Eur J Cell Biol, 2001 Jul, 80(7), 486 - 97 Targeted gene knockout of inner arm 1 in Tetrahymena thermophila; Angus SP et al.; Cilia and flagella contain at least eight different types of dynein arms . It is not entirely clear how the different types of arms are organized along the axoneme . In addition, the role each different type of dynein plays in ciliary or flagellar motility is not known . To initiate studies of dynein organization and function in cilia, we have introduced a mutation into one dynein heavy chain gene (DYH6) in Tetrahymena themophila by targeted gene knockout . We have generated mutant cells that lack wild-type copies of the DYH6 gene . We have shown that the DYH6 gene encodes one heavy chain (HC2) of Tetrahymena 18S dynein and that 18S dynein occupies the I1 position in the ciliary axoneme . We have also shown that Tetrahymena I1 is required for normal motility, normal feeding and normal doubling rate. J Mol Biol, 2001 Aug 17, 311(3), 593 - 604 Solution structure and function of a conserved protein SP14.3 encoded by an essential Streptococcus pneumoniae gene; Yu L et al.; Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity rates and has developed resistance to many antibiotics . The genome of S . pneumoniae has recently been completely sequenced revealing many genes encoding hypothetical proteins of unknown function . We have found that the gene encoding one such conserved protein, SP14.3, is essential for growth of S . pneumonia . Since it is essential, SP14.3 represents a potential target for drug discovery . Here, we describe the three-dimensional solution structure of SP14.3 as determined by NMR spectroscopy . The structure consists of two domains each with an alpha/beta-fold . The N-terminal domain contains two alpha-helices and a three-stranded beta-sheet, while the C-terminal domain is composed of one alpha-helix and a five-stranded beta-sheet . The N-terminal domain of the protein contains a highly negatively charged surface and resembles the fold of the N-terminal domain of Thermus thermophilus ribosomal protein S3 . The C-terminal domain has a protein fold similar to human small nuclear ribonucleoprotein Sm D3 and Haloarcula marismortui ribosomal protein L21E . The two domains of the protein tumble in solution overall as a whole with an overall molecular rotational correlation time (tau(m)) of 12.9 ns at 25 degrees C . The relative orientation of the two domains is not defined by the nuclear Overhauser effect data . Indeed, residual dipolar couplings and the structure calculations indicate that the relative orientation of the two domains is not rigidly oriented with respect to one another in solution . Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1539 - 48 Thermoanaerobacter yonseiensis sp . nov., a novel extremely thermophilic, xylose-utilizing bacterium that grows at up to 85 degrees C; Kim BC et al.; A novel strictly anaerobic, extremely thermophilic, spore-forming and xylose-utilizing bacterium, designated strain KB-1TP (type and patent strain), was isolated from a geothermal hot stream at Sileri on Java island, Indonesia . The cells were rod-shaped, motile and had terminal spores . The newly isolated strain stained gram-positive and the cells occurred singly or in pairs during the exponential growth phase . The temperature optimum for growth was 75 degrees C and growth occurred in the range 50-85 degrees C . The pH range for growth was 4.5-9.0, with an optimum at pH 6.5 . Strain KB-1TP grew chemo-organotrophically by fermenting a wide range of substrates such as glucose, fructose, D-xylose, lactose, maltose, sucrose, mannose, galactose, cellobiose, pullulan and soluble starch . Arabinose, xylan, cellulose, olive oil and Tween 80 were not fermented . The predominant fermentation end products after growth on glucose were lactate, acetate, ethanol, CO2 and small amounts of isovaleric acid, butyric acid, propionic acid, 1-pentanol and 2-propanol . Thiosulfate was reduced to H2S . Strain KB-1TP was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, kanamycin, vancomycin and rifampicin at concentrations of 100 microg ml(-1) . No effect was observed with chloramphenicol and neomycin at concentrations of 10 microg ml(-1) . This indicates that strain KB-1TP belongs to the bacterial domain . The G+C content of the DNA was 37 mol% . The comparison of the 165 rDNA sequence to that of closely related strains revealed that strain KB-1TP belonged to clostridial cluster V, showing highest sequence identities (92.7%) to members of the genus Thermoanaerobacter . Taking into account the physiological and molecular properties of the new isolate, it is proposed that strain KB-1TP should be classified as a new species of the genus Thermoanaerobacter, designated Thermoanaerobacter yonseiensis . The type strain, KB-1TP, has been deposited in the Korean Federation of Culture Collections (KFCC 11116P) as a patent strain and in the Deutsche Sammlung von Mikroorganismen und Zellkulturen as a type strain (= DSM 13777T). Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1425 - 35 Hydrogenobacter subterraneus sp . nov., an extremely thermophilic, heterotrophic bacterium unable to grow on hydrogen gas, from deep subsurface geothermal water; Takai K et al.; A novel extreme thermophile was isolated from a water sample derived from a deep subsurface geothermal water pool at a depth of 1500 m in the Hacchoubaru geothermal plant in Oita Prefecture, Japan . The cells were found to be straight rods, each being motile by means of a polar flagellum . Growth was observed at temperatures between 60 and 85 degrees C (optimum 78 degrees C; 120 min doubling time) and between pH 5.5 and pH 9.0 (optimum 7.5) . The isolate was a strictly aerobic heterotroph capable of utilizing a number of substrates such as yeast extract, peptone, tryptone, various carbohydrates, sugars, amino acids and organic acids . Elemental sulfur, thiosulfate, sulfide or cysteine-hydrochloride was required as an electron donor for growth . Hydrogen gas did not support growth . The G+C content of the genomic DNA was 44.7 mol% . Phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization analysis indicated that the isolate was closely related to members of the hydrogen-oxidizing, autotrophic and thermophilic genera Hydrogenobacter and Calderobacterium . However this isolate was differentiated from the previously described species of these genera on the basis of the physiological and molecular properties of the new isolate . The name Hydrogenobacter subterraneus sp . nov . is proposed; the type strain is HGP1T (= JCM 10560T = IFO 16485T). Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1335 - 41 Thermoanaerobacter tengcongensis sp . nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China; Xue Y et al.; A new, extremely thermophilic bacterium, designated strain MB4T, was isolated from a Chinese hot spring . The new isolate was an obligately anaerobic, rod-shaped, gram-negative, saccharolytic bacterium . Spore formation was not observed . Growth occurred at temperatures between 50 and 80 degrees C, with an optimum of around 75 degrees C; at pH values between 5.5 and 9.0, with an optimum of 7.0-7.5; and at salinities between 0 and 2.5% NaCl, with an optimum of around 0.2% NaCl . The organism utilized glucose, galactose, maltose, cellobiose, mannose, fructose, lactose, mannitol and starch . Acetate was the main end product from glucose fermentation . Thiosulfate and sulfur were reduced to hydrogen sulfide . Sulfate, sulfite and nitrate were not reduced . Growth was inhibited by hydrogen . The G+C content of the DNA was 33 mol% . Phylogenetic analyses based on the 16S rDNA sequence indicated that the isolate was a new member of the genus Thermoanaerobacter and formed a monophyletic unit within the Thermoanaerobacter cluster . Based on its phenotypic and phylogenetic characteristics, the isolate was proposed as a new species, Thermoanaerobacter tengcongensis . The type strain is MB4T (= Chinese Collection of Microorganisms AS 1.2430T = JCM 11007T). Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1327 - 34 Thermosipho geolei sp . nov., a thermophilic bacterium isolated from a continental petroleum reservoir in Western Siberia; Haridon SL et al.; Three strictly anaerobic, thermophilic bacteria (SL31T, SL30 and MLM39636) were isolated from a deep continental oil reservoir in Western Siberia (Russia) . Following the mid-exponential phase of growth, the non-motile rod-shaped organisms were surrounded by a sheath-like structure . As DNA-DNA hybridizations showed that these strains were highly related genomically, only strain SL31T was studied in detail . The temperature range for growth of strain SL31T was between 45 and 75 degrees C, with optimum growth at 70 degrees C . Its optimum pH and NaCl concentration for growth were pH 7.5 and 20-30 g l(-1), respectively . The novel isolate reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide . The G+C content of the genomic DNA was 30.0 mol % . As determined by 16S rDNA sequence analysis, this organism belonged to the genus Thermosipho . DNA-DNA hybridization levels between strain SL31T and type strains of the previously described species of Thermosipho were less than 10% . On the basis of physiological and molecular properties, it is proposed that this organism should be placed in a new species, Thermosipho geolei sp . nov . The novel organism represents the first species of the genus Thermosipho that has been isolated from a petroleum reservoir . The type strain is SL31T ( = DSM 13256T = JCM 10986T). J Mol Biol, 2001 May 25, 309(1), 227 - 38 Direct observation of three conformations of MutS protein regulated by adenine nucleotides; Kato R et al.; Mismatched base-pairs, which are caused by either DNA replication errors, DNA damage or genetic recombination, are repaired by the mismatch-repair system . The MutS protein, a component of the mismatch-repair system, recognizes mismatched base-pairs in DNA, and its DNA-binding activity is affected by ATP and ADP . Here, we show that the MutS protein from Thermus thermophilus HB8 can have three different conformations in solution, based on direct observations made by small-angle X-ray scattering . The conformation of MutS in solution is drastically influenced by the presence of ADP and ATP; the ATP-bound form has the most compact conformation, the ADP-bound form the most stretched, and the nucleotide-free form has a conformation intermediate between the two . Based on these findings, we conclude that the DNA-binding activity of MutS may depend on conformational changes triggered by both the binding and hydrolysis of ATP. J Biol Chem, 2001 Oct 19, 276(42), 39232 - 42 Epub 2001 Aug 06. Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus; Appleby TC et al.; The structure of 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5'-deoxy-5'-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone . The structure of unliganded SsMTAP was refined at 2.5-A resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 A . SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability . The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5'-deoxy-5'-methylthioadenosine as a substrate . The active site of SsMTAP is similar to that of E . coli PNP with 13 of 18 nearest residues being identical . The main differences are at Thr(89), which corresponds to serine in E . coli PNP, and Glu(163), which corresponds to proline in E . coli PNP . In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP . Thr(89) is near the 5'-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position . Unlike E . coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu(163) . This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding . The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates . SsMTAP has optimal activity at 120 degrees C and retains full activity after 2 h at 100 degrees C . Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability. J Bacteriol, 2001 Sep, 183(17), 5067 - 73 Functional and evolutionary relationship between arginine biosynthesis and prokaryotic lysine biosynthesis through alpha-aminoadipate; Miyazaki J et al.; Our previous studies revealed that lysine is synthesized through alpha-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27 . Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from alpha-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis . In the present study, we cloned an argD homolog of T . thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence . A knockout of the argD-like gene, now termed lysJ, in T . thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium . The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity . When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the epsilon-amino group of N(2)-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate . When N(2)-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the delta-amino group of N(2)-acetylornithine to 2-oxoglutarate 16 times more efficiently than when N(2)-acetyllysine was the amino donor . All these results suggest that lysine biosynthesis in T . thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis. Mol Microbiol, 2001 Jul, 41(2), 325 - 36 Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages; Duplessis M et al.; Phage-host interactions remain poorly understood in lactic acid bacteria and essentially in all Gram-positive bacteria . The aim of this study was to identify the phage genetic determinant (anti-receptor) involved in the recognition of Streptococcus thermophilus hosts . The complete genomic sequence of the lytic S . thermophilus phage DT1 was determined previously, and bioinformatic analysis indicated that orf18 might be the anti-receptor gene . The orf18 of six additional S . thermophilus phages was determined (DT2, DT4, MD1, MD2, MD4 and Q5) and compared with the orf18 of DT1 . The deduced ORF18 was divided into three domains . The first domain, which contains the N-terminal part of the protein, was conserved in all seven phages . The second domain was detected in only two phages and flanked by a motif called collagen-like repeats . The second domain also contained a variable region (VR1) . All seven phages had a third domain that consisted of the C-terminal section of the protein as well as another variable region (VR2) . Chimeric DT1 phages were constructed by recombination; a portion of its orf18 was replaced by the corresponding section in orf18 of the phage MD4 . All DT1 chimeric phages acquired the host range of phage MD4 . Analysis of the orf18 in the chimeric phages revealed that host specificity in phages DT1 and MD4 resulted from VR2 . This is the first report on the identification and characterization of a phage gene involved in the host recognition process of Gram-positive bacteria. EMBO J, 2001 Aug 1, 20(15), 4299 - 308 G-overhang dynamics at Tetrahymena telomeres; Jacob NK et al.; To learn more about the structure of the DNA terminus at Tetrahymena thermophila telomeres, we have devised a ligation-mediated primer extension protocol to accurately measure the length of the G-strand overhang . We show that overhang length and the identity of the 3'-terminal nucleotide are tightly regulated . The majority of overhangs terminate in the sequence 5'-TTGGGGT and >80% are either 14-15 or 20-21 nucleotides in length . No significant changes in overhang length were detected as cells traversed the cell cycle . However, changes in length distribution were observed when cells exited the cell cycle, indicating an altered balance between DNA synthesis and degradation or end protection . We also provide evidence that rDNA molecules have overhangs on both telomeres . Full-length rDNA could be cloned by a strategy that depends on overhangs being present at both ends . Moreover, analysis of leading strand telomeres revealed that a significant fraction have overhangs > or =5 nucleotides . Our results indicate that generation of the terminal telomeric DNA structure is highly regulated and requires several distinct DNA-processing events. EMBO J, 2001 Aug 1, 20(15), 4214 - 21 The kink-turn: a new RNA secondary structure motif; Klein DJ et al.; Analysis of the Haloarcula marismortui large ribosomal subunit has revealed a common RNA structure that we call the kink-turn, or K-turn . The six K-turns in H.marismortui 23S rRNA superimpose with an r.m.s.d . of 1.7 A . There are two K-turns in the structure of Thermus thermophilus 16S rRNA, and the structures of U4 snRNA and L30e mRNA fragments form K-turns . The structure has a kink in the phosphodiester backbone that causes a sharp turn in the RNA helix . Its asymmetric internal loop is flanked by C-G base pairs on one side and sheared G-A base pairs on the other, with an A-minor interaction between these two helical stems . A derived consensus secondary structure for the K-turn includes 10 consensus nucleotides out of 15, and predicts its presence in the 5'-UTR of L10 mRNA, helix 78 in Escherichia coli 23S rRNA and human RNase MRP . Five K-turns in 23S rRNA interact with nine proteins . While the observed K-turns interact with proteins of unrelated structures in different ways, they interact with L7Ae and two homologous proteins in the same way. Biotechnol Appl Biochem, 2001 Aug, 34(Pt 1), 37 - 45 Cloning, sequence analysis and functional characterization of DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus; Blondal T et al.; A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus was identified . The gene was cloned, sequenced and expressed in Escherichia coli . The gene is 2772 bp long and encodes a protein of 924 amino acids with a calculated molecular mass of 104.8 kDa . Sequence analysis showed that a generally conserved Phe residue in the O-helix is substituted by a Tyr (position 756) in the R . marinus enzyme . A Tyr in this position decreases the discrimination against dideoxynucleotides which is a major advantage in DNA sequencing . The protein was purified, characterized and showed to contain specific DNA-polymerization activity of 3100 units/mg of protein, 5'-->3' exonuclease activity and a 3'-->5' proofreading activity . Its optimum activity was at 55 degrees C and it had a half-life of 2 min at 90 degrees C . A truncated form of the enzyme lacking the 5'-->3' exonuclease domain was also expressed in E . coli . It had a specific DNA-polymerization activity of 5000 units/mg of protein and lacked the 5'-->3' exonuclease activity . Its optimum activity was at 65 degrees C and it had a half-life of 11 min at 90 degrees C . It was usable for DNA sequencing . This is the first thermostable DNA polymerase described with the O-helix Phe-->Tyr substitution. Protein Expr Purif, 2001 Aug, 22(3), 388 - 98 Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization; Sinchaikul S et al.; An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1 . The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15{EP4} . Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids . The expressed lipase was isolated and purified to homogeneity in a single chromatographic step . The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry . The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55 degrees C . It was highly stable in the temperature range of 30-65 degrees C . The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol . Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism . In addition, it was stable for 1 h at 37 degrees C in 0.1% Chaps and Triton X-100 . Int J Food Microbiol, 2001 Jul 20, 67(1-2), 153 - 5 Occurrence of Campylobacter jejuni in vegetables; Kumar A et al.; In order to understand the importance of vegetables in the transmission of thermophilic Campylobacter, 56 samples of different vegetables were screened . Out of these, 2 samples (1 spinach and 1 fenugreek) revealed the presence of Campylobacter jejuni biotype I . Both the isolates were enteropathogenic in rat ileal loop test. Arch Microbiol, 2001 Jul, 176(1-2), 29 - 36 Elucidation of the pathways of catabolic glutamate conversion in three thermophilic anaerobic bacteria; Plugge CM et al.; The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of {(13)C} glutamate NMR measurements and enzyme activity determinations . In some cases the uptake of intermediates from different pathways was studied . The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth . C . coolhaasii formed acetate, CO(2), NH(4)(+) and H(2) from glutamate . Acetate was found to be formed through the beta-methylaspartate pathway in pure culture as well as in coculture . T . acidaminovorans converted glutamate to acetate, propionate, CO(2), NH(4)(+) and H(2) . Most likely, this organism uses the beta-methylaspartate pathway for acetate formation . Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA . The metabolism of T . acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway . Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO(2), NH(4)(+) and H(2) . Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA . A minor part of the succinyl-CoA was converted to succinate and excreted. J Mol Biol, 2001 Aug 10, 311(2), 311 - 24 Detailed analysis of RNA-protein interactions within the ribosomal protein S8-rRNA complex from the archaeon Methanococcus jannaschii; Tishchenko S et al.; The crystal structure of ribosomal protein S8 bound to its target 16 S rRNA from a hyperthermophilic archaeon Methanococcus jannaschii has been determined at 2.6 A resolution . The protein interacts with the minor groove of helix H21 at two sites located one helical turn apart, with S8 forming a bridge over the RNA major groove . The specificity of binding is essentially provided by the C-terminal domain of S8 and the highly conserved nucleotide core, characterized by two dinucleotide platforms, facing each other . The first platform (A595-A596), which is the less phylogenetically and structurally constrained, does not directly contact the protein but has an important shaping role in inducing cross-strand stacking interactions . The second platform (U641-A642) is specifically recognized by the protein . The universally conserved A642 plays a pivotal role by ensuring the cohesion of the complex organization of the core through an array of hydrogen bonds, including the G597-C643-U641 base triple . In addition, A642 provides the unique base-specific interaction with the conserved Ser105, while the Thr106 - Thr107 peptide link is stacked on its purine ring . Noteworthy, the specific recognition of this tripeptide (Thr-Ser-Thr/Ser) is parallel to the recognition of an RNA tetraloop by a dinucleotide platform in the P4-P6 ribozyme domain of group I intron . This suggests a general dual role of dinucleotide platforms in recognition of RNA or peptide motifs . One prominent feature is that conserved side-chain amino acids, as well as conserved bases, are essentially involved in maintaining tertiary folds . The specificity of binding is mainly driven by shape complementarity, which is increased by the hydrophobic part of side-chains . The remarkable similarity of this complex with its homologue in the T . thermophilus 30 S subunit indicates a conserved interaction mode between Archaea and Bacteria . Lett Appl Microbiol, 2001 Aug, 33(2), 153 - 8 Partial characterization of a bacteriocin produced by Lactobacillus helveticus; Bonade A et al.; AIMS: To investigate the antimicrobial activity of a strain of Lactobacillus helveticus . METHODS AND RESULTS: The culture supernatant fluid Lact . helveticus G51 showed antimicrobial activity against thermophilic strains of Lactobacillus . Purification of the active compound was achieved after gel filtration and ion exchange chromatography . As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with a molecular mass of 12.5 kDa . The antimicrobial compound was heat labile, inactivated by proteolytic enzymes and had a bactericidal mode of action . CONCLUSION: The antimicrobial activity expressed by Lact . helveticus G51 was correlated with the production of a bacteriocin with properties that were different to other helveticins . SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on Lact . helveticus bacteriocins . The strong activity of the bacteriocin towards various thermophilic lactobacilli warrants further investigation for its potential to obtain attenuated cultures for the enhancement of the cheese-ripening process. J Am Chem Soc, 2001 Apr 18, 123(15), 3412 - 7 Reversible