Lett Appl Microbiol, 2004, 38(5), 415 - 22 Gene expression studies of Thermus thermophilus promoters PdnaK, Parg and Pscs-mdh; Park HS et al.; AIMS: To obtain data concerning gene expression in Thermus thermophilus and demonstrate the use of the beta-galactosidase gene from Thermus sp . A4 as a convenient reporter gene . METHODS AND RESULTS: Thermus thermophilus PPKU was constructed, in which the beta-gal gene was deleted from the chromosome . Two inducible promoters PdnaK (regulating the DnaK heat shock-inducible protein) and Parg (regulating expression of an arginine-inducible protein) and a carbon-regulated promoter, Pscs-mdh (regulating expression of succinyl-coA and malate dehydrogenase) were cloned upstream of the beta-gal reporter gene derived from Thermus sp . A4 to construct vectors pTEX7, pTEX8 and pTEX9, respectively . The amount of beta-galactosidase activity produced by the PdnaK promoter in pTEX7 was substantially above the background level of 0.3 U mg(-1), and increased from 5.2 to 10.4 U mg(-1) after heat-shock induction indicating that significant amounts of DnaK are produced even when T . thermophilus is grown at its optimum temperature . The Parg promoter was found to be maximally induced by 10-30 mm arginine, but was inhibited by higher concentrations . The Pscs-mdh promoter was maximally active in the presence of malate while lower levels of activity were observed in the presence of succinate, pyruvate, glutamate, glucose and the presence of yeast extract or peptone . CONCLUSIONS: These results demonstrate that several inducible and regulated promoters are available for genetic studies in Thermus and that beta-galactosidase can be used as a convenient reporter gene for studies of transcriptional regulation in Thermus . SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of characterized inducible and regulated promoters will facilitate the development of improved gene expression vectors for Thermus . The demonstration that beta-galactosidase activity in T . thermophilus PPKU can be used to allow reliable screening for beta-gal-positive transformant colonies on agar plates will add to the convenience of performing genetic manipulations in T . thermophilus . Future studies of transcriptional regulation in Thermus will benefit from the beta-gal host-vector system reported here. Lett Appl Microbiol, 2004, 38(5), 400 - 5 Use of RAPD-PCR and TTGE for the evaluation of biodiversity of whey cultures for Grana Padano cheese; Andrighetto C et al.; AIMS: This work was carried out in order to evaluate the microbial diversity of whey cultures collected from different Grana Padano cheese plants in Veneto region (north-east Italy) by means of RAPD-PCR and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analysis . METHODS AND RESULTS: Lactobacillus helveticus was the dominant species among isolated thermophilic lactobacilli . RAPD-PCR with primers M13 and D8635 resulted a suitable method for typing Lact . helveticus at strain level . Thirteen different Lact . helveticus biotypes were detected in the seven whey cultures studied with one biotype present in all the whey cultures . Besides Lact . helveticus, Lact . delbrueckii subsp . lactis was the main microbial species detected by TTGE . CONCLUSIONS: RAPD-PCR resulted very useful in studying Lact . helveticus biodiversity; furthermore, TTGE analysis allowed to detect the dominant thermophilic microflora characteristic of Grana Padano cheese whey cultures . IMPACT OF THE STUDY: By the combined used of RAPD-PCR and TTGE it could be possible to follow the behaviour in strain or species composition of whey cultures during time. Biotechnol Prog, 2004 Mar-Apr, 20(2), 630 - 5 Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports; Palomo JM et al.; A lipase from Bacillus thermocatenulatus (BTL2) cloned in E . coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton . Only one band was detected by SDS-PAGE . The pure enzyme was immobilized using different methodologies . BTL2 adsorbed on a hydrophobic support (octadecyl-Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity . The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl-Sepabeads-BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 degrees C or 30% of dioxane and 45 degrees C after several days of incubation . The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively . The adsorption of this thermophilic lipase on octadecyl-Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 degrees C. Curr Microbiol, 2004 Apr, 48(4), 295 - 9 Molecular analysis of the xylFGH operon, coding for xylose ABC transport, in Thermoanaerobacter ethanolicus; Erbeznik M et al.; A xylose ABC (ATP-binding cassette) transport operon, xylFGH, was cloned from Thermoanaerobacter ethanolicus, a thermophilic ethanol-producing eubacterium . The cistrons code for a periplasmic D-xylose-binding protein (XylF, partial sequence of 250 amino acids), ATP-binding protein (XylG, 505 amino acids), and integral membrane protein (XylH, 388 amino acids) . These results, together with previous work, indicate that duplicate copies of both xylF and xylH are present in the T . ethanolicus chromosome, suggesting ancient gene duplication or lateral gene transfer events . XylG resembles other eubacterial monosaccharide ABC-ATPases in that its two nucleotide-binding domains (NBDs) are highly homologous, yet significantly different with respect to putative catalytic residues . Unlike most other integral membrane ABC transport proteins, XylH apparently contains 11 or 12 transmembrane segments (TMS) and is similar to a small group of ABC permeases that defy the "2 x 6" helix paradigm . This is the first report of a monosaccharide ABC transport operon in a thermophilic anaerobic eubacterium. Appl Biochem Biotechnol, 2004 Spring, 113-116, 497 - 508 Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus; Kadar Z et al.; This study addressed the utilization of an industrial waste stream, paper sludge, as a renewable cheap feedstock for the fermentative production of hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus . Hydrogen, acetate, and lactate were produced in medium in which paper sludge hydrolysate was added as the sole carbon and energy source and in control medium with the same concentration of analytical grade glucose and xylose . The hydrogen yield was dependent on lactate formation and varied between 50 and 94% of the theoretical maximum . The carbon balance in the medium with glucose and xylose was virtually 100% . The carbon balance was not complete in the paper sludge medium because the measurement of biomass was impaired owing to interfering components in the paper sludge hydrolysate . Nevertheless, >85% of the carbon could be accounted for in the products acetate and lactate . The maximal volumetric hydrogen production rate was 5 to 6 mmol/(L x h), which was lower than the production rate in media with glucose, xylose, or a combination of these sugars (9-11 mmol/{L x h}) . The reduced hydrogen production rate suggests the presence of inhibiting components in paper sludge hydrolysate. Appl Biochem Biotechnol, 2004 Spring, 113-116, 1003 - 12 Application of xylanase from Thermomyces lanuginosus IOC-4145 for enzymatic hydrolysis of corncob and sugarcane bagasse; Damaso MC et al.; Xylanases have significant current and potential uses for several industries including paper and pulp, food, and biofuel . For the biofuel industry, xylanases can be used to aid in the conversion of lignocellulose to fermentable sugars (e.g., xylose) . We investigated the thermophilic fungus Thermomyces lanuginosus was yielded for xylanase production and found that the highest activity (850 U/mL) was yielded after 96 h of semisolid fermentation . The enzyme was used for hydrolyzing agricultural residues with and without pretreatment . Such residues were characterized in relation to the maximum xylose content by total acid hydrolysis . The highest xylose yields realized by enzymatic hydrolysis were 24 and 52%, achieved by using 3000 U/g (dried material) of sugarcane bagasse and corncob, respectively, which received both alkali and thermal pretreatment. Syst Appl Microbiol, 2004 Feb, 27(1), 10 - 7 Sugar utilisation and conservation of the gal-lac gene cluster in Streptococcus thermophilus; van den Bogaard PT et al.; The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus . These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium . In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S . thermophilus strains isolated from a variety of sources . For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined . The sequence, organisation and flanking regions of the S . thermophilus gal-lac gene cluster were found to be highly conserved among all strains . The vast majority of the S . thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose . Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains . These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess. Can J Microbiol, 2004 Jan, 50(1), 1 - 17 Developments in the use of Bacillus species for industrial production; Schallmey M et al.; Bacillus species continue to be dominant bacterial workhorses in microbial fermentations . Bacillus subtilis (natto) is the key microbial participant in the ongoing production of the soya-based traditional natto fermentation, and some Bacillus species are on the Food and Drug Administration's GRAS (generally regarded as safe) list . The capacity of selected Bacillus strains to produce and secrete large quantities (20-25 g/L) of extracellular enzymes has placed them among the most important industrial enzyme producers . The ability of different species to ferment in the acid, neutral, and alkaline pH ranges, combined with the presence of thermophiles in the genus, has lead to the development of a variety of new commercial enzyme products with the desired temperature, pH activity, and stability properties to address a variety of specific applications . Classical mutation and (or) selection techniques, together with advanced cloning and protein engineering strategies, have been exploited to develop these products . Efforts to produce and secrete high yields of foreign recombinant proteins in Bacillus hosts initially appeared to be hampered by the degradation of the products by the host proteases . Recent studies have revealed that the slow folding of heterologous proteins at the membrane-cell wall interface of Gram-positive bacteria renders them vulnerable to attack by wall-associated proteases . In addition, the presence of thiol-disulphide oxidoreductases in B . subtilis may be beneficial in the secretion of disulphide-bond-containing proteins . Such developments from our understanding of the complex protein translocation machinery of Gram-positive bacteria should allow the resolution of current secretion challenges and make Bacillus species preeminent hosts for heterologous protein production . Bacillus strains have also been developed and engineered as industrial producers of nucleotides, the vitamin riboflavin, the flavor agent ribose, and the supplement poly-gamma-glutamic acid . With the recent characterization of the genome of B . subtilis 168 and of some related strains, Bacillus species are poised to become the preferred hosts for the production of many new and improved products as we move through the genomic and proteomic era. Protein Eng Des Sel, 2004 Mar, 17(3), 285 - 91 Epub 2004 Mar 29. Zinc binding drives the folding and association of the homo-trimeric gamma-carbonic anhydrase from Methanosarcina thermophila; Simler BR et al.; Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed beta-helical subunits of which bind three catalytic Zn(2+) ions at symmetry-related subunit interfaces . The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of approximately 70 kcal (mol trimer)(-1) at standard state . Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer . The monomer is estimated to have a stability of 4.0 +/- 0.3 kcal (mol monomer)(-1) . The association to form apo-trimer contributes 13.2 +/- 0.4 kcal (mol trimer)(-1), a value confirmed by analytical ultracentrifugation measurements . Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer . The literature value for the free energy of binding of one Zn(2+) ion to a canonical active site, 16.4 kcal mol(-1), is consistent with the presumption that the >45 kcal (mol trimer)(-1) generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam. Appl Microbiol Biotechnol, 2004 Jun, 64(6), 806 - 15 Epub 2004 Mar 27. Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants; Yuan J et al.; To study the role of the hinge region in nisin and to obtain mutants that exhibit altered or new biological activities and functional properties, we changed certain amino acids in the hinge region by performing site-directed mutagenesis with the nisinZ structural gene ( nisZ) . The results showed that the nisinZ mutants had decreased antimicrobial activities against Micrococcus flavus NCIB8166 and Streptococcus thermophilus . Interestingly, compared with wild nisinZ, mutant N20K nisinZ and M21K nisinZ displayed antimicrobial activity against gram-negative Shigella, Pseudomonas and Salmonella; and they had a higher solubility than wild-type nisinZ . At pH 8, the solubilities of N20K nisinZ and M21K nisinZ were, respectively, three-fold higher and five-fold higher than that of nisinZ . Mutant N20Q nisinZ and M21G nisinZ were considerably more stable than nisinZ at higher temperatures and neutral or alkaline pH . These mutants provided information that the central hinge region in nisinZ plays an important role in providing the conformational flexibility required for the antimicrobial activity on the membrane . Our finding documented that it may well be worth considering the construction of the new nisin mutants with changed inhibitory activity against a wide range of gram-negative bacteria and the improvement of functional properties by site-directed mutagenesis. Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 176 - 80 First characterization of co-chaperonin protein 10 from hyper-thermophilic Aquifex aeolicus; Guidry J et al.; All known co-chaperonin protein 10 (cpn10) molecules are heptamers of seven identical subunits that are linked together by beta-strand interactions . Here, we report the first characterization of a cpn10 protein from a thermophilic organism: Aquifex aeolicus . Primary-structure alignment of A . aeolicus cpn10 (Aaecpn10) shows high homology with mesophilic cpn10 sequences, except for a unique 25-residue C-terminal extension not found in any other cpn10 . Recombinant Aaecpn10 adopts a heptameric structure in solution at pH values above 4 (20 degrees C) . Both monomers and heptamers are folded at 20 degrees C, although the thermal stability of the monomers (pH 3; Tm approximately 58 degrees C) is lower than that of the heptamers (pH 7; Tm approximately 115 degrees C) . Aaecpn10 functions in a GroEL-dependent in vitro activity assay . Taken together, Aaecpn10 appears similar in secondary, tertiary, and quaternary structure, as well as in many biophysical features, to its mesophilic counterparts despite a functional temperature of 90 degrees C. J Mol Biol, 2004 Apr 9, 337(5), 1149 - 60 Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus; Tahirov TH et al.; The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit . The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8% . The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively . The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site . A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases . Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity . However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme . In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base . While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state . The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 306 - 9 Metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus: dynamic enzyme-enzyme interactions involved in the formation of the channelling complex; Massant J et al.; Protection of thermolabile metabolites and coenzymes is a somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles . Detailed information about the mechanisms used by thermophiles to protect these thermolabile metabolites and coenzymes is still scarce . A case in point is CP (carbamoyl phosphate), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate . Recently we obtained the first evidence for a physical interaction between two hyperthermophilic enzymes for which kinetic evidence had suggested that these enzymes channel a highly thermolabile and potentially toxic intermediate . By physically interacting with each other, CKase (carbamate kinase) and OTCase (ornithine carbamoyltransferase) prevent thermodenaturation of CP in the aqueous cytoplasmic environment . The CP channelling complex involving CKase and OTCase or ATCase (aspartate carbamoyltransferase), identified in hyperthermophilic archaea, provides a good model system to investigate the mechanism of metabolic channelling and the molecular basis of protein-protein interactions in the physiology of extreme thermophiles. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 293 - 7 Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts; Bergquist PL et al.; Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical . We have tested two fungal systems for the bulk production of enzymes from thermophiles . The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium . Our second system involves the filamentous fungus Trichoderma reesei . Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 ( cbh1 ) promoter and recombinant plasmid DNAs introduced into various high-secreting T . reesei strains using biolistic particle delivery . In some cases (e.g . the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes . The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains . A proteomics approach has been taken to identify strongly expressed proteins produced by T . reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins . Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions . The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 290 - 2 Application of thermophilic enzymes in commercial biotransformation processes; Taylor IN et al.; Biocatalysis is a useful tool in the provision of chiral technology and extremophilic enzymes are just one component in that toolbox . Their role is not always attributable to their extremophilic properties; as with any biocatalyst certain other criteria should be satisfied . Those requirements for a useful biocatalyst will be discussed including issues of selectivity, volume efficiency, security of supply, technology integration, intellectual property and regulatory compliance . Here we discuss the discovery and commercialization of an L-aminoacylase from Thermococcus litoralis, the product of a LINK project between Chirotech Technology and the University of Exeter . The enzyme was cloned into Escherichia coli to aid production via established mesophilic fermentation protocols . A simple downstream process was then developed to assist in the production of the enzyme as a genetically modified-organism-free reagent . The fermentation and downstream processes are operated at the 500 litre scale . Characterization of the enzyme demonstrated a substrate preference for N-benzoyl groups over N-acetyl groups . The operational parameters have been defined in part by substrate-concentration tolerances and also thermostability . Several examples of commercial biotransformations will be discussed including a process that is successful by virtue of the enzyme's thermotolerance. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 283 - 9 Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicellulose; Sommer P et al.; A limited number of bacteria, yeast and fungi can convert hemicellulose or its monomers (xylose, arabinose, mannose and galactose) into ethanol with a satisfactory yield and productivity . In the present study we tested a number of thermophilic enrichment cultures, and new isolates of thermophilic anaerobic bacterial strains growing optimally at 70-80 degrees C for their ethanol production from D-xylose . The new isolates came from different natural and man-made systems such as hot springs, paper pulp mills and brewery waste water . The test was composed of three different steps; (i) test for conversion of D-xylose into ethanol; (ii) test for viability and ethanol production in pretreated wheat straw hemicellulose hydrolysate; (iii) test for tolerance against high D-xylose concentrations . A total of 86 enrichment cultures and 58 pure cultures were tested and five candidates were selected which successfully fulfilled the criteria defined for the screening test. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 279 - 82 Exploring and exploiting starch-modifying amylomaltases from thermophiles; Kaper T et al.; Starch is a staple food present in water-insoluble granules in many economically important crops . It is composed of two glucose polymers: the linear alpha-1,4-linked amylose and amylopectin with a backbone of alpha-1,4-glycosidic bonds and alpha-1,6-linked side chains . To dissolve starch completely in water it needs to be heated; when it cools down too much the starch solution forms a thermo-irreversible gel . Amylomaltases (EC 2.4.1.25) are enzymes that transfer a segment of an alpha-1,4-D-glucan to a new 4-position in an acceptor, which may be glucose or another alpha-1,4-D-glucan . Acting upon starch, amylomaltases can produce cycloamylose or a thermoreversible starch gel, both of which are of commercial interest. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 276 - 8 Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles; Satyanarayana T et al.; The extensive efforts to screen thermophilic fungi and bacteria, isolated from various environmental samples, have resulted in the selection of Thermomucor indicae-seudaticae, Geobacillus thermoleovorans NP33 and G . thermoleovorans NP54 for the production of glucoamylase, amylopullulanase and alpha-amylase, respectively . Submerged and solid-state fermentation processes were optimized for maximizing the secretion of glucoamylase by T . indicae-seudaticae . The production of amylopullulanase and alpha-amylase by NP33 and NP54 in submerged fermentation was also optimized . Glucoamylase was optimally active at pH 7.0 and 60 degrees C and was shown to saccharify soluble as well as raw starches . Amylopullulanase and alpha-amylase exhibited optima at pH 7.0 and 100 degrees C and saccharified starch efficiently . Differential inhibition and action on mixed substrates clearly suggested that there are two separate active sites for alpha-amylase and pullulanase activities of amylopullulanase . Both alpha-amylase and amylopullulanase are high maltose-forming and Ca(2+)-independent . These amylolytic enzymes have been shown to be useful in starch saccharification alone and in combination. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 273 - 5 Oxygen and carbon dioxide kinetic challenges for thermophilic mineral bioleaching processes; de Kock SH et al.; Agitated bacterial tank bioleaching reactors are currently sparged with air to satisfy both oxygen and CO(2) requirements of microbial cells . Under high-sulphide loading conditions, as is the case with high-grade metal sulphide concentrates, the microbial and chemical demand for oxygen is significantly increased during the bioleaching process . Sparging with enriched oxygen gas may offer an alternative process option to increased agitation and sparged aeration, to overcome the mass transfer difficulties at elevated temperatures where thermophilic Archaea, rather than Bacteria, are used . In the case of air sparging, the DO (dissolved oxygen) concentration in tank reactors could not be increased to a point where it would become inhibitory due to the limited oxygen content of air (20.9% O(2)) . The use of enriched oxygen in such reactors at large scale does, however, pose its own set of process risks . The first aim of this investigation was, therefore, to determine the effects of various DO concentrations, in both the limiting and inhibitory ranges, on the microbial activity of Sulfolobus sp . U40813, a typical thermophilic mineral-leaching archaeon . Secondly, the effect of CO(2) concentration on the rate of ferrous iron oxidation was investigated . Both the oxygen and CO(2) kinetics were examined in controlled batch cultures at 78 degrees C, using ferrous sulphate and potassium tetrathionate as energy sources . The optimal DO concentration for iron oxidation was found to be between 1.5 and 4.1 mg.l(-1) . The use of elevated DO concentrations (above 4.1 mg.l(-1)) inhibited the ferrous oxidation rates . The optimal gas CO(2) concentration for ferrous iron oxidation was found to be in the range 7-17% (v/v) . The iron oxidation rates were, however, severely limited at CO(2) concentrations less than 7%, indicating that the CO(2) supply was limiting in this range and inhibited the microbial growth rate. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 236 - 9 DNA replication in thermophiles; Majernik AI et al.; DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR . The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase) . One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria . These proteins also appear to be simpler versions of those found in eukaryotes . For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination . Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 231 - 5 Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea; Leduc D et al.; The hyperthermophilic anaerobic archaeon Pyrococcus abyssi, which lacks thymidine kinase, incorporates label from extracellular uracil, but not from thymidine, into its DNA . This implies that P . abyssi must synthesize dTMP (thymidylate), an essential precursor for DNA synthesis, de novo . However, iterative similarity searches of the three completed Pyrococcus genomes fail to detect candidate genes for canonical thymidylate synthase ThyA, suggesting the presence of alternative pathways for dTMP synthesis . Indeed, by identifying a novel class of flavin-dependent thymidylate synthases, ThyX, we have recently proven that two distinct pathways for de novo synthesis of dTMP are operational in the microbial world . While both thyX and thyA can be found in hyperthermophilic micro-organisms, the phylogenetic distribution of thyX among hyperthermophiles is wider than that of thyA . In this contribution, we discuss the differences in the distinct mechanisms of dTMP synthesis, with a special emphasis on hyperthermophilic micro-organisms. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 227 - 30 Archaeal histones: structures, stability and DNA binding; Reeve JN et al.; Structures, stability and DNA-binding properties have been established for archaeal histones from mesophiles, thermophiles and hyperthermophiles . Most archaeal histones are simply histone folds that are stabilized by dimer formation . Archaeal histones and the histone folds of the eukaryotic nucleosome core histones share a common ancestry and bind and wrap DNA similarly using conserved residues . The histone-fold residues that stabilize dimer-dimer interactions within an archaeal histone core contribute to determining archaeal histone-DNA affinity. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 218 - 21 16 S rDNA primers and the unbiased assessment of thermophile diversity; Baker GC et al.; Our understanding of thermophile diversity is based predominantly on PCR studies of community DNA . "Universal" and domain-specific rRNA gene PCR primers have historically been used for the assessment of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic groups . In a reassessment of the published primers commonly used for "universal" and archaeal 16 S rDNA sequence amplification we note that substantial variations in specificity exist . An unconsidered choice of primers may therefore lead to significant bias in determination of microbial community composition . In particular, Archaea-specific primer sequences typically lack specificity for the Korarchaeota and Nanoarchaea and are often biased towards certain clades . New primer pairs specifically designed for "universal" archaeal 16 S rDNA sequence amplification, with homology to all four archaeal groups, have been designed . Here we present the application of these new primers for preparation of 16 S libraries from thermophile communities. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 214 - 7 Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus; McMullan G et al.; Thermophilic bacteria belonging to Bacillus genetic group 5 have been reclassified as being members of Geobacillus gen . nov., with G . stearothermophilus as the type strain . Geobacillus species, literally meaning earth or soil Bacillus, are widely distributed and readily isolated from natural and man-made thermophilic biotopes . Work within our group has however shown that an abundance of genetically distinct Geobacillus isolates can be obtained from temperate Irish soils . As with many thermophiles there is considerable interest in potential industrial application of these bacteria and their gene products . This review describes two novel applications for Geobacillus isolates, firstly in the metabolism of the herbicide glyphosate and secondly in the metabolism of quorum-sensing signal molecules from Gram-negative bacteria . Finally the current state of the art is described for Bacillus genomics, with details given of three independent genome-sequencing projects of Geobacillus isolates. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 209 - 13 Distribution and molecular investigation of highly thermophilic bacteria associated with cool soil environments; Rahman TJ et al.; In this study, both molecular and culture-based methods were used to characterize thermophilic bacteria associated with the subsurface soil environment in Northern Ireland . A total of 53 thermophilic, aerobic, sporulating and non-sporulating bacteria were isolated from subsurface soil samples obtained from two sites . They were screened by amplified ribosomal DNA restriction analysis prior to 16 S rRNA gene sequencing . The majority of the sequences were associated with Geobacillus thermoleovorans (50%) and Geobacillus caldoxylosilyticus (34.6%) . Isolates F10, F20 and Tf exhibited only 93% similarity with Geobacillus toebii strain F70 . Hence they may represent a new species of the genus Geobacillus. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 193 - 8 Alkalithermophiles; Wiegel J et al.; Alkalithermophiles are an exciting subset of extremophilic organisms and represent extremophiles that are adapted to two extreme conditions, i.e . to a combination of alkaline and thermobiotic growth conditions . Among the anaerobic alkalithermophiles are representatives of both Bacteria and Archaea within a wide variety of physiological types and systematic groups, although a great majority belongs to the Firmicutes . Alkaliphiles have been isolated from a variety of niches including mesobiotic and neutrophilic soils and sediments . Interestingly anaerobic isolates from mesobiotic and neutrophilic niches exhibit shorter doubling times than isolates from thermobiotic niches; some anaerobic alkalithermophiles exhibit extremely fast growth rates, i.e . doubling times as short as 10 min . Their adaptation to both high pH and high temperature draws our attention not only because they are potential sources of industrially valuable enzymes but also because of their adaptive mechanisms to external environmental parameters . They could thus function as model organisms for extraterrestrial life in some environments and for theories on the origin of life . Alkalithermophiles, as far we know, do not represent the most thermophilic nor the most alkaliphilic of micro-organisms but represent the most alkaliphilic ones among the thermophiles and vice versa . We believe that the presently known species are only the tip of the iceberg and therefore that they do not represent the true boundaries under which life can thrive in respect to high temperature in alkaline environments. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 172 - 4 Sulphate metabolism among thermophiles and hyperthermophiles in natural aquatic systems; Roychoudhury AN; Although controversial, the idea that hydrothermal systems may have been the site for prebiotic synthesis of organic molecules and origin of life is widely supported . For the nascent life to survive, it must have had some sort of metabolic mechanism for generating energy . However, little is known of the specific metabolic pathways utilized by the early life forms or the effect of high temperatures on their activity . Recent research on natural high temperature aquatic environments, though limited because of difficult field logistics and experimental problems, is revolutionizing our understanding of possible energy-generating redox pathways, such as sulphate reduction . An abridged review of research on thermophilic sulphate reduction is presented here . Because of a complex interplay between microbiological and geochemical entities involved, and the uncertainties that modern hydrothermal systems are proxy for biogeochemical conditions on early Earth, great caution is required for interpretation and extrapolation of data from these studies to primordial times . Furthermore, a general lack of integrated geological and microbiological studies towards a common understanding of origin and sustenance of life on Earth is starkly evident from this review. Biochem Soc Trans, 2004 Apr, 32(Pt 2), 165 - 7 Finding extraterrestrial sites for thermophiles; Naylor T; Virtually our entire knowledge of the universe comes from two sorts of measurement of the electromagnetic radiation from the stars and galaxies within it; either their flux through relatively wide bandpasses (photometry), or measurements of the shape and wavelength of relatively narrow lines via spectroscopy . These techniques are now being used to discover planets outside our solar system, and perhaps in the next 10 years will begin to characterize them . If a serious search is to be made for extraterrestrial thermophiles, we need predictions for the effects of thermophiles on their host planets that are observable with these techniques . In this paper I shall outline what sorts of observation are likely to be used in the next 15 years for extra-solar planet work . All of the journal articles quoted here can be found through and often also accessed as preprints at http://uk.arxiv.org/form/astro%20ph?MULTI=form%20+/-%20interface. Biophys J, 2004 Apr, 86(4), 2438 - 44 Probing the Q-proton pathway of ba3-cytochrome c oxidase by time-resolved Fourier transform infrared spectroscopy; Koutsoupakis C et al.; In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme . Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site . In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases . The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool . We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature . The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)) . The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed. Ecotoxicol Environ Saf, 2004 Mar, 57(3), 375 - 82 Evaluating the toxicity of Triton X-100 to protozoan, fish, and mammalian cells using fluorescent dyes as indicators of cell viability; Dayeh VR et al.; Three viability assays using fluorescent dyes effectively detected a loss of viability in cultures of three mammalian cell lines (H4IIE, Caco2, and HepG-2), two fish cell lines (RTgill-W1 and RTL-W1), and a ciliated protozoan, Tetrahymena thermophila, after exposure to Triton X-100, used as a model toxicant . The dyes were Alamar Blue (AB), neutral red (NR), and propidium iodide, which respectively monitored energy metabolism, lysosomal activity, and membrane integrity . A fourth fluorescent dye, 5-carboxyfluorescein diacetate acetoxymethyl ester, was problematic . For 2-h Triton X-100 exposures, mammalian cell lines were as susceptible as piscine cell lines, whereas T . thermophila was approximately twofold less sensitive as detected with AB and NR . Despite being less sensitive, cytotoxicity tests on T . thermophila could be done in spring water, which means that unlike animal cells they could be directly exposed to most industrial effluents without osmolality adjustments . Therefore, T . thermophila could be a useful complement to animal cells as alternatives to fish in toxicity testing. Acta Crystallogr D Biol Crystallogr, 2004 Apr, 60(Pt 4), 761 - 3 Epub 2004 Mar 23. Crystallization and preliminary crystallographic analysis of 2-keto-3-deoxygluconate kinase from Thermus thermophilus; Inagaki E et al.; 2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate . Two crystal forms of KDGK from Thermus thermophilus were obtained by vapour-diffusion and microbatch methods . Crystals in the form of triangular plates (TtKDGK-1) were obtained that belong to space group P3, with unit-cell parameters a = b = 145.83, c = 74.63 A, and diffract to 3.2 A . These crystals exhibited nearly perfect hemihedral twinning . Assigning six subunits of TtKDGK to the asymmetric unit of the crystal corresponds to a 46.2% solvent content . A single plate-like crystal (TtKDGK-2) belonged to space group P6(3), with unit-cell parameters a = b = 84.83, c = 168.49 A, and diffracts to 2.25 A . This crystal exhibits only partial hemihedral twinning, with a twin fraction of 24.4% . Diffraction-quality crystals of TtKDGK with bound ATP (TtKDGK-ATP), a = b = 84.72, c = 321.61 A and with bound KDG plus the ATP analogue AMP-PNP (TtKDGK-ATP-KDG), with unit-cell parameters a = b = 84.32, c = 168.7 A, were also prepared and characterized. Acta Crystallogr D Biol Crystallogr, 2004 Apr, 60(Pt 4), 727 - 9 Epub 2004 Mar 23. Crystallization and preliminary crystallographic analysis of the circadian clock protein KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1; Iwase R et al.; KaiB is a component of the circadian clock oscillator in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms . KaiB consists of 108 amino-acid residues and has a molecular weight of 12 025 Da . KaiB and Cys-substituted KaiB mutants from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were expressed as GST-fusion proteins in Escherichia coli, purified and crystallized . The crystals of wild-type KaiB belong to the monoclinic space group P2(1), with unit-cell parameters a = 89.6, b = 71.2, c = 106.8 A, beta = 100.1 degrees . While the native crystals diffract to 3.7 A, osmium derivatives, which show an approximately 4 A shrinkage in the b axis, diffract to 2.6 A . The crystals of the singly Cys-substituted mutant T64C with Hg, which show different morphology, diffract to 2.5 A and belong to the monoclinic space group P2, with unit-cell parameters a = 63.7, b = 33.4, c = 93.7 A, beta = 100.1 degrees . Anomalous difference Patterson maps of the Os- and Hg-derivative crystals had significant peaks in their Harker sections, suggesting that both derivatives are suitable for structure determination. J Struct Biol, 2004 Apr-May, 146(1-2), 130 - 40 Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation; Kenniston JA et al.; ClpX and related AAA+ ATPases of the Clp/Hsp100 family are able to denature native proteins . Here, we explore the role of protein stability in ClpX denaturation and subsequent ClpP degradation of model substrates bearing ssrA degradation tags at different positions . ClpXP degraded T . thermophilus RNase-H* with a C-terminal ssrA tag very efficiently, despite the very high global stability of this thermophilic protein . In fact, global thermodynamic stability appears to play little role in susceptibility to degradation, as a far less stable RNase-H*-ssrA mutant was degraded more slowly than wild type by ClpXP and a completely unfolded mutant variant was degraded less than twice as fast as the wild-type parent . When ssrA peptide tags were covalently linked to surface cysteines at positions 114 or 140 of RNase-H*, the conjugates were proteolyzed very slowly . This resistance to degradation was not caused by inaccessibility of the ssrA tag or an inability of ClpXP to degrade proteins with side-chain linked ssrA tags . Our results support a model in which ClpX denatures proteins by initially unfolding structural elements attached to the degradation tag, suggest an important role for the position of the degradation tag and direction of force application, and correlate well with the mapping of local protein stability within RNase-H* by native-state hydrogen exchange. J Mol Biol, 2004 Mar 5, 336(5), 1075 - 86 Modular structure in developmentally eliminated DNA in Tetrahymena may be a consequence of frequent insertions and deletions; Huvos P; The work reported here describes insertion-deletion (Indel) polymorphisms in two internally eliminated sequences (IESs, that are deleted during development in Tetrahymena): a 1.8 kb Indel at one end of the 1.1 kb H1 IES and a 0.5 kb Indel inside the 1.4 kb calmodulin (C) IES . These two IESs are located in the proximity of the H1 histone and calmodulin genes, respectively, and are among the ten IESs that have been fully sequenced out of an estimated total of 6000 . Three hundred base-pairs of the 1.8 kb H1 Indel are retained in the macronucleus . Both the +Indel and the -Indel variants of the H1 and C IESs that occur in different strains are eliminated during development . Thus, a drastic change involving over half of the deleted sequence and 300 bp of flanking sequence does not disable developmental elimination of the H1 IES, which may indicate a lack of requirement for specific sequences on the Indel side of the IES . The H1 Indel is a composite of three sequence elements: a unique segment and two other sections containing members of different repeat families . One of these, a 0.5 kb repetitive component, is 75% similar to another 0.5 kb sequence that constitutes the C Indel, a sequence present in the middle of the calmodulin IES in some strains, but not in others . Therefore, the C Indel sequence is likely to have been part of a mobile unit, even though it has no obvious features of a transposon . However, sequences similar to the C Indel are present in about 100 copies in the genome . The results suggest that IESs may consist, at least in part, of relatively short modules of repeated sequences that are the source of insertion-deletion polymorphisms among strains of Tetrahymena thermophila. J Mol Biol, 2004 Mar 5, 336(5), 1061 - 73 A member of a repeat family is the source of an insertion-deletion polymorphism inside a developmentally eliminated sequence of Tetrahymena thermophila; Huvos P; In Tetrahymena thermophila, the development of a transcriptionally active macronucleus from a transcriptionally inert micronucleus is accompanied by the elimination of numerous DNA segments, called internally eliminated sequences (IESs), many of which belong to dispersed repetitive sequence families . To examine the relationship between the insertion and deletion events expected to occur during evolution of the repeats and the developmental elimination process, IESs were compared among different Tetrahymena strains . A 600 base-pair DNA segment, the R Indel, was discovered inside the R IES, one of the ten sequenced IESs out of an estimated 6000 total in the Tetrahymena genome . The R Indel was found in strains B3 and C2 but not in several other strains examined, indicating that the Indel was probably present in a progenitor of strains B3 and C2 . The R Indel was found to belong to a moderately large sequence family of about 200 members; however, BLAST searches did not reveal meaningful similarities with other mobile elements . Sequence comparisons revealed that a 300 base-pair stretch, very closely related to the first half of the R Indel, was present inside the previously described B IES, another of the ten sequenced IESs . This is the first example of shared sequences between two of the known IESs. J Mol Biol, 2004 Apr 2, 337(4), 1011 - 33 Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography; Nakai T et al.; The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme . E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component . In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)) . Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site . Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA . The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP . The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha . As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form . These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA . ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively. FEMS Microbiol Lett, 2004 Mar 19, 232(2), 145 - 52 Phylogenetic diversity of sulfate-reducing prokaryotes in active deep-sea hydrothermal vent chimney structures; Nakagawa T et al.; The phylogenetic diversity of sulfate-reducing prokaryotes occurring in active deep-sea hydrothermal vent chimney structures was characterized based on the deduced amino acid sequence analysis of the polymerase chain reaction-amplified dissimilatory sulfite reductase (DSR) gene . The DSR genes were successfully amplified from microbial assemblages of the chimney structures, derived from three geographically and geologically distinct deep-sea hydrothermal systems in the Central Indian Ridge (CIR), in the Izu-Bonin Arc (IBA), and the Okinawa Trough (OT), respectively . Phylogenetic analysis revealed seven major phylogenetic groups . More than half of the clones from the CIR chimney structure were related to DSR amino acid sequences of the hyperthermophilic archaeal members of the genus Archaeoglobus, and those of environmental DSR clones within the class Thermodesulfobacteria . From the OT chimney structure, a different group was obtained, which comprised a novel, deep lineage associated with the DSRs of the thermophilic sulfate-reducing bacterium Thermodesulfovibrio . Most of the DSR clones from the IBA chimney structure were phylogenetically associated with the delta-proteobacterial sulfate-reducing bacteria represented by the genus Desulfobulbus . Sequence analysis of DSR clones demonstrated a diverse sulfate-reducing prokaryotic community in the active deep-sea hydrothermal chimney structures. Water Res, 2004 Apr, 38(7), 1707 - 14 Emission of volatile organic compounds during composting of municipal solid wastes; Komilis DP et al.; The objective of this study was