate inhibits formation of the ADP-fluoroaluminate complex . When introduced after adding 200 microM ADP plus Mg2+ to alpha3beta3gamma, but before adding Al3+ and F-, phosphate accelerated formation of the ADP-fluoroaluminate complex 3-fold . Sulfite accelerated formation of the ADP-fluoroaluminate complex 9-fold when 200 microM ADP plus Mg2+ was added to alpha3beta3gamma before adding Al3+ and F- . The accelerations induced by phosphate or sulfite in the presence of excess ADP and Mg2+ suggest noncatalytic to catalytic site cooperativity . When Al3+ and F- were added to the (alphaD261N)3beta3gamma subcomplex containing MgADP in a single catalytic site, the ADP-fluoroaluminate complex formed at least 10-fold more slowly than observed with wild-type under the same conditions . Therefore, the catalytic site containing MgADP recognizes the alphaD261N substitution when noncatalytic sites are empty . Cross-linking alpha to gamma or beta to gamma by oxidizing the (alphaA396C)3beta3(gammaA22C) and alpha3(betaD390C)3(gammaS90C) subcomplexes, respectively, abolishes cooperative formation of ADP-fluoroaluminate complexes in two catalytic sites . ADP-fluoroaluminate complex formation is restricted to a single catalytic site in the oxidized double mutants . The alpha3beta3delta subcomplex does not form an inhibitory ADP-fluoroaluminate complex under any of the conditions examined for the alpha3beta3gamma subcomplexes. Biochemistry, 1997 Mar 18, 36(11), 3084 - 94 Crystal structure analysis of the activation of histidine by Thermus thermophilus histidyl-tRNA synthetase; Aberg A et al.; The crystal structure at 2.7 A resolution of histidyl-tRNA synthetase (HisRS) from Thermus thermophilus in complex with its amino acid substrate histidine has been determined . In the crystal asymmetric unit there are two homodimers, each subunit containing 421 amino acid residues . Each monomer of the enzyme consists of three domains: (1) an N-terminal catalytic domain containing a six-stranded antiparallel beta-sheet and the three motifs common to all class II aminoacyl-tRNA synthetases, (2) a 90-residue C-terminal alpha/beta domain which is common to most class IIa synthetases and is probably involved in recognizing the anticodon stem-loop of tRNA(His), and (3) a HisRS-specific alpha-helical domain inserted into the catalytic domain, between motifs II and III . The position of the insertion domain above the catalytic site suggests that it could clamp onto the acceptor stem of the tRNA during aminoacylation . Two HisRS-specific peptides, 259-RGLDYY and 285-GGRYDG, are intimately involved in forming the binding site for the histidine, a molecule of which is found in the active site of each monomer . The structure of HisRS in complex with histidyl adenylate, produced enzymatically in the crystal, has been determined at 3.2 A resolution . This structure shows that the HisRS-specific Arg-259 interacts directly with the alpha-phosphate of the adenylate on the opposite side to the usual conserved motif 2 arginine . Arg-259 thus substitutes for the divalent cation observed in seryl-tRNA synthetase and plays a crucial catalytic role in the mechanism of histidine activation. Int J Food Microbiol, 1997 Mar 18, 35(1), 49 - 56 Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR; Tilsala-Timisjarvi A et al.; Spacer regions between the 16S and 23S rRNA genes of different dairy and probiotic lactic acid bacteria were amplified by the polymerase chain reaction (PCR) with conserved primers and the nucleotide sequences of these spacer regions were determined . Amplification/oligonucleotide primer pairs were designed for Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus and Streptococcus thermophilus based on the differences in the nucleotide sequences of the 16S-23S rRNA spacer regions . Also a primer pair identifying both Lb . paracasei and Lb . rhamnosus was designed . In addition to conventional PCR in a heat block a rapid PCR method in an Air Thermocycler (ATC) with glass capillaries was used. FEMS Microbiol Lett, 1997 Mar 15, 148(2), 209 - 16 Characterization of the xylanases from the new isolated thermophilic xylan-degrading Bacillus thermoleovorans strain K-3d and Bacillus flavothermus strain LB3A; Sunna A et al.; Three strictly aerobic strains (K-1, K-3d and K-4) were isolated from a hot-spring in Kobe, Japan, and a facultative anaerobic strain LB3A was isolated from sediments collected from the alkaline Lake Bogoria, Kenya . All strains were thermophilic and capable of growth on xylan . On the basis of morphological, physiological and phylogenetic studies the new aerobic isolates resemble the thermophilic species Bacillus thermoleovorans while the facultative anaerobic isolate LB3A resembles the facultative anaerobic thermophilic species Bacillus flavothermus . When grown on xylan as sole carbon source, all isolates produce thermoactive xylanases, Xylanases from strains K-3d and LB3A are active at temperatures between 40 and 90 degrees C and pH values between 5.0 and 9.0 . Applying SDS-PAGE the crude xylanase complex of isolate K-3d was shown to be composed of two active bands, with molecular masses of 40 and 69 kDa . The crude xylanase complex of isolate LB3A, on the other hand, is composed of at least four activity bands with molecular masses ranging from 80 to 130 kDa . Due to the product pattern of xylan hydrolysis both enzymes are classified as endoxylanases . The xylanolytic enzyme system of isolate K-3d produces xylotriose, xylotetraose and larger xylooligosacharides, whereas the xylanases from isolate LB3A release xylotetraose as the major product of hydrolysis. J Mol Biol, 1997 Mar 14, 266(5), 1016 - 