pressure deformation of a thermophilic cytochrome P450 enzyme (CYP119) and its active-site mutants; Tschirret-Guth RA et al.; The pressure stability of the thermophilic CYP119 from Sulfolobus solfataricus and its active-site Thr213 and Thr214 mutants was investigated . At 20 degrees C and pH 6.5, the protein undergoes a reversible P450-to-P420 inactivation with a midpoint at 380 MPa and a reaction volume change of -28 mL/mol . The volume of activation of the process was -9.5 mL/mol . The inactivation transition was retarded, and the absolute reaction volume was decreased by increasing temperature or by mutations that decrease the size of the active-site cavity . High pressure affected the tryptophan fluorescence yield, which decreased by about 37% at 480 MPa . The effect was reversible and suggested considerable contraction of the protein . Aerobic decomposition of iron-aryl complexes of the CYP119 T213A mutant under increasing hydrostatic pressure resulted in variation of the N-arylprotoporphyrin-IX regioisomer (N(B):N(A):N(C):N(D)) adduct pattern from 39:47:07:07 at 0.1 MPa to 23:36:14:27 at 400 MPa . Preincubation of the protein at 400 MPa followed by complex formation and decomposition gave the same regioisomer distribution as untreated protein . The results indicate that the protein is reversibly inactivated by pressure, in contrast to the irreversible inactivation of P450(cam) and other P450 enzymes, and that this inactivation process is modulated by changes in the active-site cavity dimensions. J Struct Biol, 2001 Apr, 134(1), 88 - 92 Crystallization and preliminary X-ray studies of V(1)-ATPase of Thermus thermophilus HB8 complexed with Mg-ADP; Ishii N et al.; Crystals have been grown of the V(1)-ATPase sector of the V-type ATP synthase complex (V(0)V(1)) from the thermophilic eubacterium Thermus thermophilus HB8 . These crystals are grown by the vapor diffusion method in the presence of 5 mM Mg-ADP, from solutions containing 100 mM sodium acetate and 2 M sodium formate, pH 5.5 . The crystals diffracted X rays beyond 3.4 A in resolution on a synchrotron radiation source . The crystals belong to the trigonal space group P3, with unit cell dimensions of a = b = 89.0 A, c = 179.2 A, and gamma = 120 degrees . The unit cell presumably contains one molecule of V(1)-ATPase and the V(m) value is calculated as 3.0 A(3)/Da . Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1177 - 9 Epub 2001 Jul 23. Crystallization and preliminary X-ray diffraction data of the second and archaebacterial-type aspartyl-tRNA synthetase from Thermus thermophilus; Charron C et al.; The archaebacterial-type aspartyl-tRNA synthetase (AspRS2) from the thermophilic eubacterium Thermus thermophilus was crystallized using the hanging-drop vapour-diffusion method . Crystals grew at pH 9.5 in the presence of PEG 8000 and NaCl . A native diffraction data set has been collected at 2.5 A resolution using synchrotron radiation and cryocooling . Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 57.3, b = 121.9, c = 166.9 A and V(M) = 3.03 A(3) Da(-1) . There is one dimer of M(r) 96 000 per asymmetric unit . A molecular-replacement analysis gave solutions for the rotation and translation functions. Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1150 - 2 Epub 2001 Jul 23. Crystals of a mutant form of ribosomal protein L22 rendering bacterial ribosomes resistant to erythromycin; Davydova N et al.; A mutant form of Thermus thermophilus ribosomal protein L22 responsible for erythromycin resistance has been overexpressed in Escherichia coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique . While several different crystallization conditions were found, only one set of conditions yielded crystals suitable for X-ray diffraction analysis . These crystals grow as thick plates, with unit-cell parameters a = 31.8, b = 86.59, c = 38.96 A, beta = 104.47 degrees . The crystals belong to the space group P2(1) and diffract to 1.8 A resolution . On the basis of density calculations, two monomers are predicted per asymmetric unit (V(M) = 2.06 A(3) Da(-1)), with a solvent content of 40%. Protein Sci, 2001 Aug, 10(8), 1539 - 48 High stability of a ferredoxin from the hyperthermophilic archaeon A . ambivalens: involvement of electrostatic interactions and cofactors; Moczygemba C et al.; The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (T(m)) of 122 degrees C (pH 7) . To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs . Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (DeltaH{T(m)}) and T(m) from which an upper limit for the heat capacity of unfolding (DeltaC(P)) was determined to be 3.15 +/- 0.1 kJ/(mole * K) . Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A . ambivalens ferredoxin at pH 7 (20 degrees C) observed ({GuSCN}(1/2) = 3.1 M; DeltaG(U){H(2)O} = 79 +/- 8 kJ/mole) . The protein is, however, less stable at low pH: At pH 2.5, T(m) is 64 +/- 1 degrees C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U){H(2)O} = 20 +/- 1 kJ/mole) . These results support that electrostatic interactions contribute significantly to the stability . Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface . In addition, ferredoxin incorporates two iron-sulfur clusters and a zinc ion that all coordinate deprotonated side chains . The zinc remains bound in the unfolded state whereas the iron-sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation. Curr Issues Mol Biol, 2000 Jan, 2(1), 1 - 7 Universal TA cloning; Zhou MY et al.; TA cloning is one of the simplest and most efficient methods for the cloning of PCR products . The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase . Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs . The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs . The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species . This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another . Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts . With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency . The universal TA cloning method is thus both convenient and labor-saving. J Ind Microbiol Biotechnol, 2001 Apr, 26(4), 203 - 9 Thermophilic aerobic treatment of a synthetic wastewater in a membrane-coupled bioreactor; LaPara TM et al.; Synthetic wastewater containing alpha-lactose and gelatin was treated in a thermophilic membrane-coupled bioreactor (MBR) . Thermophilic (>45 degrees C) treatment represents a potentially advantageous process for high-temperature as well as high-strength industrial wastewaters susceptible to reactor autoheating . Thermophilic systems, however, generally support a nonflocculating biomass that resists conventional methods of cell separation from the treated wastewater . MBRs were applied to thermophilic treatment systems because bacterial cells can be retained regardless of cell aggregation . Thermophilic aerobic MBRs were successfully operated at high levels of biocatalyst and produced a better effluent quality than analogous thermophilic bioreactors without cell recycle . At a hydraulic residence time (HRT) of 13.1 h, the chemical oxygen demand (COD) of the membrane eluate improved from 760 mg l(-1) (without cell recycle) to 160 mg l(-1) (with cell recycle) . Bacterial community shifts were detected by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) -amplified 16S rRNA gene fragments - 6 of 13 bands disappeared within 2 days of MBR operation . A concomitant 40-50% reduction in physiological indicators of cell reactivity (RNA:protein; ATP:protein) was also observed . The specific activity of beta-galactosidase and aminopeptidase, however, increased by 10-25%, indicating that there is a definite advantage to MBR operation at the highest biomass level possible . Nucleotide sequence analysis of 16S rDNA clones identified phylotypes from the low-G+C Gram-positive division and the beta- and gamma-subdivisions of Proteobacteria. J Bioenerg Biomembr, 2001 Feb, 33(1), 1 - 8 A new type-II NADH dehydrogenase from the archaeon Acidianus ambivalens: characterization and in vitro reconstitution of the respiratory chain; Gomes CM et al.; A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens . This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron-sulfur clusters . Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids . Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein(-1)/min(-1)) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain . The apparent Km for NADH was found to be approximately 6 microM, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site . Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment . A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa3 type quinol oxidase . This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase. J Am Chem Soc, 2001 Jun 6, 123(22), 5325 - 36 Pulsed EPR/ENDOR characterization of perturbations of the Cu(A) center ground state by axial methionine ligand mutations; Slutter CE et al.; The effect of axial ligand mutation on the Cu(A) site in the recombinant water soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase ba(3) has been investigated . The weak methionine ligand was replaced by glutamate and glutamine which are stronger ligands . Two constructs, M160T0 and M160T9, that differ in the length of the peptide were prepared . M160T0 is the original soluble fragment construct of cytochrome ba(3) that encodes 135 amino acids of subunit II, omitting the transmembrane helix that anchors the domain in the membrane . In M160T9 nine C-terminal amino acids are missing, including one histidine . The latter has been used to reduce the amount of a secondary T2 copper which is most probably coordinated to a surface histidine in M160T0 . The changes in the spin density in the Cu(A) site, as manifested by the hyperfine couplings of the weakly and strongly coupled nitrogens, and of the cysteine beta-protons, were followed using a combination of advanced EPR techniques . X-band ( approximately 9 GHz) electron-spin-echo envelope modulation (ESEEM) and two-dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy were employed to measure the weakly coupled (14)N nuclei, and X- and W-band (95 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy for probing the strongly coupled (14)N nuclei and the beta-protons . The high field measurements were extremely useful as they allowed us to resolve the T2 and Cu(A) signals in the g( perpendicular) region and gave (1)H ENDOR spectra free of overlapping (14)N signals . The effects of the M160Q and M160E mutations were: (i) increase in A( parallel)((63,65)Cu), (ii) larger hyperfine coupling of the weakly coupled backbone nitrogen of C153, (iii) reduction in the isotropic hyperfine interaction, a(iso), of some of the beta-protons making them more similar, (iv) the a(iso) value of one of the remote nitrogens of the histidine residues is decreased, thus distinguishing the two histidines, and finally, (v) the symmetry of the g-tensor remained axial . These effects were associated with an increase in the Cu-Cu distance and subtle changes in the geometry of the Cu(2)S(2) core which are consistent with the electronic structural model of Gamelin et al . (Gamelin, D . R.; Randall, D . W.; Hay, M . T.; Houser, R . P.; Mulder, T . C.; Canters, G . W.; de Vries, S.; Tolman, W . B.; Lu, Y.; Solomon, E . I . J . Am . Chem . Soc . 1998, 120, 5246-5263). J Eukaryot Microbiol, 2001 Jul-Aug, 48(4), 414 - 24 Purification of GVBD-inducing protein from the ciliate Tetrahymena thermophila; Sugii M et al.; Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes . Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel . Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis . This band resolved into two bands on SDS-PAGE: 60 and 112 kDa . The 60 kDa protein was the active fraction inducing GVBD . Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance . An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13suc1-beads could remove cdc2 homologues from T . thermophila supernatant but could not remove the GVBD-inducing activity . The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division . The cyclic appearance of the 60-kDa protein in the T . thermophila cell cycle suggests that this protein has a cell cycle function. Appl Biochem Biotechnol, 2001 May, 94(2), 97 - 109 Proteolysis of mesophilic and thermophilic alpha-amylases: a comparative study; Khajeh K et al.; A comparative study was performed on limited and extensive proteolysis of mesophilic (from Bacillus amyloliquefaciens {BAA}) and thermophilic (from Bacillus licheniformis {BLA}) alpha-amylases using trypsin . As expected, the thermophilic enzyme showed greater resistance to digestion by the protease . While the catalytic potential of BLA was enhanced on proteolysis, that of BAA was diminished owing to this process . Combined with greater catalytic activity, a lower thermal stability was observed for BLA on proteolytic treatment . For both enzymes, the extent of proteolytic cleavage was reduced in the presence of various stabilizing agents . The digestion patterns are explained in terms of available information in the literature on the structure of these proteins, especially in relation to segmental mobility. J Mol Biol, 2001 Jul 20, 310(4), 827 - 43 Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: purification, crystallization and structure determination; Clemons WM Jr et al.; We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus . Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction . Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution . Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage . Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model . Extremophiles, 2001 Jun, 5(3), 193 - 8 Innovative fermentation strategies for the production of extremophilic enzymes; Schiraldi C et al.; A new type of microfiltration (MF) bioreactor, developed in our laboratory, was investigated for use in improving efficiency of the production of extremophilic enzymes . In spite of the difficulties in cultivating hyperthermophiles, we achieved, in 300 h fermentation, more than 38 g/l dry weight of Sulfolobus solfataricus using a MF technique, and we demonstrated that the activity of alcohol dehydrogenase (ADH), as the reporter enzyme, was not affected by cell density . However, hyperthermophile cultivation is difficult to scale up because of evaporation and the very low growth rate . Thus, to achieve high productivity we cultivated, in the MF bioreactor, recombinant mesophilic hosts engineered for the production of two thermophilic enzymes, namely, trehalosyldextrin-forming enzyme (SsTDFE) and trehalose-forming enzyme (SsTFE) from Sulfolobus solfataricus . The traditional Luria-Bertani broth used for recombinant Escherichia coli growth was replaced with a semidefined medium . The latter was used in both the batch and the MF experiments, and the ratio of complex components (e.g., yeast extract and tryptone) to a simple carbon source (glycerol) was decreased during the fed-batch phase to further decrease the medium cost in view of industrial applications . The bioprocess developed was able to improve productivity 500 fold for rSsTFE and 60 fold for rSsTDFE with respect to the wild type cultivated in MF mode . Comparisons with another recombinant enzyme, alpha-glucosidase (rSsalphagly), from Sulfolobus solfataricus produced in our MF bioreactor are reported. Extremophiles, 2001 Jun, 5(3), 183 - 92 Molecular and biochemical characterization of the recombinant amidase from hyperthermophilic archaeon Sulfolobus solfataricus; d'Abusco AS et al.; We have cloned, sequenced, and overexpressed in Escherichia coli the amidase gene from the hyperthermophilic archaeon Sulfolobus solfataricus (strain MT4) . The recombinant thermophilic protein was expressed as a fusion protein with an N-terminus six-histidine-residue affinity tag . The enzyme, the first characterized archaeal amidase, is a monomer of 55,784 daltons, enantioselective, and active on 2- to 6-carbon aliphatic amides and on many aromatic amides, over the pH range 4-9 and at temperatures from 60 degrees to 95 degrees C . The S . solfataricus amidase belongs to the class of amidases that share a characteristic signature, GGSS(S/ G)GS, located in the central region of the protein, and which show remarkable variability in their individual substrate specificities, can hydrolyze aliphatic or aromatic substrates, and share a large invariance of their primary structure. Extremophiles, 2001 Jun, 5(3), 153 - 9 Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus; Cannio R et al.; A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts . This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S . solfataricus, i.e., up to 82 degrees C . Furthermore, it may be transferred to S . solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA . The hph(mut) and the wild-type gene products were expressed at higher levels in E . coli and purified by specific affinity chromatography on immobilized hygromycin B . Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency. FEMS Microbiol Ecol, 2001 Jul, 36(2-3), 235 - 243 Evidence for the presence of thermophilic Fe(III)-reducing microorganisms in deep-sea hydrothermal vents at 13 degrees N (East Pacific Rise); Slobodkin A et al.; Microorganisms capable of dissimilatory Fe(III) reduction in the temperature range of 52-90 degrees C were enriched from outer and inner parts of chimney-like structures, tubes of the polychaetous annelid Alvinella sp., and hydrothermal fluids collected at 13 degrees N hydrothermal vent sites on the East Pacific Rise at a depth of 2650 m . Numbers of culturable Fe(III)-reducing thermophiles estimated by the serial dilution technique varied from 10 to 10(7) cells per cm(3) of sample . Phylogenetic analysis of bacterial and archaeal PCR-amplified 16S rDNA genes obtained from Fe(III)-reducing enrichments and separated by denaturing gradient gel electrophoresis revealed sequences related to Deferibacter, Thermotogales (Bacteria) and Thermococcus (Archaea) for which the capacity for Fe(III) reduction had been reported . This was confirmed by isolating a hyperthermophilic iron reducer that belongs to the genus Thermococcus . Other bacterial thermophiles found in the enrichments were related to so far uncultured members of the Clostridiaceae, and epsilon-subdivision of the Proteobacteria. Mikrobiologiia, 2001 May-Jun, 70(3), 293 - 9 {Growth and carbohydrate metabolism of sulfobacilli}; Karavaiko GI et al.; The moderately thermophilic acidophilic bacteria Sulfobacillus thermosulfidooxidans, strain 1269, S . thermosulfidooxidans subsp . "asporogenes," strain 41, and the thermotolerant strain S . thermosulfidooxidans subsp . "thermotolerans" K1 prefer mixotrophic growth conditions (the concomitant presence of ferrous iron, thiosulfate, and organic compounds in the medium) . In heterotrophic and autotrophic growth conditions, these sulfobacilli can grow over only a few culture transfers . In cell-free extracts of these sulfobacilli, key enzymes of the Embden-Meyerhof-Parnas, pentose-phosphate, and Entner-Doudoroff pathways were found . The role of a particular pathway depended on the cultivation conditions . All of the enzymes assayed were most active under mixotrophic conditions in the presence of Fe2+ and glucose, suggesting the operation of all of the three major pathways of carbohydrate metabolism under these conditions . However, the operation of the Entner-Doudoroff pathway in strain 41 was restricted under mixotrophic conditions . After the first culture transfer from mixotrophic to heterotrophic conditions, the utilization of glucose occurred only via the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways . After the first culture transfer from mixotrophic to autotrophic conditions, the activity of carbohydrate metabolism enzymes decreased in all of the strains studied; in strain K1, only the glycolytic pathway remained operative . The high activity of fructose-bisphosphate aldolase, remaining in strain 41 cells under these conditions, suggests the involvement of this enzyme in the reactions of the Calvin cycle or of gluconeogenesis. Nature, 2001 Jul 12, 412(6843), 145 - 9 An off-axis hydrothermal vent field near the Mid-Atlantic Ridge at 30 degrees N; Kelley DS et al.; Evidence is growing that hydrothermal venting occurs not only along mid-ocean ridges but also on old regions of the oceanic crust away from spreading centres . Here we report the discovery of an extensive hydrothermal field at 30 degrees N near the eastern intersection of the Mid-Atlantic Ridge and the Atlantis fracture zone . The vent field--named 'Lost City'--is distinctly different from all other known sea-floor hydrothermal fields in that it is located on 1.5-Myr-old crust, nearly 15 km from the spreading axis, and may be driven by the heat of exothermic serpentinization reactions between sea water and mantle rocks . It is located on a dome-like massif and is dominated by steep-sided carbonate chimneys, rather than the sulphide structures typical of 'black smoker' hydrothermal fields . We found that vent fluids are relatively cool (40-75 degrees C) and alkaline (pH 9.0-9.8), supporting dense microbial communities that include anaerobic thermophiles . Because the geological characteristics of the Atlantis massif are similar to numerous areas of old crust along the Mid-Atlantic, Indian and Arctic ridges, these results indicate that a much larger portion of the oceanic crust may support hydrothermal activity and microbial life than previously thought. J Biol Chem, 2001 Oct 5, 276(40), 37482 - 90 Epub 2001 Jul 10. Residues at the active site of the esterase 2 from Alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature; Manco G et al.; The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulfonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate . The aim of this work was to design, by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms . Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations . Among five variants with single and double amino acid substitutions, three were obtained, M211S, R215L, and M211S/R215L, that changed the catalytic efficiency profile in the desired direction . Kinetic characterization of mutants and wild type showed that this change was achieved by an increase in k(cat) and a decrease in K(m) values with respect to the parental enzyme . The M211S/R215L specificity constant for p-nitrophenyl decanoate substrate was 6-fold higher than the wild type . However, variants M211T, M211S, and M211V showed strikingly increased activity as well as maximal activity with monoacylesters with four carbon atoms in the acyl chain, compared with the wild type . In the case of mutant M211T, the k(cat) for p-nitrophenyl butanoate was 2.4-fold higher . Overall, depending on the variant and on the substrate, we observed improved catalytic activity at 70 degrees C with respect to the wild type, which was a somewhat unexpected result for an enzyme with already high k(cat) values at high temperature . In addition, variants with altered specificity toward the acyl-chain length were obtained . The results were interpreted in the context of the EST2 three-dimensional structure and a proposed catalytic mechanism in which k(cat), e.g . the limiting step of the reaction, was dependent on the acyl chain length of the ester substrate. Chemosphere, 2001 Jul, 44(2), 289 - 99 Biodegradation of aliphatic-aromatic copolyesters: evaluation of the final biodegradability and ecotoxicological impact of degradation intermediates; Witt U et al.; The biological degradation behaviour of the aliphatic-aromatic copolyester Ecoflex was investigated with regard to the degree of degradation and the intermediates formed during the degradation process . The individual thermophilic strain Thermomonospora fusca, isolated from compost material, was used for the degradation experiments in a defined synthetic medium at 55 degrees C . After 22 days of degradation more than 99.9% of the polymer had depolymerized and with regard to the degradation of the diacid and diol components of Ecoflex only the monomers of the copolyesters (1,4-butanediol, terephthalate and adipate) could be detected by gas chromatography/mass spectroscopy (GC-MS) measurements in the medium . In interrupted degradation experiments predominantly the monoesters of adipic acid and terephthalic acid with 1,4-butanediol were observed in addition to the