to identify and quantify volatile and semi-volatile organic compounds (VOCs) produced during composting of the organic fraction of municipal solid wastes (MSW) . A laboratory experiment was conducted using organic components of MSW that were decomposed under controlled aerobic conditions . Mixed paper primarily produced alkylated benzenes, alcohols and alkanes . Yard wastes primarily produced terpenes, alkylated benzenes, ketones and alkanes, while food wastes primarily produced sulfides, acids and alcohols . Among 13 aromatic VOCs found in MSW composting facilities, toluene, ethylbenzene, 1,4-dichlorobenzene, p-isopropyl toluene, and naphthalene were in the largest amounts . Unseeded mixed paper, seeded mixed paper, seeded yard wastes, unseeded yard wastes, seeded food wastes and unseeded food wastes produced approximately 6.5, 6.1, 2.1, 0.83, 2.5 and 0.33 mg of 13 volatile and semi-volatile aromatic organic compounds combined, respectively, per dry kg . All VOCs were emitted early during the composting process and their production rates decreased with time at thermophilic temperatures. Water Res, 2004 Apr, 38(7), 1653 - 62 Mesophilic and thermophilic temperature co-phase anaerobic digestion compared with single-stage mesophilic- and thermophilic digestion of sewage sludge; Song YC et al.; The performance of thermophilic and mesophilic temperature co-phase anaerobic digestions for sewage sludge, using the exchange process of the digesting sludge between spatially separated mesophilic and thermophilic digesters, was examined, and compared to single-stage mesophilic and thermophilic anaerobic digestions . The reduction of volatile solids from the temperature co-phase anaerobic digestion system was dependent on the sludge exchange rate, but was 50.7-58.8%, which was much higher than 46.8% of single-stage thermophilic digestion, as well as 43.5% of the mesophilic digestion . The specific methane yield was 424-468 mL CH(4) per gram volatile solids removed, which was as good as that of single-stage mesophilic anaerobic digestion . The process stability and the effluent quality in terms of volatile fatty acids and soluble chemical oxygen demand of the temperature co-phase anaerobic digestion system were considerably better than those of the single-stage mesophilic anaerobic processes . The destruction of total coliform in the temperature co-phase system was 98.5-99.6%, which was similar to the single-stage thermophilic digestion . The higher performances on the volatile solid and pathogen reduction, and stable operation of the temperature co-phase anaerobic system might be attributable to the well-functioned thermophilic digester, sharing nutrients and intermediates for anaerobic microorganisms, and selection of higher substrate affinity anaerobic microorganisms in the co-phase system, as a result of the sludge exchange between the mesophilic and thermophilic digesters. Biochem J, 2004 Jul 1, 381(Pt 1), 249 - 55 Reversion of protein aggregation mediated by Sso7d in cell extracts of Sulfolobus solfataricus; Guagliardi A et al.; In eukaryotic cells and in Escherichia coli, reversion of protein aggregation is mediated by the network of chaperones belonging to Hsp70 and Hsp100 families {Weibezahn, Bukau and Mogk (2004) Microb . Cell Fact . 3, 1-12} . The thermophilic prokaryotes of the archaea domain lack homologues of these chaperone families, and the mechanisms they use to rescue aggregated proteins are unknown {Macario, Malz and Conway de Macario (2004) Front . Biosci . 9, 1318-1332} . In the present study, we show that stable protein aggregates can be detected in extracts of starved cells of the thermophilic archaeon Sulfolobus solfataricus, and that the protein Sso7d interacts with the aggregates and mediates the disassembly of the aggregates and the re-activation of insolubilized beta-glycosidase in the presence of ATP hydrolysis . Furthermore, we report that heat-induced protein aggregates in extracts of exponential cells of S . solfataricus contain Sso7d that rescues insolubilized proteins in the presence of ATP hydrolysis . Results of these experiments performed in cell extracts are consistent with an in vivo role of Sso7d in reverting protein aggregation. J Biol Chem, 2004 May 21, 279(21), 21732 - 9 Epub 2004 Mar 15. The Nudix hydrolase Ndx1 from Thermus thermophilus HB8 is a diadenosine hexaphosphate hydrolase with a novel activity; Iwai T et al.; The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8 . This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins . Ndx1 was overexpressed in Escherichia coli and purified . Ndx1 was stable up to 95 degrees C and at extreme pH . Size exclusion chromatography indicated that Ndx1 was monomeric in solution . Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always generated ATP as the product . Diadenosine hexaphosphate (Ap(6)A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap(6)A, with a K(m) of 1.4 microm and a k(cat) of 4.1 s(-1) . These results indicate that Ndx1 is a (di)adenosine polyphosphate hydrolase . Ndx1 activity required the presence of the divalent cations Mn(2+), Mg(2+), Zn(2+), and Co(2+), whereas Ca(2+), Ni(2+), and Cu(2+) were not able to activate Ndx1 . Fluoride ion inhibited Ndx1 activity via a non-competitive mechanism . Optimal activity for Ap(6)A was observed at around pH 8.0 and about 70 degrees C . We found two important residues with pK(a) values of 6.1 and 9.6 in the free enzyme and pK(a) values of 7.9 and 10.0 in the substrate-enzyme complex . Kinetic studies of proteins with amino acid substitutions suggested that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis . Trp-26 was likely involved in enzyme-substrate interactions based on fluorescence measurements . Based on these results, the mechanism of substrate recognition and catalysis are discussed. Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 467 - 74 Isolation from oil reservoirs of novel thermophilic anaerobes phylogenetically related to Thermoanaerobacter subterraneus: reassignment of T . subterraneus, Thermoanaerobacter yonseiensis, Thermoanaerobacter tengcongensis and Carboxydibrachium pacificum to Caldanaerobacter subterraneus gen . nov., sp . nov., comb . nov . as four novel subspecies; Fardeau ML et al.; Novel thermophilic, anaerobic, Gram-positive, rod-shaped bacteria, strains SL9 and OCA1, were isolated from oilfields in France and Australia, respectively . Both strains, together with Thermoanaerobacter yonseiensis KB-1(T) (=DSM 13777(T)), Thermoanaerobacter tengcongensis MB4(T) (=DSM 15242(T)) and Carboxydibrachium pacificum JM(T) (=DSM 12653(T)), possessed genomic (DNA-DNA hybridization studies) and phylogenetic similarities with Thermoanaerobacter subterraneus SEBR 7858(T) (=DSM 13054(T)), which was isolated recently from an oilfield reservoir in south-west France . Marked phenotypic differences exist between the three oilfield isolates (T . subterraneus, strain OCA1 and strain SL9): they include temperature range for growth and substrates used . Differences were also observed in the DNA G+C contents of all organisms . Similarly to T . subterraneus, strains SL9 and OCA1, and also T . yonseiensis, T . tengcongensis and Carboxydibrachium pacificum, produced acetate and L-alanine as major end products of glucose metabolism {0.8-1.0 mol L-alanine produced (mol glucose consumed)(-1)} and reduced thiosulfate, but not sulfate, to sulfide . Because of these significant metabolic and phylogenetic differences between the oilfield isolates (T . subterraneus, strain OCA1 and strain SL9), T . yonseiensis, T . tengcongensis and Carboxydibrachium pacificum and other Thermoanaerobacter species, it is proposed to reassign them as a novel genus and species, Caldanaerobacter subterraneus gen . nov., sp . nov., comb . nov., with the creation of four novel subspecies, Caldanaerobacter subterraneus subsp . subterraneus subsp . nov., comb . nov., Caldanaerobacter subterraneus subsp . yonseiensis subsp . nov., comb . nov., Caldanaerobacter subterraneus subsp . tengcongensis subsp . nov., comb . nov . and Caldanaerobacter subterraneus subsp . pacificus subsp . nov., comb . nov. J Mol Biol, 2004 Mar 26, 337(3), 761 - 70 Crystal structure of the GTP-binding protein Obg from Thermus thermophilus HB8; Kukimoto-Niino M et al.; Obg comprises a unique family of high-molecular mass GTPases conserved from bacteria to eukaryotes . Bacterial Obg is essential for cellular growth, sporulation, and differentiation . Here, we report the crystal structure of the full-length form of Obg from Thermus thermophilus HB8 at 2.07 A resolution, in the nucleotide-free state . It reveals a three-domain arrangement, composed of the N-terminal domain, the guanine nucleotide-binding domain (G domain), and the C-terminal domain . The N-terminal and G domains have the Obg fold and the Ras-like fold, respectively . These global folds are similar to those of the recently published structure of the C-terminal domain-truncated form of Obg from Bacillus subtilis . On the other hand, the C-terminal domain of Obg was found to have a novel fold (the OCT fold) . A comparison of the T.thermophilus and B.subtilis nucleotide-free Obg structures revealed significant conformational changes in the switch-I and switch-II regions of the G domain . Notably, the N-terminal domain is rotated drastically, by almost 180 degrees, around the G domain axis . In the T.thermophilus Obg crystal, the nucleotide-binding site of the G domain interacts with the C-terminal domain of the adjacent molecule . These data suggest a possible domain rearrangement of Obg, and a potential role of the C-terminal domain in the regulation of the nucleotide-binding state. Curr Microbiol, 2004 Jan, 48(1), 51 - 6 Identification of an iron-binding protein of the Dps family expressed by Streptococcus thermophilus; Nicodeme M et al.; Streptococcus thermophilus PB18 can grow between 20 degrees and 52 degrees C and is resistant to various stresses such as heat, acidic or cold shock . During cold shock, a protein of 21.5 kDa was previously shown to be induced in S . thermophilus . In addition to its cold-shock induction, 2D-PAGE revealed that the 21.5-kDa protein was also expressed during the stationary phase of growth . The recent access to the genome sequence of S . thermophilus LMG18311 allowed the identification of a 173-amino acid protein displaying a strong homology between the 21.5-kDa protein and members of the Dps family of proteins . Specific staining of non-denaturing polyacrylamide gel electrophoresis (ND-PAGE) followed by two-dimensional PAGE (2D-PAGE) showed that the 21.5-kDa protein was an iron-binding protein. Z Naturforsch {C}, 2004 Jan-Feb, 59(1-2), 99 - 103 Improvement of carotenoid-synthesizing yeast Rhodotorula rubra by chemical mutagenesis; Frengova GI et al.; A mutant Rhodotorula rubra with enhanced carotenoid-synthesizing activity for synthesizing total carotenoids and beta-carotene was obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis . When co-cultivated with yogurt starter bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus) in whey ultrafiltrate it produced 15.7 mg total carotenoids l(-1) culture fluid or 946 microg total carotenoids g(-1) dry cells of which 71% was beta-carotene . Grown as a monoculture in glucose substrate, the mutant shown 1.4 times lower carotenoid-synthesizing activity, and the relative share of beta-carotene in the total carotenoids was lower (63%) . The individual pigments torulene and torularhodin were identified, whose mass fractions were (29% and 7%) and (24% and 4%), respectively, for the mutant grown as a monoculture and as a mixed culture with the yogurt bacteria. Virology, 