31 Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus; Wallon G et al.; The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts . A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability . The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme . The structure of the E . coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S . typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8% . The three structures were compared to elucidate the basis of the higher thermostability of the T . thermophilus enzyme . A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability . The structure of this mutant was analyzed . The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues . The mutation in the hydrophobic core of T . thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed. Science, 1997 Mar 7, 275(5305), 1478 - 81 Block in anaphase chromosome separation caused by a telomerase template mutation; Kirk KE et al.; Telomeres are essential for chromosome stability, but their functions at specific cell-cycle stages are unknown . Telomeres are now shown to have a role in chromosome separation during mitosis . In telomeric DNA mutants of Tetrahymena thermophila, created by expression of a telomerase RNA with an altered template sequence, division of the germline nucleus was severely delayed or blocked in anaphase . The mutant chromatids failed to separate completely at the midzone, becoming stretched to up to twice their normal length . These results suggest a physical block in mutant telomere separation. Appl Environ Microbiol, 1997 Mar, 63(3), 969 - 72 Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana Galperin MY, Noll KM, Romano AH. Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate . T . neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase . Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose . Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy . These data show that beta-D-galactosides are taken up by T . neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose . Thus, the phenomenon of catabolite repression is present in T . neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present . Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system. Biol Chem, 1997 Mar-Apr, 378(3-4), 331 - 6 Structural features correlated with the extreme thermostability of 1{4Fe-4S} ferredoxin from the hyperthermophilic bacterium Thermotoga maritima; Macedo-Ribeiro S et al.; Understanding the molecular mechanisms behind extreme temperature stability is of relevance for the protein folding problem and for designing proteins for industrial and medical applications . A powerful approach for understanding the structural basis of thermostability is the comparison of high resolution structures of homologous proteins from mesophiles and thermophiles . The 1.75 A crystal structure of Thermotoga maritima 1{4Fe-4S} ferredoxin was compared with those of mesophilic ferredoxins . Detailed analysis of structural differences reveals that thermostability is achieved without large changes of the overall polypeptide chain folding . The most striking differences include the formation of additional hydrogen bonding networks involving both side-chain and main-chain atoms . These networks are mainly connecting turns and strongly fix the N-terminus to the central core of the protein, increasing the overall rigidity of Thermotoga maritima ferredoxin . Other possibly stabilizing factors are the shortening of a solvent exposed surface loop, the increased content of alanines in the second alpha-helix, and the replacement of three residues close to the iron-sulfur cluster, which are in energetically unfavourable conformations in other ferredoxins, by glycines. Protein Eng, 1997 Mar, 10(3), 237 - 48 Stability of aspartate aminotransferase from Sulfolobus solfataricus; Arnone MI et al.; Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C . The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling . Gel filtration analysis indicated that SsAspAT unfolds with an N2 reversible 2D mechanism . In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure . At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism . Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants . Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the delta G degrees (H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter delta n, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and delta Cp is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol . These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT . In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network. Protein Eng, 1997 Mar, 10(3), 249 - 54 Stabilization of Escherichia coli isopropylmalate dehydrogenase by single amino acid substitution; Aoshima M et al.; To determine the key position for the unusual stability of isopropylmalate dehydrogenase from extreme thermophiles (Thermus thermophilus and T . aquaticus), sequence comparisons were carried out . As a result, a motif which is characteristic to the thermophilic dehydrogenases was found between two highly conserved stretches . The sequence motif was introduced into a mesophilic (Escherichia coli) isopropylmalate dehydrogenase, one by one . Contrary to our expectation, introduction of the whole motif led the mesophilic enzyme to be more unstable whereas substitution of only one amino acid residue in the motif thermostabilized the enzyme . From the 3D structure of the enzyme, a mechanism for the thermostabilization is speculated. J Biochem (Tokyo), 1997 Mar, 121(3), 448 - 55 Contribution of a salt bridge to the thermostability of DNA binding protein HU from Bacillus stearothermophilus