monomers . In toxicological tests with Daphnia magna and Photobacterium phosphoreum no significant toxicological effect was observed, neither for the monomeric intermediates nor for the oligomeric intermediates . From a risk assessment it can be concluded that there is no indication for an environmental risk when aliphatic-aromatic copolyesters of the Ecoflex-type are introduced into composting processes. Water Sci Technol, 2001, 43(11), 251 - 8 Full-scale evaluation of heat balance for autothermal thermophilic aerobic treatment of food processing wastewater; Chiang CF et al.; A full-scale autothermal thermophilic aerobic treatment (ATAT) of food-processing wastewater was evaluated in this study . The wastewater was rich in oil and grease at concentrations of 1,500-2,000 mg/L . The system has been operated for more than one and a half years since the startup . Under steady state conditions, the ATAT process was capable of spontaneous reaction at temperatures of 45-55 degrees C without the addition of external heat . Treatment efficiency was as high as 95% in COD reduction at a volumetric COD loading of 4.1 kg/m3-d . A mathematical heat balance model was developed based on the theoretical considerations of heat sources and losses for the ATAT process . A computer algorithm was established to evaluate specific heat potential (Hs) of the wastewater under steady state conditions . Six months of steady-state data were used for the evaluation . The result shows that on average the wastewater had a specific heat potential (Hs) of 4,720 kcal/kg-COD removed and the biological heat contributed 41.4% of the total heat input . A net heat flux of 4,270 kcal/min and volumetric heat intensity (Hv) of 38.0 kcal/L was necessary to maintain reaction temperature at 48.2 degrees C for the ATAT process . The full-scale ATAT process showed the typical characteristics of high removal rate, low sludge yield, and poor solids settleability for thermophilic aerobic treatment reported in the literature. Lett Appl Microbiol, 2001 Jul, 33(1), 45 - 9 Detection of extracellular bound proteinase in EPS-producing lactic acid bacteria cultures on skim milk agar; Pailin T et al.; AIMS: Skim milk agar was developed to investigate extracellular cell-bound proteinase in yogurt cultures, Streptococcus thermophilus and Lactobacillus bulgaricus . METHODS AND RESULTS: The Lact . bulgaricus cultures produced more extracellular cell-bound proteinase than did Strep . thermophilus cultures . Strong positive correlations between the size of the exopolysaccharide (EPS) layer and extracellular cell-bound proteinase were found for both Streptococcus and Lactobacillus cultures . CONCLUSION: Strong positive linear relationships existed between the EPS size and colony size and the diameter of clear zone and colony size for Streptococcus cultures, whereas weak positive linear relationships were observed for Lactobacillus cultures . SIGNIFICANCE AND IMPACT OF THE STUDY: These data are useful to validate the relationship between extracellular proteinase and the EPS size of LAB . Also, a convenient medium to detect the presence of extracellular cell-bound proteinase of LAB is valuable for dairy industries. J Appl Microbiol, 2001 Jul, 91(1), 147 - 53 Production of growth-inhibiting factors by Lactobacillus delbrueckii; van de Guchte M et al.; AIMS: The detection of growth-inhibiting factors produced by Lactobacillus delbrueckii . METHODS AND RESULTS: A bioscreen assay was developed to study the effect of Lact . delbrueckii culture supernatant fluids on the growth of phylogenically or functionally related bacteria in broth cultures . Several growth-inhibiting factors could be distinguished based on differential effects on different test strains, separation by ultrafiltration and sensitivity to heat, proteinase treatment or catalase addition . CONCLUSION: Lactobacillus delbrueckii strain VI1007 was found to produce at least three growth-inhibiting factors, other than lactic acid, when grown under microaerobic conditions in MRS broth . These included H2O2 and a bacteriocin-like, heat- and proteinase-sensitive bactericidal molecule or complex with a molecular weight greater than 50 kDa . A third factor inhibited the growth of Streptococcus thermophilus . SIGNIFICANCE AND IMPACT OF THE STUDY: The assay system used allows the detection of subtle interactions between strains, that are likely to be of ecological importance in mixed cultures but would go unnoticed in classical agar diffusion tests. J Mol Biol, 2001 Jul 13, 310(3), 577 - 89 Genetic analysis of an archaeal Holliday junction resolvase in Escherichia coli; Bolt EL et al.; The study of genes and proteins in heterologous model systems provides a powerful approach to the analysis of common processes in biology . Here, we show how the bacterium Escherichia coli can be exploited to analyse genetically and biochemically the activity and function of a Holliday junction resolving enzyme from an archaeal species . We have purified and characterised a member of the newly discovered Holliday junction cleaving (Hjc) family of resolvases from the moderately thermophilic archaeon Methanobacterium thermoautotrophicum and demonstrate that it promotes DNA repair in resolvase-deficient ruv mutants of E . coli . The data presented provide the first direct evidence that such archaeal enzymes can promote DNA repair in vivo, and support the view that formation and resolution of Holliday junctions are key to the interplay between DNA replication, recombination and repair in all organisms . We also show that Hjc promotes DNA repair in E . coli in a manner that requires the presence of the RecG branch migration protein . These results support models in which RecG acts at a replication fork stalled at a lesion in the DNA, catalysing fork regression and forming a Holliday junction that can then be acted upon by Hjc . Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8709 - 13 Epub 2001 Jul 03. An antisense approach to phenotype-based gene cloning in Tetrahymena; Chilcoat ND et al.; We report a pioneering approach using Tetrahymena thermophila that permits rapid identification of genes based on their null or hypomorphic phenotypes . This technique involves cell transformation with a library of plasmids that encode 26S ribosomal subunits containing short insertions . The insertions correspond to antisense sequences for a large number of genes . The majority of cells each acquires a single antisense sequence, which silences a single genomic locus . Because the insertion site within the ribosomal sequence is known, the silenced gene is easily amplified . We demonstrate that this approach can be used to identify genes required for dense core granule exocytosis. J Biochem (Tokyo), 2001 Jul, 130(1), 89 - 98 Substrate recognition mechanism of thermophilic dual-substrate enzyme; Ura H et al.; Aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ttAspAT), has been believed to be specific for an acidic substrate . However, stepwise introduction of mutations in the active-site residues finally changed its substrate specificity to that of a dual-substrate enzyme . The final mutant, {S15D, T17V, K109S, S292R} ttAspAT, is active toward both acidic and hydrophobic substrates . During the course of stepwise mutation, the activities toward acidic and hydrophobic substrates changed independently . The introduction of a mobile Arg292* residue into ttAspAT was the key step in the change to a "dual-substrate" enzyme . The substrate recognition mechanism of this thermostable "dual-substrate" enzyme was confirmed by X-ray crystallography . This work together with previous studies on various enzymes suggest that this unique "dual-substrate recognition" mechanism is a feature of not only aminotransferases but also other enzymes. Environ Sci Technol, 2001 Jun 15, 35(12), 2612 - 9 Thermophilic biotrickling filtration of ethanol vapors; Cox HH et al.; The treatment of ethanol vapors in biotrickling filters for air pollution control was investigated . Two reactors were operated in parallel, one at ambient temperature (22 degrees C) and one at high temperature (53 degrees C) . After a short adaptation phase, the removal of ethanol was similar in both reactors . At a bed contact time of 57 s, the elimination capacity exceeded 220 g m(-3) h(-1) at both temperatures . The experiments performed revealed that the process was most likely limited by biodegradation in the biofilm . The high-temperature biotrickling filter exhibited a higher degree of ethanol mineralization to CO2 (60 vs 46% at ambient temperature); hence, a lower rate of biomass accumulation was observed . Plating and cultivation of biofilm samples revealed that the high-temperature biotrickling filter hosted a process culture composed of both mesophilic and thermotolerant or thermophilic microorganisms, whereas the ambient-temperature reactor lacked microorganisms capable of growing at high temperature . Consequently, the performance of the control biotrickling filter was significantly affected by a short incursion at 53 degrees C . The upper temperature limit for treatment was 62 degrees C . Overall, the results of this study open new possibilities for biotrickling filtration of hot gases. Vet Res, 2001 May-Aug, 32(3-4), 311 - 21 Antimicrobial resistance of thermophilic Campylobacter; Aarestrup FM et al.; Campylobacter has become the leading cause of zoonotic enteric infections in developed and developing countries world-wide . Antimicrobial resistance has emerged among Campylobacter mainly as a consequence of the use of antimicrobial agents in food animal production . Resistance to drugs of choice for the treatment of infections, macrolides and fluoroquinolones has emerged as a clinical problem and interventions to reduce this are recommended . Resistance to fluoroquinolones and macrolides is mediated by chromosomal mutations . Resistance to other relevant antimicrobial agents, mediated by acquired resistance genes, has not become widespread so far . However, resistance genes originating from both Gram-positive and Gram-negative bacterial species have been found, showing the potential for acquired resistance to emerge in Campylobacter. Nat Genet, 2001 Jul, 28(3), 281 - 5 Universal trees based on large combined protein sequence data sets; Brown JR et al.; Universal trees of life based on small-subunit (SSU) ribosomal RNA (rRNA) support the separate mono/holophyly of the domains Archaea (archaebacteria), Bacteria (eubacteria) and Eucarya (eukaryotes) and the placement of extreme thermophiles at the base of the Bacteria . The concept of universal tree reconstruction recently has been upset by protein trees that show intermixing of species from different domains . Such tree topologies have been attributed to either extensive horizontal gene transfer or degradation of phylogenetic signals because of saturation for amino acid substitutions . Here we use large combined alignments of 23 orthologous proteins conserved across 45 species from all domains to construct highly robust universal trees . Although individual protein trees are variable in their support of domain integrity, trees based on combined protein data sets strongly support separate monophyletic domains . Within the Bacteria, we placed spirochaetes as the earliest derived bacterial group . However, elimination from the combined protein alignment of nine protein data sets, which were likely candidates for horizontal gene transfer, resulted in trees showing thermophiles as the earliest evolved bacterial lineage . Thus, combined protein universal trees are highly congruent with SSU rRNA trees in their strong support for the separate monophyly of domains as well as the early evolution of thermophilic Bacteria. FEMS Microbiol Lett, 2001 May 1, 198(2), 177 - 82 Identification of the DNA-binding protein, HrcA, of Streptococcus thermophilus; Martirani L et al.; HrcA is a negative transcriptional factor controlling the expression of the stress-specific operons dnaK and groESL in several bacteria . Although the HrcA structural gene has been identified in various organisms, studies at the protein level have been so far limited and mostly restricted to Bacillus subtilis . We have identified the HrcA protein of Streptococcus thermophilus and show here that it is a dimer with a native molecular mass of 74.5 kDa and a sequence-specific DNA-binding activity . Partially denatured and inactive S . thermophilus HrcA recovered its binding activity in the presence of the GroEL chaperone. FEMS Microbiol Lett, 2001 May 1, 198(2), 135 - 40 Mesophilic cyanobacteria producing thermophilic restriction endonucleases; Piechula S et al.; When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations . Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65-80 degrees C, which is far above the lethal temperature for the host microorganism (40 degrees C) . These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively . The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase . Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely: PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsalI), PpaAII, PpeI (ApaI) . Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers . Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60 degrees C) which also extended the optimal growth temperature of the hosts . We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer . Restriction modification systems may be an excellent tool for investigation of that phenomenon. Plant Cell Physiol, 2001 Jun, 42(6), 599 - 607 Functional analysis of psbV and a novel c-type cytochrome gene psbV2 of the thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1; Katoh H et al.; Cytochrome c-550 is an extrinsic protein associated with photosystem II (PSII) in cyanobacteria and lower eukaryotic algae and plays an important role in the water-splitting reaction . The gene (psbV) for cytochrome c-550 was cloned from the thermophilic cyanobacteria Thermosynechococcus (formerly Synechococcus) elongatus and T . (formerly Synechococcus) vulcanus . In both genomes, located downstream of psbV were a novel gene (designated psbV2) for a c-type cytochrome and petJ for cytochrome c-553 . The deduced product of psbV2 showed composite similarities to psbV and petJ . Phenotype of psbV-disruptant in Thermosynechococcus was practically the same as that reported in Synechocystis sp . PCC 6803 . Either psbV or psbV2 gene of T . elongatus was expressed in the psbV-disruptant of Synechocystis sp . PCC 6803, which resulted in recovery of the photoautotrophic growth . However, the enhanced requirement of Ca(2+) or Cl- ions in the psbV-disruptant of Synechocystis was suppressed by expression of psbV but not by expression of psbV2 . Thus, it is concluded that psbV2 can partly replace the role of psbV in PSII . The close tandem arrangement of psbV/psbV2/petJ implies that psbV2 was created by gene duplication and intergenic recombination during evolution. Enzyme Microb Technol, 2001 Jul 5, 29(1), 90 - 98 Isolation and characterization of a thermostable endo-beta-glucanase active on 1,3-1,4-beta-D-glucans from the aerobic fungus talaromyces emersonii CBS 814.70; Murray PG et al.; A novel endoglucanase active on 1,3-1,4-beta-D-glucans was purified to apparent homogeneity from submerged cultures of the moderately thermophilic aerobic fungus Talaromyces emersonii CBS 814.70 . The enzyme is a single subunit glycoprotein with M(r) and pI values of 40.7 +/- 0.3 kDa and 4.4, respectively, and an estimated carbohydrate content of 77% (w/w) . The purified beta-glucanase displayed activity over broad ranges of pH and temperature, yielding respective optima values of pH 4.8 and 80 degrees C . This enzyme was markedly thermostable with 15% of the original activity remaining after incubation for 15 min at 100 degrees C . Substrate specificity studies revealed the identity of the enzyme to be a 1,3-1,4-beta-D-glucanase . Identical K(m) values (13.38 mg.ml(-1)) were obtained with lichenan and BBG, while the V(max) value with lichenan (142.9 IU.mg(-1)) was approximately twice the value obtained with BBG (79.3 IU.mg(-1)) . Time-course hydrolysis of barley-beta-glucan did not proceed linearly with respect to time indicating an 'endo' or more processive action for the enzyme . HPAEC fractionation of the products of hydrolysis yielded a range of oligosaccharides, with cellobiose, cellotriose and cellotetraose being the predominant oligosaccharide products. Prep Biochem Biotechnol, 2001 May, 31(2), 103 - 12 Factors influencing the stability of alpha-helices and beta-strands in thermophilic ribonuclease H; Gromiha MM; Understanding the influence of structural parameters is crucial to enhance the thermal stability of proteins . In this work, the stability (deltaG) of residues in different secondary structures of Ribonuclease H (RNase H) has been analyzed with 48 amino acid properties . The properties reflecting hydrophobicity show a good correlation with stability . Further, the linear distribution of surrounding hydrophobicity in alpha-helices, obtained from the three dimensional structure of thermophilic RNase H, agrees well with experimental deltaG values . Moreover, the stability parameters correlate better in alpha-helices than those did in beta-strand segments . Multiple regression analysis, incorporating combinations of three properties from among all possible combinations of the 48 properties, increased the correlation coefficient to 0.77. Appl Environ Microbiol, 2001 Jul, 67(7), 3140 - 8 Natural transformation in mesophilic and thermophilic bacteria: identification and characterization of novel, closely related competence genes in Acinetobacter sp . strain BD413 and Thermus thermophilus HB27; Friedrich A et al.; The mesophile Acinetobacter sp . strain BD413 and the extreme thermophile Thermus thermophilus HB27 display high frequencies of natural transformation . In this study we identified and characterized a novel competence gene in Acinetobacter sp . strain BD413, comA, whose product displays significant similarities to the competence proteins ComA and ComEC in Neisseria and Bacillus species . Transcription of comA correlated with growth phase-dependent transcriptional regulation of the recently identified pilin-like factors of the transformation machinery . This finding strongly suggests that comA is part of a competence regulon . Examination of the genome sequence of T . thermophilus HB27 led to detection of a comA/comEC-like open reading frame (ORF) which is flanked by an ORF whose product shows significant similarities to the Bacillus subtilis competence protein ComEA . To examine whether these two ORFs, designated comEC and comEA, are implicated in natural transformation of T . thermophilus HB27, both were disrupted by using a thermostable kanamycin resistance marker . Natural transformation in comEC mutants was reduced 1,000-fold, whereas in comEA mutants the natural transformation phenotype was completely eliminated . These results strongly suggest that both genes, comEC and comEA, are required for natural transformation in T . thermophilus HB27 . Several transmembrane alpha-helices are predicted based on the amino acid sequences of ComA in Acinetobacter sp . strain BD413 and ComEC in T . thermophilus HB27, which suggests that ComA and ComEC are located in the inner membrane and function in DNA transport through the cytoplasmic membrane. Environ Microbiol, 2001 May, 3(5), 295 - 303 Thermophilic methanogens in rice field soil; Fey A et al.; The soil temperature in flooded Italian rice fields is generally lower than 30 degrees C . However, two temperature optima at approximately 41 degrees C and 50 degrees C were found when soil slurries were anoxically incubated at a temperature range of 10-80 degrees C . The second temperature optimum indicates the presence of thermophilic methanogens in the rice field soil . Experiments with 14C-labelled bicarbonate showed that the thermophilic CH4 was exclusively produced from H2/CO2 . Terminal restriction fragment length polymorphism (T-RFLP) of archaeal SSU rRNA gene fragments revealed a dramatic change in the archaeal community structure at temperatures > 37 degrees C, with the euryarchaeotal rice cluster I becoming the dominant group (about 80%) . A clone library of archaeal SSU rRNA gene fragments generated at 49 degrees C was also dominated (10 out of 11 clones) by rice cluster I . Our results demonstrate that Italian rice field soil contains thermophilic methanogenic activity that was most probably a result of members of the as yet uncultivated euryarchaeotal rice cluster I. Nature, 2001 Jun 21, 411(6840), 909 - 17 Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution; Jordan P et al.; Life on Earth depends on photosynthesis, the conversion of light energy from the Sun to chemical energy . In plants, green algae and cyanobacteria, this process is driven by the cooperation of two large protein-cofactor complexes, photosystems I and II, which are located in the thylakoid photosynthetic membranes . The crystal structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus described here provides a picture at atomic detail of 12 protein subunits and 127 cofactors comprising 96 chlorophylls, 2 phylloquinones, 3 Fe4S4 clusters, 22 carotenoids, 4 lipids, a putative Ca2+ ion and 201 water molecules . The structural information on the proteins and cofactors and their interactions provides a basis for understanding how the high efficiency of photosystem I in light capturing and electron transfer is achieved. Acta Crystallogr D Biol Crystallogr, 2001 Jul, 57(Pt 7), 1036 - 7 Epub 2001 Jun 21. Crystallization and X-ray diffraction measurements of a thermophilic archaeal recombinant amidase from Sulfolobus solfataricus MT4; Nastopoulos V et al.; Recombinant amidase is a 55.8 kDa enzyme from the thermophilic archaeon Sulfolobus solfataricus MT4 that catalyses the hydrolysis of aliphatic amides of 2-6 C atoms as well as many aromatic amides . Single crystals of purified amidase were obtained by the hanging-drop method at 294 K . Diffraction data for the native protein (2.55 A resolution) and a putative derivative (2.20 A) have been collected at low temperature using synchrotron radiation . The crystals belong to the rhombohedral space group R3 . Structure determination by multiple isomorphous replacement is in progress . It is expected that structural information from this signatured thermostable amidase will increase our knowledge of the molecular mechanisms employed to maintain high-temperature stability in thermophilic proteins. Acta Crystallogr D Biol Crystallogr, 2001 Jul, 57(Pt 7), 1030 - 1 Epub 2001 Jun 21. Preliminary crystallographic study of Thermus aquaticus glycerol kinase; Huang HS et al.; Glycerol kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving glycerol metabolism . GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method . The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A . Native data were collected to 2.87 A resolution on a Cu Kalpha rotating-anode X-ray source. Acta Crystallogr D Biol Crystallogr, 2001 Jul, 57(Pt 7), 1008 - 12 Epub 2001 Jun 21. Using surface-bound rubidium ions for protein phasing; Korolev S et al.; Rubidium is a monovalent metal that can be used as a counterion in protein solutions . X-ray anomalous scattering from rubidium ions bound to the protein surface was used for phasing of the crystal structure of the hsp60 apical domain from Thermus thermophilus . Multiple-wavelength anomalous dispersion (MAD) data were collected from a crystal obtained from a solution containing 0.2 M rubidium salt . One molecule of protein (147 amino acids) binds one well ordered and one poorly ordered Rb atom . Phases calculated with the program SHARP were sufficient for automatic tracing and side-chain assignment using the program ARP/wARP . The data show that bound rubidium ions can be used to determine protein structures and to study the interaction of monovalent metal ions with proteins and other macromolecules. Acta Crystallogr D Biol Crystallogr, 2001 Jul, 57(Pt 7), 968 - 76 Epub 2001 Jun 21. Structure of ribosomal protein TL5 complexed with RNA provides new insights into the CTC family of stress proteins; Fedorov R et al.; The crystal structure of Thermus thermophilus ribosomal protein TL5 in complex with a fragment of Escherichia coli 5S rRNA has been determined at 2.3 A resolution . The protein consists of two domains . The structure of the N-terminal domain is close to the structure of E . coli ribosomal protein L25, but the C-terminal domain represents a new fold composed of seven beta-strands connected by long loops . TL5 binds to the RNA through its N-terminal domain, whereas the C-terminal domain is not included in this interaction . Cd(2+) ions, the presence of which improved the crystal quality significantly, bind only to the protein component of the complex and stabilize the protein molecule itself and the interactions between the two molecules in the asymmetric unit of the crystal . The TL5 sequence reveals homology to the so-called general stress protein CTC . The hydrophobic cores which stabilize both TL5 domains are highly conserved in CTC proteins . Thus, all CTC proteins may fold with a topology close to that of TL5. J Bacteriol, 2001 Jul, 183(14), 4382 - 5 Spontaneous erythromycin resistance mutation in a 23S rRNA gene, rrlA, of the extreme thermophile Thermus thermophilus IB-21; Gregory ST et al.; Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons . The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism . This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of Thermus. J Bacteriol, 2001 Jul, 183(14), 4244 - 50 Role of arginines in coenzyme A binding and catalysis by the phosphotransacetylase from Methanosarcina thermophila; Iyer PP et al.; Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH(3)COOPO(3)(2-) + CoASH <==> CH(3)COSCoA + HPO(4)(2-) . The role of arginine residues was investigated for the phosphotransacetylase from Methanosarcina thermophila . Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA . Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3'-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3'-phosphate of CoA . Arg 133 is postulated to interact with the 5'-phosphate of CoA . Large decreases in k(cat) and k(cat)/K(m) for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis . Large decreases in k(cat) and k(cat)/K(m) were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity . Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding . Chemical modification of the wild-type enzyme by 2,3-butanedione and substrate protection by CoA indicated that at least one reactive arginine is in the active site and is important for activity . The inhibition pattern of the R87Q variant indicated that Arg 87 is modified, which contributes to the inactivation; however, at least one additional active-site arginine is modified leading to enzyme inactivation, albeit at a lower rate. Biochim Biophys Acta, 2001 Jul 2, 1506(1), 31 - 46 Fluorescent probes for non-invasive bioenergetic studies of whole cyanobacterial cells; Teuber M et al.; Fluorescent DeltapH and DeltaPsi indicators have been screened for the non-invasive monitoring of bioenergetic processes in whole cells of the cyanobacterium Synechocystis sp . PCC 6803 . Acridine yellow and Acridine orange proved to be the best DeltapH indicators for the investigation of thylakoid and cytoplasmic membrane energization: While Acridine yellow indicated only cytosolic energization, Acridine orange showed signals from both the thylakoid lumen and the cytosol that could be separated kinetically . Both indicators were applied successfully to monitor cellular energetics, such as the interplay of linear and cyclic photosynthetic electron transport, osmotic adaptation and solute transport across the cytoplasmic membrane . In contrast, useful membrane potential indicators were more difficult to find, with Di-4-ANEPPS and Brilliant cresyl blue being the only promising candidates for further studies . Finally, Acridine yellow and Acridine orange could also be applied successfully for the thermophilic cyanobacterium Synechococcus elongatus . Different from Synechocystis sp . PCC 6803, where both respiration and ATP hydrolysis could be utilized for cytoplasmic membrane energization, proton extrusion at the cytoplasmic membrane in Synechococcus elongatus was preferentially driven by ATP hydrolysis. J Dairy Sci, 2001 Jun, 84(6), 1367 - 74 Natural exopolysaccharides enhance survival of lactic acid bacteria in frozen dairy desserts; Hong SH et al.; Viable lactic acid-producing bacteria in frozen dairy desserts can be a source of beta-galactosidase for persons who absorb lactose insufficiently . However, freezing kills many of the cells, causing loss of enzymatic activity . Cultures selected for high beta-galactosidase activities and high survival rates in the presence of bile were examined for survivability during freezing in reduced-fat ice cream . Encapsulated S . thermophilus strains survived better than their nonencapsulated mutants in reduced-fat ice cream after freezing and frozen storage at -29 degrees C for 16 d (28 vs . 19%) . However, a small nonencapsulated strain of Lactobacillus delbrueckii sp . bulgaricus survived better than the large encapsulated strain in reduced-fat ice cream . Factors that improved survival of encapsulated S . thermophilus 1068 in ice cream were 1) harvest of cells in the late-log phase of growth at 37 degrees C rather than at 40, 42.5, or 45 degrees C; 2) overrun at 50% rather than 100%; and 3) storage at -17 degrees C rather than -23 or -29 degrees C . Survival of strain ST1068 was unaffected by 1) neutralization of acid during growth or 2) substitution of nitrogen for air in building overrun. J Dairy Sci, 2001 Jun, 84(6), 1335 - 40 Influence of milk-clotting enzyme concentration on the alphas1-casein hydrolysis during soft cheeses ripening; Hynes ER et al.; We studied the influence of the dose of milk-clotting enzyme on alphas1-CN degradation, soluble nitrogen production, and sensory profile for an Argentinean soft cheese: Cremoso Argentino . Five different types of cheeses were produced: 1) control cheeses with normal technology, 2) cheeses with inactivated milk-clotting enzyme, 3) cheeses with inactivated milk-clotting enzyme, without starter (acidified with glucono delta lactone), 4) cheeses with a half dose of milk-clotting enzyme, and 5) cheeses with a double dose of milk-clotting enzyme . Proteolysis was assessed by isoelectric focusing electrophoresis of the insoluble fraction at pH 4.6, followed by densitometric quantification . Soluble nitrogen at pH 4.6, expressed as a percentage of total nitrogen and defined as ripening index was also performed . A sensorial panel evaluated the cheeses at the end of ripening . The hydrolysis level of alphas1-CN depended on the milk-clotting enzyme dose used in cheese making . Cheeses without active coagulant did not show degradation at the end of ripening, while cheeses with half and whole doses showed proportional degradations to coagulant dose . Cheese with a double dose of coagulant did not show higher alphas1-CN hydrolysis than normal cheese . No difference was found between cheeses with and without microbiological starter, indicating that the selected culture, composed of thermophilic strains, was unable to attack the whole casein . A high linear correlation was found between ripening index and the relation Sensorial characteristics of cheeses agree with objective analysis . Cheeses without active coagulant were hard and crumbly, while cheeses with normal dose were soft and creamy. Boll Chim Farm, 2001 Mar-Apr, 140(2), 83 - 9 Synthesis of 1,2,4-triazino{5,6-b} indoles bearing 1,2,4-triazine moiety; Morsy JM et al.; Some new 3-(5,6-Diphenyl-1,2,4-triazin-3-yl)-5-substituted- 1,2,4-triazino{5,6-b}indole derivatives (6-12) have been obtained via treatment of both (4 and 5) with p-nitro-benzoyl chloride, ammonium thiocyante, formaldehyde-methanol, acrylonitrile and thiosemi-carbazide . The former structure of the new products was established by the help of elemental analyses, as well as spectral data . Some of their showed pronounced effect on the Cellobiase produced by Thermomyces lanuginosus and Chaetomium thermophilum. J Am Chem Soc, 2001 Jun 27, 123(25), 5861 - 6 Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases; Tu C et al.; Catalysis of (18)O exchange between CO(2) and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H(2)(18)O from the active site . This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound (18)O-labeled hydroxide, and was observed in a site-specific mutant of carbonic anhydrase II in which a prominent proton shuttle residue His64 was replaced by alanine, which does not support proton transport . Upon increasing the concentration of bicarbonate, the rate of release of H(2)(18)O increased in a saturable manner to a maximum of 4 x 10(5) s(-)(1), consistent with proton transfer from bicarbonate to the Co(II)-bound hydroxide . The same mutant of carbonic anhydrase containing Zn(II) had the rate of release of H(2)(18)O smaller by 10-fold, but rate of interconversion of CO(2) and HCO(3)(-) about the same as the Co(II)-containing enzyme . These data as well as solvent hydrogen isotope effects suggest that the bicarbonate transferring the proton is bound to the cobalt in the enzyme . The enhancement of (18)O exchange caused by increasing bicarbonate concentration during catalysis by the Zn(II)-containing carbonic anhydrase from the archaeon Methanosarcina thermophila suggests that a very similar mechanism for proton donation by bicarbonate occurs with this wild-type enzyme. Lett Appl Microbiol, 2001 Jun, 32(6), 433 - 7 Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure; Marshall VM et al.; AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs) . METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD) . Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure . CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups . Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria. Lett Appl Microbiol, 2001 Jun, 32(6), 412 - 8 Inducible and constitutive expression using new plasmid and integrative expression vectors for Thermus sp; Kayser KJ et al.; AIMS: To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus . METHODS AND RESULTS: Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette . Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli . An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85 degrees C) from the dnaK gene of T . flavus, and the malate dehydrogenase gene (mdh) from T . flavus was cloned and expressed in both E . coli and T . thermophilus HB27 . CONCLUSION: This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species . The mdh gene can be used as a high temperature (85 degrees C) reporter gene for Thermus sp . The dnaK promoter is thermo-inducible . Significance and Impact of the Study: The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp . The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species. J Appl Microbiol, 2001 Jun, 90(6), 928 - 42 Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced in whey; Mauriello G et al.; AIMS: Seventy-eight strains of lactic acid bacteria belonging to five genera and showing six different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and Cit (citrate degradation) characters were investigated for their main flavouring properties with the aim to detect variability among and within the groups . METHODS AND RESULTS: High resolution gas chromatography-mass spectrometry analysis of neutral volatile compounds produced in whey showed that, considering both neo-formation compounds and substances quantified in the whey cultures at different concentrations in comparison to the extract from sterile whey, the groups of lactococci, enterococci, thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles than thermophilic lactobacilli and leuconostocs . Applying principal component analysis (PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci showed a broad diversity, while lactococci included rather similar strains as well as strains with special flavouring properties . Applying PCA to thermophilic streptococci and enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to mesophilic and thermophilic lactobacilli, the strains gathered consistently with their systematic position . CONCLUSION: The study evidenced strains producing some volatile compounds responsible for food flavouring . Flavouring properties were variable among the systematic groups and in some cases different within