2004 Mar 15, 320(2), 229 - 42 The prophages of Lactobacillus johnsonii NCC 533: comparative genomics and transcription analysis; Ventura M et al.; Two non-inducible, but apparently complete prophages were identified in the genome of the sequenced Lactobacillus johnsonii strain NCC 533 . The 38- and 40-kb-long prophages Lj928 and Lj965 represent distinct lineages of Sfi11-like pac-site Siphoviridae unrelated at the DNA sequence level . The deduced structural proteins from Lj928 demonstrated aa sequence identity with Lactococcus lactis phage TP901-1, while Lj965 shared sequence links with Streptococcus thermophilus phage O1205 . With the exception of tRNA genes, inserted between DNA replication and DNA packaging genes, the transcription of the prophage was restricted to the genome segments near both attachment sites . Transcribed genes unrelated to phage functions were inserted between the phage repressor and integrase genes; one group of genes shared sequence relatedness with a mobile DNA element in Staphylococcus aureus . A short, but highly transcribed region was located between the phage lysin and right attachment site; it lacked a protein-encoding function in one prophage. Structure (Camb), 2004 Mar, 12(3), 361 - 70 Coenzyme binding in F420-dependent secondary alcohol dehydrogenase, a member of the bacterial luciferase family; Aufhammer SW et al.; F(420)-dependent secondary alcohol dehydrogenase (Adf) from methanogenic archaea is a member of the growing bacterial luciferase family which are all TIM barrel enzymes, most of which with an unusual nonprolyl cis peptide bond . We report here on the crystal structure of Adf from Methanoculleus thermophilicus at 1.8 A resolution in complex with a F(420)-acetone adduct . The knowledge of the F(420) binding mode in Adf provides the molecular basis for modeling F(420) and FMN into the other enzymes of the family . A nonprolyl cis peptide bond was identified as an essential part of a bulge that serves as backstop at the Re-face of F(420) to keep it in a bent conformation . The acetone moiety of the F(420)-acetone adduct is positioned at the Si-face of F(420) deeply buried inside the protein . Isopropanol can be reliably modeled and a hydrogen transfer mechanism postulated . His39 and Glu108 can be identified as key players for binding of the acetone or isopropanol oxygens and for catalysis. Biochem J, 2004 Jun 15, 380(Pt 3), 677 - 84 Theoretical model of the three-dimensional structure of a sugar-binding protein from Pyrococcus horikoshii: structural analysis and sugar-binding simulations; Marabotti A et al.; The three-dimensional structure of a sugar-binding protein from the thermophilic archaea Pyrococcus horikoshii has been predicted by a homology modelling procedure and investigated for its stability and its ability to bind different sugars . The model was created by using as templates the three-dimensional structures of a maltodextrin-binding protein from Pyrococcus furiosus, a trehalose-maltose-binding protein from Thermococcus litoralis and a maltodextrin-binding protein from Escherichia coli . According to the suggestions from the CASP (Critical Assessment of Structure Prediction) meetings, the homology modelling strategy was applied by assessing an accurate multiple sequence alignment, based on the high structural conservation in the family of ATP-binding cassette transporters to which all these proteins belong . The model has been deposited in the Protein Data Bank with the code 1R25 . According to the origin of the protein, several characteristics in the organization of the secondary-structure elements and in the distribution of polar and non-polar amino acids are very similar to those of thermophilic proteins, compared with proteins from mesophilic organisms, and are analysed in detail . Finally, a simulation of the binding of several sugars in the binding site of this protein is presented, and interactions with amino acids are highlighted in detail. Mol Biol Evol, 2004 Jul, 21(7), 1242 - 51 Epub 2004 Mar 10. Coenzyme A biosynthesis: reconstruction of the pathway in archaea and an evolutionary scenario based on comparative genomics; Genschel U; Coenzyme A (CoA) holds a central position in cellular metabolism and therefore can be assumed to be an ancient molecule . Starting from the known E . coli and human enzymes required for the biosynthesis of CoA, phylogenetic profiles and chromosomal proximity methods enabled an almost complete reconstruction of archaeal CoA biosynthesis . This includes the identification of strong candidates for archaeal pantothenate synthetase and pantothenate kinase, which are unrelated to the corresponding bacterial or eukaryotic enzymes . According to this reconstruction, the topology of CoA synthesis from common precursors is essentially conserved across the three domains of life . The CoA pathway is conserved to varying degrees in eukaryotic pathogens like Giardia lamblia or Plasmodium falciparum, indicating that these pathogens have individual uptake-mechanisms for different CoA precursors . Phylogenetic analysis and phyletic distribution of the CoA biosynthetic enzymes suggest that the enzymes required for the synthesis of phosphopantothenate were recruited independently in the bacterial and archaeal lineages by convergent evolution, and that eukaryotes inherited the genes for the synthesis of pantothenate (vitamin B5) from bacteria . Homologues to bacterial enzymes involved in pantothenate biosynthesis are present in a subset of archaeal genomes . The phylogenies of these enzymes indicate that they were acquired from bacterial thermophiles through horizontal gene transfer . Monophyly can be inferred for each of the enzymes catalyzing the four ultimate steps of CoA synthesis, the conversion of phosphopantothenate into CoA . The results support the notion that CoA was initially synthesized from a prebiotic precursor, most likely pantothenate or a related compound. Mutat Res, 2004 Mar 22, 547(1-2), 41 - 7 Directly fishing out subtle mutations in genomic DNA with histidine-tagged Thermus thermophilus MutS; Wang J et al.; Tth MutS, a mismatch repair protein from Thermus thermophilus, was reported to effectively recognize all eight possible types of base pair mismatches and insertions or deletions up to three base pairs at a wide temperature range up to 60 degrees C . Here a procedure for directly fishing out subtle unknown mutations in bacterial genome with Tth MutS was described . Wild type genomic DNA and mutant one were mixed, digested with restriction enzymes, denatured and re-annealed . Hetero-duplex DNA carrying mispaired bases were bound to Tth MutS and recovered through Ni-NTA His-Bind((R)) Resin . The recovered DNA was cloned into plasmids, producing a mini-library with inserts of the mutated regions . Further DNA sequencing and genetic complementation demonstrated that the method was extremely efficient in fishing out the mutations from total genomic DNA . Using this method, the mutations existed in a Psedomonas aeruginosa mutant strain were screened, indicating that A/G transitions at nt 181 and nt 314 in chloramphenicol acetyltransferase (catB7) gene conferred this strain with a high chloramphenicol dosage resistant . Compared with those reported previously, this protocol can screen the mixed mutations more easily. J Appl Microbiol, 2004, 96(4), 810 - 8 An Aneurinibacillus sp . strain AM-1 produces a proline-specific aminopeptidase useful for collagen degradation; Murai A et al.; AIMS: We have been for a species of thermophilic bacteria that can effectively decompose collagen and collagen peptides that tend to be hard-to-degrade proteins because of their high content of proline residues . This study focused upon the enzymatic degradation of prolyl peptides by thermophilic bacteria . METHODS AND RESULTS: A strain, AM-1, producing a proline-specific aminopeptidase was isolated using a medium containing gelatin that was taken from soil samples collected at Arima Hot Spring located near Kobe, Japan . The strain showed the strongest level of hydrolysing activity toward prolyl-p-nitroanilide, and the activity proved to be thermostable . Phylogenetic analysis based on 16S rDNA sequences revealed that the isolated strain AM-1 was closest to Aneurinibacillus thermoaerophilus DSM10154T in its characteristics . Analysis of the purified proline-specific aminopeptidase suggested that the enzyme is an aminopeptidase containing metal that includes important disulphide bond(s) . The strain AM-1 aminopeptidase has more similarities with leucyl aminopeptidases, but its activity level differs greatly with prolyl peptides . CONCLUSIONS: The proline-specific aminopeptidase from strain AM-1 is the first from the genus Aneurinibacillus and may be a new type of aminopeptidase for hydrolysing prolyl peptide . This enzyme also contributed to the degradation of collagen when used in combination with another collagenolytic protease . SIGNIFICANCE AND IMPACT OF THE STUDY: The proline-specific aminopeptidase obtained from strain AM-1 may be used in the treatment of wastewater containing collagen that is encountered in the meat industries, and for decreasing bitter peptides in milk products. J Appl Microbiol, 2004, 96(4), 641 - 7 Microbiological monitoring in the biodegradation of sewage sludge and food waste; Ivanov VN et al.; AIM: To study the microbiology of intensive, in-vessel biodegradation of a mixture of sewage sludge and vegetable food waste . METHODS AND RESULTS: The biodegradation was performed in a closed reactor with the addition of a starter culture of Bacillus thermoamylovorans SW25 under conditions of controlled aeration, stirring, pH and temperature (60 degrees C) . The content of viable bacterial cells, determined by flow cytometry, increased from 5 x 108 g-1 of dry matter to 61 x 108 g-1 for 6 days of the process and then dropped to the initial value at the end of the process . The reductions of organic matter, 16S rRNA of methanogens and coenzyme F420 fluorescence during 10 days of the treatment were 67, 54 and 87% of the initial values, respectively . The biodegradability of the organic matter decreased during the 10 days of the treatment from 3.8 to 1.3 mg CO2 g-1 of organic matter per day . The treatment of sewage sludge and food waste at 60 degrees C did not remove enterobacteria, which are the agents of intestinal infections, from the material . The percentage of viable enterobacterial cells, determined by fluorescent in situ hybridization (FISH) with Enterobacteriaceae-specific oligonucleotide probe and flow cytometry, varied from 1 to 14% of the viable bacterial cells . CONCLUSIONS: The mixture of sewage sludge and food waste can be degraded by the aerobic thermophilic bacteria; the starter culture of Bacillus thermoamylovorans SW25 can be used to perform this process; and enterobacteria can survive under treatment of sewage sludge and food waste at 60 degrees C for 13 days . SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that FISH with an oligonucleotide probe can be used to study not only the growth but also the degradation of biomass . Obtained results could be used to design the bioconversion of sewage sludge and food waste into organic fertilizer. Genet Mol Res, 2003 Dec 30, 2(4), 383 - 93 Preferred amino acids and thermostability; Farias ST et al.; Most organisms grow at temperatures from 20 to 50 degrees C, but some prokaryotes, including Archaea and Bacteria, are capable of withstanding higher temperatures, from 60 to >100 degrees C . Their biomolecules, especially proteins, must be sufficiently stable to function under these extreme conditions; however, the basis for thermostability remains elusive . We investigated the preferential usage of certain groupings of amino acids and codons in thermally adapted organisms, by comparative proteome analysis, using 28 complete genomes from 18 mesophiles (M), 4 thermophiles (T), and 6 hyperthermophiles (HT) . Whenever the percent of glutamate (E) and lysine (K) increased in the HT proteomes, the percent of glutamine (Q) and histidine (H) decreased, so that the E + K/Q + H ratio was >4.5; it was <2.5 in the M proteomes, and 3.2 to 4.6 in T . The E + K/Q + H ratios for chaperonins, potentially thermostable proteins, were higher than their proteome ratios, whereas for DNA ligases, which are not necessarily thermostable, they followed the proteome ratios . Analysis of codon usage revealed that HT had more AGR codons for Arg than they did CGN codons, which were more common in mesophiles . The E + K/Q + H ratio may provide a useful marker for distinguishing HT, T and M prokaryotes, and the high percentage of the amino acid couple E + K, consistently associated with a low percentage of the pair Q + H, could contribute to protein thermostability . The preponderance of AGR codons for Arg is a signature of all HT so far analyzed . The E + K/Q + H ratio and the codon bias for Arg are apparently not related to phylogeny . HT members of the Bacteria show the same values as the HT members of the Archaea; the values for T organisms are related to their lifestyle (intermediate temperature) and not to their domain (Archaea) and the values for M are similar in Eukarya, Bacteria and Archaea. Appl Microbiol Biotechnol, 2004 Oct, 65(5), 600 - 5 Epub 2004 Mar 06. Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485; Desai SG et al.; The gene encoding L-lactate dehydrogenase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned, sequenced, and used to obtain an L-ldh deletion mutant strain (TD1) following a site-specific double-crossover event as confirmed by PCR and Southern blot . Growth rates and final cell densities were similar for strain TD1 and the wild-type grown on glucose and xylose . Lactic acid was below the limit of detection (0.3 mM) for strain TD1 on both glucose and xylose at all times tested, but was readily detected for the wild-type strain, with average final concentrations of 8.1 and 1.8 mM on glucose and xylose, respectively . Elimination of lactic acid as a fermentation product was accompanied by a proportional increase in the yields of acetic acid and ethanol . The results reported here represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids, and support the methodological feasibility of this goal. Appl Environ Microbiol, 2004 Mar, 70(3), 1858 - 64 ThyA as a selection marker in construction of food-grade host-vector and integration systems for Streptococcus thermophilus; Sasaki Y et al.; We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker . Two thyA genes, thyA(St) and thyA(Lb), were cloned from S . thermophilus and Lactobacillus delbrueckii subsp . bulgaricus, respectively . Thymidine-requiring mutants of S . thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host . Food-grade vectors were constructed by using either thyA(St) or thyA(Lb) as the selection marker . Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance . By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA(Lb) as the selection marker via a double-crossover event . The results obtained show that thyA is an efficient and safe selection marker for S . thermophilus that is suitable for food applications. Appl Environ Microbiol, 2004 Mar, 70(3), 1735 - 43 Antisense RNA targeting of primase interferes with bacteriophage replication in Streptococcus thermophilus; Sturino JM et al.; The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages . Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S . thermophilus phage kappa 3 provided significant protection from kappa 3 and two other Sfi21-type phages . Expression of pri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of kappa 3 to 3 x 10(-3) and reduced its burst size by 20% . Mutant phages capable of overcoming antisense inhibition were not recovered . Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene . Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 x 10(-3) . The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5' region of the gene . The impacts of primase-targeted antisense RNAs on phage development were examined . The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized . Targeting a key genome replication function with antisense RNA provided effective phage protection in S . thermophilus. Appl Environ Microbiol, 2004 Mar, 70(3), 1570 - 5 Characteristics of a new enantioselective thermostable dipeptidase from Brevibacillus borstelensis BCS-1 and its application to synthesis of a D-amino-acid-containing dipeptide; Baek DH et al.; A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the D-Glu auxotroph Escherichia coli WM335 on a plate containing D-Ala-D-Glu . Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M(r) of 35,000 . The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes . The enzyme was purified to homogeneity from recombinant E . coli WM335 harboring the dipeptidase gene from B . borstelensis BCS-1 . Investigation of the enantioselectivity (E) to the P(1) and P(1)' site of Ala-Ala revealed that the ratio of the specificity constant (k(cat)/K(m)) for L-enantioselectivity to the P(1) site of Ala-Ala was 23.4 +/- 2.2 {E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(D,D)}, while the D-enantioselectivity to the P(1)' site of Ala-Ala was 16.4 +/- 0.5 {E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(L,L)} at 55 degrees C . The enzyme was stable up to 55 degrees C, and the optimal pH and temperature were 8.5 and 65 degrees C, respectively . The enzyme was able to hydrolyze L-Asp-D-Ala, L-Asp-D-AlaOMe, Z-D-Ala-D-AlaOBzl, and Z-L-Asp-D-AlaOBzl, yet it could not hydrolyze D-Ala-L-Asp, D-Ala-L-Ala, D-AlaNH(2), and L-AlaNH(2.) The enzyme also exhibited beta-lactamase activity similar to that of a human renal dipeptidase . The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-L-Asp-D-AlaOBzl. Biotechnol Lett, 2004 Jan, 26(1), 45 - 9 Isolation of a new thermohalophilic Thermus thermophilus strain from hot spring, able to grow on a renewable source of polysaccharide; Romano I et al.; A thermohalophilic strain, Samu-Sal, isolated from hot springs of the Mount Grillo (Baia, Naples, Italy) at a depth of 60 m, according to its genotypic analyses is related to Thermus genus and should be classified as a new strain of Thermus thermophilus . Strain Samu-SA1 grew using, as sole carbon source, a polysaccharide extracted from waste industrial tomato process with a yield of 3.5 g l(-1) . Strain Samu-SA1 synthesized several alpha- and beta-glycosidases. Biotechnol Lett, 2004 Jan, 26(1), 15 - 9 Microbial degradation of poly(D-3-hydroxybutyrate) by a new thermophilic streptomyces isolate; Calabia BP et al.; A new thermophilic microorganism capable of degrading poly(D-3-hydroxybutyrate) (PHB) was isolated from soil . A phylogenetic analysis based on 16S rDNA sequences indicated that the new isolate belongs to genus Streptomyces . PHB film and powder were completely degraded after 6 and 3 d cultivation, respectively at 50 degrees C . Scanning micrographs showed adherence of the microbial cells to the entire film surface, indicating that biodegradation occurs by colonization of the PHB surface . The film was degraded both by microbial attack and by the action of an extracellular enzyme secreted by the microorganism . The strain can also degrade poly(ethylene succinate), poly(ester carbonate), polycaprolactone and poly(butylene succinate), but to a lesser extent. Biochemistry (Mosc), 2004 Feb, 69(2), 154 - 63 Effect of nucleotide replacements in tRNAPhe on positioning of the acceptor end in the complex with phenylalanyl-tRNA synthetase; Vasil'eva IA et al.; The effect of replacement of tRNA(Phe) recognition elements on positioning of the 3'-terminal nucleotide in the complex with phenylalanyl-tRNA synthetase (PheRS) from T . thermophilus in the absence or presence of phenylalanine and/or ATP has been studied by photoaffinity labeling with s(4)U76-substituted analogs of wild type and mutant tRNA(Phe) . The double mutation G34C/A35U shows the strongest disorientation in the absence of low-molecular-weight substrates and sharply decreases the protein labeling, which suggests an initiating role of the anticodon in generation of contacts responsible for the acceptor end positioning . Efficiency of photo-crosslinking with the alpha- and beta-subunits in the presence of individual substrates is more sensitive to nucleotide replacements in the anticodon (G34 by A or A36 by C) than to changes in the general structure of tRNA(Phe) (as a result of replacement of the tertiary pair G19-C56 by U19-G56 or of U20 by A) . The degree of disorders in the 3'-terminal nucleotide positioning in the presence of both substrates correlates with decrease in the turnover number of aminoacylation due to corresponding mutations . The findings suggest that specific interactions of the enzyme with the anticodon mainly promote the establishment (controlled by phenylalanine) of contacts responsible for binding of the CCA-end and terminal nucleotide in the productive complex, and the general conformation of tRNA(Phe) determines, first of all, the acceptor stem positioning (controlled by ATP) . The main recognition elements of tRNA(Phe), which optimize its initial binding with PheRS, are also involved in generation of the catalytically active complex providing functional conformation of the acceptor arm. Biochemistry (Mosc), 2004 Feb, 69(2), 143 - 53 Role of low-molecular-weight substrates in functional binding of the tRNAPhe acceptor end by phenylalanyl-tRNA synthetase; Vasil'eva IA et al.; The functional roles of phenylalanine and ATP in productive binding of the tRNA(Phe) acceptor end have been studied by photoaffinity labeling (cross-linking) of T . thermophilus phenylalanyl-tRNA synthetase (PheRS) with tRNA(Phe) analogs containing the s(4)U residue in different positions of the 3'-terminal single-stranded sequence . Human and E . coli tRNA(Phe)s used as basic structures differ by efficiency of the binding and aminoacylation with the enzyme under study . Destabilization of the complex with human tRNA(Phe) caused by replacement of three recognition elements decreases selectivity of labeling of the alpha- and beta-subunits responsible for the binding of adjacent nucleotides of the CCA-end . Phenylalanine affects the positioning of the base and ribose moieties of the 76th nucleotide, and the recorded effects do not depend on structural differences between bacterial and eukaryotic tRNA(Phe)s . Both in the absence and presence of phenylalanine, ATP more effectively inhibits the PheRS labeling with the s(4)U76-substituted analog of human tRNA(Phe) (tRNA(Phe)-s(4)U76) than with E . coli tRNA(Phe)-s(4)U76: in the first case the labeling of the alpha-subunits is inhibited more effectively; the labeling of the beta-subunits is inhibited in the first case and increased in the second case . The findings analyzed with respect to available structural data on the enzyme complexes with individual substrates suggest that the binding of phenylalanine induces a local rearrangement in the active site and directly controls positioning of the tRNA(Phe) 3'-terminal nucleotide . The effect of ATP on the acceptor end positioning is caused by global structural changes in the complex, which modulate the conformation of the acceptor arm . The rearrangement of the acceptor end induced by small substrates results in reorientation of the 