determined by site-directed mutagenesis; Kawamura S et al.; We have employed site-directed mutagenesis to evaluate the contribution of the amino acid residues, Glu34, Arg37, and Lys38, to the thermostability of the thermophilic protein, Bacillus stearothermophilus DNA binding protein HU (BstHU), relative to the mesophilic homologue, Bacillus subtilis HU (BsuHU) . Mutants BstHU-E34D and BstHU-K38N, in which Glu34 and Lys38 were individually replaced by the corresponding residues, Asp34 and Asn38, in BsuHU, exhibited decreased thermostabilities by 2.08 and 2.17 kJ/mol, respectively, whereas mutant BstHU-R37K with Lys instead of Arg at position 37 showed no change in thermostability . These results suggested that Glu34 and Lys38 contribute to the thermostability of BstHU by forming a possible salt bridge on the hydrophilic surface . The role of Glu34 as a salt bridge partner was corroborated by generating BstHU mutant protein BstHU-E34Q, in which the Glu residue was changed to Gln; this substitution clearly decreased the stability of the protein by 2.71 kJ/mol . The contribution of this favorable salt bridge to the thermostability was further investigated as the salt and pH dependencies of the stabilities of the wild-type BstHU, BstHU-K38N, and BstHU-E34D . As for salt dependency, the stability of the wild-type relative to those of the two mutants decreased with an increase in ionic strength, indicating that the electrostatic interaction between Glu34 and Lys38 indeed significantly contributes to the thermostability of BstHU . As for pH dependency, the difference in thermostability between the wild-type BstHU and mutant BstHU-K38N showed that the mutant protein was as stable as the wild-type protein at pH 2.0, however, at neutral pH, the mutant protein was less stable than the wild-type protein . In contrast, the difference between the melting temperatures of mutant BstHU-E34D and the wild-type did not change as a function of pH . This suggested that the Glu34 residue may play an important role in the thermostability not only as a partner in the salt bridge with Lys38, but also by stabilizing the alpha-helix from residue 18 to 35 in BstHU . On the basis of the present results, together with the previous results {Kawamura et al . (1996) Biochemistry 35, 1195-1200}, three mutations, Glu15 to Gly, Asp34 to Glu, and Asn38 to Lys, were simultaneously introduced into mesophilic protein BsuHU to demonstrate their contributions to thermostability . This combination of multiple thermostabilizing mutations generated a more stable mutant protein HU, showing a Tm value of 64.5 degrees C compared to 48.6 degrees C for the wild-type BsuHU. Eur J Biochem, 1997 Mar 1, 244(2), 441 - 8 Effect of nucleotides on the thermal stability and on the deuteration kinetics of the thermophilic F0F1 ATP synthase; Villaverde J et al.; Differential scanning calorimetry has been used to characterize the influence of specific nucleotide binding on the thermal unfolding of the F0F1-type ATP synthase from the thermophilic Bacillus PS3 (TF0F1) . The calorimetric trace shows an irreversible and kinetically controlled endothermic transition for TF0F1 in the absence of nucleotides . The thermal denaturation occurs at a transition temperature (t(m)) of 81.7 degrees C . The remarkable thermostability of this enzyme was decreased upon tight binding of Mg2+ x ATP to noncatalytic sites, whereas binding of Mg2+ x ADP increased the temperature at which thermal denaturation occurred . At high temperatures, an exothermic transition due to aggregation processes was also affected by nucleotide binding . With the aim to correlate these thermal effects with possible structural differences among the various forms of TF0F1, Fourier transform infrared spectroscopy was carried out . Hydrogen/deuterium exchange was clearly affected by specific nucleotide occupancy . As illustrated by the total extent of protons exchanged, our results demonstrate that more peptide groups are exposed to the medium in the presence of Mg2+ x ATP than in the presence of Mg2+ x ADP . Therefore, consistent with microcalorimetric data, binding of Mg2+ x ADP induces conformational changes which shield amide protons to more buried hydrogen-bonded structures, whereas binding of Mg2+ x ATP results in a more open or flexible structure. Mol Microbiol, 1997 Mar, 23(5), 1033 - 42 A selenium-dependent and a selenium-independent formylmethanofuran dehydrogenase and their transcriptional regulation in the hyperthermophilic Methanopyrus kandleri; Vorholt JA et al.; The genome of Methanopyrus kandleri was found to harbour a gene, fwuB, predicted to encode the catalytic subunit of a tungsten formylmethanofuran dehydrogenase with an active site selenocysteine, and a second gene, fwcB, encoding a tungsten formylmethanofuran dehydrogenase with an active site cysteine . Northern blot and primer-extension analysis revealed that both genes were differentially transcribed . During growth of the methanogen on medium supplemented with selenium only fwuB was transcribed, whereas transcription of both fwuB and fwcB was observed on selenium-deprived medium . Growth of M . kandleri was stimulated by tungstate and selenite but not by molybdate . The findings indicate that the hyperthermophilic archaeon contains two tungsten isoenzymes of formylmethanofuran dehydrogenase, one of which is a novel selenium enzyme . They also indicate that the hyperthermophilic methanogen probably does not contain a molybdenum formylmethanofuran dehydrogenase which appears to be present only in thermophilic and mesophilic methanogens. Microb Pathog, 1997 Mar, 22(3), 155 - 64 Hydrophobic characterization of