the same bacterial group . SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry. J Appl Microbiol, 2001 Jun, 90(6), 901 - 8 Factors influencing attachment of thermophilic bacilli to stainless steel; Parkar SG et al.; AIMS: This project aimed to investigate the mechanism of attachment of the vegetative cells and spores of thermophilic bacilli to stainless steel with a view to devising strategies to limit biofilm development and survival . METHODS AND RESULTS: Spores and vegetative cells of bacterial isolates were exposed to protein denaturing agents (sodium dodecyl sulphate (SDS) and trypsin) and polysaccharide removing agents (sodium metaperiodate, trichloroacetic acid (TCA) and lysozyme) . Treatment with sodium metaperiodate, TCA and lysozyme increased the number of vegetative cells attaching in many of the strains studied, while SDS and trypsin decreased attachment . Spores attached to stainless steel in greater numbers than vegetative cells, and the various treatments had less effect on this attachment than for vegetative cells . Viability of the cells or spores was not an important factor in attachment, as cells and spores rendered non-viable also attached to stainless steel in similar numbers . Coating the stainless steel with skim milk proteins decreased the attachment of both vegetative cells and spores . There was no correlation between the degree of attachment and the amount of extracellular polysaccharide (EPS) produced by each strain, surface hydrophobicity or zeta potential of vegetative cells or spores, though spores were found to be more hydrophobic than vegetative cells . CONCLUSIONS: The results suggest that biofilm formation by these thermophilic bacilli is probably a multifactorial process, and that cell-surface proteins play a very important role in the initial process of attachment during the formation of biofilms by these bacteria . SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved cleaning systems to control biofilms of thermophilic bacilli in dairy manufacturing plants. J Eukaryot Microbiol, 2001 May-Jun, 48(3), 332 - 7 The I-antigens of Ichthyophthirius multifiliis are GPI-anchored proteins; Clark TG et al.; The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host . Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena . Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists . Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well . Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase . Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic . Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C . Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell . Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins. J Eukaryot Microbiol, 2001 May-Jun, 48(3), 266 - 79 Development in electrofused conjugants of tetrahymena thermophila; Cole ES et al.; Electric shock can create parabiotic fusions of living Tetrahymena cells . In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs . In particular, we electrofused pairs from normal {diploid x diploid} matings with vegetatively dividing cells in G- or M-phase of the cell cycle . We also fused {diploid x diploid} conjugants with mating pairs involving an aneuploid partner {diploid x "star"}, which typically undergo an abortive conjugal pathway termed genomic exclusion . Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: 1) the "abort" signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the "terminal commitment point", a developmental stage in normal {diploid x diploid} pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division) . Finally we demonstrate that a conjugal-arrest-activity varies with the vegetative cell cycle, reaching its highest level of activity during M-phase and dropping just after cytokinesis. FEMS Microbiol Lett, 2001 Jun 12, 200(1), 103 - 9 Bacterial community analysis of Indonesian hot springs; Baker GC et al.; We report the first attempts to describe thermophilic bacterial communities in Indonesia's thermal springs using molecular phylogenetic analyses . 16S rRNA genes from laboratory cultures and DNA directly amplified from three hot springs in West Java were sequenced . The 22 sequences obtained were assignable to the taxa Proteobacteria, Bacillus and Flavobacterium, including a number of clades not normally associated with thermophily. FEMS Microbiol Lett, 2001 Jun 12, 200(1), 85 - 90 Insertional mutagenesis of an industrial strain of Streptococcus thermophilus; Labarre C et al.; Random mutagenesis of an industrial strain of Streptococcus thermophilus was achieved through an adapted version of a two-plasmid system . The mutagenesis strategy is based on random integration of derivatives of the non-replicative (Rep(-)) plasmid pORI19 by means of homologous recombination following a temperature shift that eliminates replication of the temperature-sensitive (Rep(ts)) helper plasmid pVE6007 . In this way mutants were generated which were affected in bacteriophage sensitivity or sucrose metabolism . Homologues were identified of a protein related to folate metabolism from a bacteriophage-resistant mutant and of two subunits of an oligopeptide transport system from a mutant deficient in sucrose utilisation. Biochim Biophys Acta, 2001 Jun 11, 1547(2), 214 - 20 Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond; Kabashima T et al.; From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability . Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis . The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS-PAGE with and without 2-mercaptoethanol . The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate . However, the thermal stability of the S185C mutant was found to be 30 degrees C higher than that of wild-type . Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme. EMBO J, 2001 Jun 15, 20(12), 3251 - 61 Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila; Saveliev SV et al.; DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated . A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process . New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate . The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15-16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form. Biochemistry (Mosc), 2001 May, 66(5), 520 - 3 Comparative study of thermal degradation of iron-sulfur proteins in spinach chloroplasts and membranes of thermophilic cyanobacteria: mössbauer spectroscopy; Novakova AA et al.; Mossbauer spectra of chloroplasts isolated from spinach plants grown in a mineral medium enriched with 57Fe and Mossbauer spectra of native membranes of the thermophilic cyanobacterium Synechococcus elongatus contain a broad asymmetric doublet typical of the iron-sulfur proteins of Photosystem (PS) I . Exposure of chloroplasts to temperatures of 20-70 degrees C significantly modifies the central part of the spectra . This spectral change is evidence of decreased magnitude of the quadrupole splitting . However, the thermally induced doublet (DeltaQ = 3.10 mm/sec and delta = 1.28 mm/sec) typical of hydrated forms of reduced (divalent) inorganic iron is not observed in spinach chloroplasts . This doublet is usually associated with degradation of active centers of ferredoxin, a surface-exposed protein of PS I . The Mossbauer spectra of photosynthetic membranes of spinach chloroplasts and cyanobacteria were compared using the probability distribution function of quadrupole shift (1/2 quadrupole splitting DeltaQ) of trivalent iron . The results of calculation of these functions for the two preparations showed that upon increasing the heating temperature there was a decrease in the probability of the presence of native iron-sulfur centers FX, FA, and FB (quadrupole shift range, 0.43-0.67 mm/sec) in heated preparations . This process was also accompanied by an increase in the probability of appearance of clusters of trivalent iron . This increase was found to be either gradual and continuous or abrupt and discrete in photosynthetic membranes of cyanobacteria or spinach chloroplasts, respectively . The probability of the presence of the iron-sulfur centers FX, FA, and FB in chloroplasts abruptly decreases to virtually to zero within the temperature range critical for inhibition of electron transport through PS I to oxygen . In cyanobacteria, both thermal destruction of iron-sulfur centers of PS I and functional degradation of PS I are shifted toward a higher temperature . The results of this study suggest that the same mechanism of thermal destruction of the PS I core occurs in both thermophilic and mesophilic organisms: destruction of iron-sulfur centers FX, FA, and FB, release of oxidized (trivalent) iron, and its accumulation in membrane-bound iron-oxo clusters. Biophys Chem, 2001 Jun 15, 91(1), 71 - 7 Important inter-residue contacts for enhancing the thermal stability of thermophilic proteins; Gromiha MM; Proteins from thermophilic organisms exhibit high thermal stability, but have structures that are very similar to their mesophilic homologues . In order to gain insight into the basis of thermostability, we have analyzed the medium- and long-range contacts in mesophilic and thermophilic proteins of 16 different families . We found that the thermophiles prefer to have contacts between residues with hydrogen-bond-forming capability . Apart from hydrophobic contacts, more contacts are observed between polar and non-polar residues in thermophiles than mesophiles . Residue-wise analysis showed that Tyr has good contacts with several other residues, and Cys has considerably higher long-range contacts in thermophiles compared with mesophiles . Furthermore, the residues occurring in the range of 31-34 residues apart in the sequence contribute significant long-range contacts to the stability of thermophilic proteins. Plasmid, 2001 May, 45(3), 171 - 83 Isolation and characterization of a Streptococcus thermophilus plasmid closely related to the pMV158 family; Turgeon N et al.; Twenty-two Streptococcus thermophilus strains used for milk fermentations were analyzed for their plasmid content and 13 of them (59%) were found to contain one or two plasmids . Fifteen S . thermophilus plasmids were divided into four groups using DNA homology . Ten plasmids were classified within group A and they shared homologies with all the previously sequenced S . thermophilus plasmids . Three plasmids (group B) hybridized with each other and two plasmids only hybridized with themselves (groups C and D) . Single-stranded DNA was detected within strains containing plasmids of groups A, C, and D, indicating that they replicate via a rolling-circle mode . The only plasmid of group C, named pSMQ172, was further characterized . This 4230-bp plasmid replicates in Escherichia coli, Lactococcus lactis, and Streptococcus salivarius and does not confer phage resistance . Comparisons with databases showed that pSMQ172 was related to pMV158 of Streptococcus agalactiae and to pSSU1 of Streptococcus suis . These results suggest that genetic exchanges may have occurred between pathogenic and nonpathogenic streptococci. Cell Biol Int, 2001, 25(6), 509 - 19 Cell death in Tetrahymena thermophila: new observations on culture conditions; Christensen ST et al.; We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation . The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish . At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30 min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds . The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface . Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium . In addition, plastic caps in well-filled vials release substances, which promote cell survival . The fate of low-density cultures is related to certain 'physical' conditions, in addition to the availability of oxygen within closed culture systems . Biochemistry, 2001 Jun 19, 40(24), 7165 - 73 Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus; Gerstner RB et al.; Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA . This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex . Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions {350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)}, a complex with an association constant of