3'-OH-group of the terminal ribose from the catalytic subunit onto the noncatalytic one, and this may explain the unusual stereospecificity of aminoacylation in this system. Proteins, 2004 Mar 1, 54(4), 693 - 704 Molecular modeling study of the editing active site of Escherichia coli leucyl-tRNA synthetase: two amino acid binding sites in the editing domain; Lee KW et al.; Aminoacyl-tRNA synthetases (aaRSs) strictly discriminate their cognate amino acids . Some aaRSs accomplish this via proofreading and editing mechanisms . Mursinna and coworkers recently reported that substituting a highly conserved threonine (T252) with an alanine within the editing domain of Escherichia coli leucyl-tRNA synthetase (LeuRS) caused LeuRS to cleave its cognate aminoacylated leucine from tRNA(Leu) (Mursinna et al., Biochemistry 2001;40:5376-5381) . To achieve atomic level insight into the role of T252 in LeuRS and the editing reaction of aaRSs, a series of molecular modeling studies including homology modeling and automated docking simulations were carried out . A 3D structure of E . coli LeuRS was constructed via homology modeling using the X-ray structure of Thermus thermophilus LeuRS as a template because the E . coli LeuRS structure is not available from X-ray or NMR studies . However, both the X-ray T . thermophilus and homology-modeled E . coli structures were used in our studies . Amino acid binding sites in the proposed editing domain, which is also called the connective polypeptide 1 (CP1) domain, were investigated by automated docking studies . The root mean square deviation (RMSD) for backbone atoms between the X-ray and homology-modeled structures was 1.18 A overall and 0.60 A for the editing (CP1) domain . Automated docking studies of a leucine ligand into the editing domain were performed for both structures: homology structure of E . coli LeuRS and X-ray structure of T . thermophilus LeuRS for comparison . The results of the docking studies suggested that there are two possible amino acid binding sites in the CP1 domain for both proteins . The first site lies near a threonine-rich region that includes the highly conserved T252 residue, which is important for amino acid discrimination . The second site is located in a flexible loop region surrounded by residues E292, A293, M295, A296, and M298 . The important T252 residue is at the bottom of the first binding pocket . Proteins, 2004 Mar 1, 54(4), 648 - 56 Missense mutations in transmembrane domains of proteins: phenotypic propensity of polar residues for human disease; Partridge AW et al.; Previous experiments on the cystic fibrosis transmembrane conductance regulator suggested that non-native polar residues within membrane domains can compromise protein structure/function . However, depending on context, replacement of a native residue by a non-native residue can result either in genetic disease or in benign effects (e.g., polymorphisms) . Knowledge of missense mutations that frequently cause protein malfunction and subsequent disease can accordingly reveal information as to the impact of these residues in local protein environments . We exploited this concept by performing a statistical comparison of disease-causing mutations in protein membrane-spanning domains versus soluble domains . Using the Human Gene Mutation Database of 240 proteins (including 80 membrane proteins) associated with human disease, we compared the relative phenotypic propensity to cause disease of the 20 naturally occurring amino acids when removed from-or inserted into-native protein sequences . We found that in transmembrane domains (TMDs), mutations involving polar residues, and ionizable residues in particular (notably arginine), are more often associated with protein malfunction than soluble proteins . To further test the hypothesis that interhelical cross-links formed by membrane-embedded polar residues stabilize TMDs, we compared the occurrence of such residues in the TMDs of mesophilic and thermophilic prokaryotes . Results showed a significantly higher proportion of ionizable residues in thermophilic organisms, reinforcing the notion that membrane-embedded electrostatic interactions play critical roles in TMD stability . Proteins, 2004 Mar 1, 54(4), 616 - 21 Extreme free energy of stabilization of Taq DNA polymerase; Schoeffler AJ et al.; We have examined the chemical denaturations of the Klentaq and Klenow large-fragment domains of the Type 1 DNA polymerases from Thermus aquaticus (Klentaq) and Escherichia coli (Klenow) under identical solution conditions in order to directly compare the stabilization energetics of the two proteins . The high temperature stability of Taq DNA polymerase is common knowledge, and is the basis of its use in the polymerase chain reaction . This study, however, is aimed at understanding the thermodynamic basis for this high-temperature stability . Chemical denaturations with guanidine hydrochloride report a folding free energy (DeltaG) for Klentaq that is over 20 kcal/mol more favorable than that for Klenow under the conditions examined . This difference between the stabilization free energies of a homologous mesophilic-thermophilic protein pair is significantly larger than generally observed . This is due in part to the fact that the stabilization free energy for Klentaq polymerase, at 27.5 kcal/mol, is one of the largest ever determined for a monomeric protein . Large differences in the chemical midpoints of the unfolding (Cm) and the dependences of the unfolding free energy on denaturant concentration in the transition region (m-value) between the two proteins are also observed . Measurements of the sedimentation coefficients of the two proteins in the native and denatured states report that both proteins approximately double in hydrodynamic size upon denaturation, but that Klentaq expands somewhat more than Klenow . Chembiochem, 2004 Mar 5, 5(3), 280 - 90 Different roles of electrostatics in heat and in cold: adaptation by citrate synthase; Kumar S et al.; Electrostatics plays a major role in heat adaptation by thermophilic proteins . Here we ask whether electrostatics similarly contributes to cold adaptation in psychrophilic proteins . We compare the sequences and structures of citrate synthases from the psychrophile Arthobacter Ds2-3R, from chicken, and from the hyperthermophile Pyrococcus furiosus . The three enzymes share similar packing, burial of nonpolar surface area, and main-chain hydrogen bonding . However, both psychrophilic and hyperthermophilic citrate synthases contain more charged residues, salt bridges, and salt-bridge networks than the mesophile . The electrostatic free-energy contributions toward protein stability by individual charged residues show greater variabilities in the psychrophilic citrate synthase than in the hyperthermophilic enzyme . The charged residues in the active-site regions of the psychrophile are more destabilizing than those in the active-site regions of the hyperthermophile . In the hyperthermophilic enzyme, salt bridges and their networks largely cluster in the active-site regions and at the dimer interface . In contrast, in the psychrophile, they are more dispersed throughout the structure . On average, salt bridges and their networks provide greater electrostatic stabilization to the thermophilic citrate synthase at 100 degrees C than to the psychrophilic enzyme at 0 degrees C . Electrostatics appears to play an important role in both heat and cold adaptation of citrate synthase . However, remarkably, the role may be different in the two types of enzyme: In the hyperthermophile, it may contribute to the integrity of both the protein dimer and the active site by possibly countering conformational disorder at high temperatures . On the other hand, in the psychrophile at low temperatures, electrostatics may contribute to enhance protein solvation and to ensure active-site flexibility. J Ind Microbiol Biotechnol, 2004 Mar, 31(3), 115 - 21 Epub 2004 Mar 03. Synthesis of carotenoids by Rhodotorula rubra GED8 co-cultured with yogurt starter cultures in whey ultrafiltrate; Simova ED et al.; Two cultures, a yeast ( Rhodorula rubra GED8) and a yogurt starter ( Lactobacillus bulgaricus 2-11+ Streptococcus thermophilus 15HA), were selected for associated growth in whey ultrafiltrate (WU) and active synthesis of carotenoids . In associated cultivation with the yogurt culture L bulgaricus 2-11+S . thermophilus 15HA under intensive aeration (1.3 l(-1)min(-1) air-flow rate) in WU (45 g lactose l(-1)), initial pH 5.5, 30 degrees C, the lactose-negative strain R . rubra GED8 synthesized large amounts of carotenoids (13.09 mg l(-1 )culture fluid) . The carotenoid yield was approximately two-fold higher in association with a mixed yogurt culture than in association with pure yogurt bacteria . The major carotenoid pigments comprising the total carotenoids were beta-carotene (50%), torulene (12.3%) and torularhodin (35.2%) . Carotenoids with a high beta-carotene content were produced by the microbial association 36 h earlier than by Rhodotorula yeast species . No significant differences were notd in the ratio between the pigments synthesized by R . rubra GED8+ L . bulgaricus 2-11, R . rubra GED8+ S . thermophilus 15HA, and R.rubra GED8+yogurt culture, despite the fact that the total carotenoid concentrations were lower in the mixed cultures with pure yogurt bacteria. Appl Microbiol Biotechnol, 2004 Jun, 64(5), 605 - 10 Epub 2004 Feb 28. Occurrence, biochemistry and possible biotechnological application of the 3-hydroxypropionate cycle; Ishii M et al.; The 3-hydroxypropionate cycle, a pathway for autotrophic carbon dioxide fixation, is reviewed with special emphasis on the biochemistry of CO2 fixing enzymes in Acidianus brierleyi, a thermophilic and acidophilic archeon . In the 3-hydroxypropionate cycle, two enzymes, acetyl-CoA carboxylase and propionyl-CoA carboxylase, catalyze CO2 fixation . It has been shown in A . brierleyi, and subsequently in Metallosphaera sedula, that acetyl-CoA carboxylase is promiscuous, acting equally well on acetyl-CoA and propionyl-CoA . The subunit structure of the acyl-CoA carboxylase was shown to be alpha4beta4gamma4 . Gene cloning revealed that the genes encoding the three subunits are adjacent to each other . accC encodes the beta-subunit (59 kDa subunit, biotin carboxylase subunit), accB encodes the gamma-subunit (20 kDa subunit, biotin carboxyl carrier protein), and pccB encodes the alpha-subunit (62 kDa subunit, carboxyltransferase subunit) . Sequence analyses showed that accC and accB are co-transcribed and that pccB is transcribed separately . Potential biotechnological applications for the 3-hydroxypropionate cycle are also presented. Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 574 - 6 Epub 2004 Feb 25. Expression, purification and preliminary X-ray characterization of histidinol phosphate phosphatase; Omi R et al.; Histidinol phosphate phosphatase (HisPPase) catalyzes the eighth step of histidine biosynthesis, in which L-histidinol phosphate undergoes dephosphorylation to give histidinol . A recombinant form of the histidinol phosphate phosphatase from Thermus thermophilus HB8 has been expressed in Escherichia coli, purified and crystallized in two crystal forms by the hanging-drop vapour-diffusion technique . Crystal form I belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 84.8, b = 97.2, c = 74.9 A, and crystal form II belongs to the orthorhombic space group C222(1), with unit-cell parameters a = 76.9, b = 157.6, c = 116.7 A . The crystals probably contain two monomers in the asymmetric unit, with V(M) values of 2.57 A(3) Da(-1) for form I and 2.96 A(3) Da(-1) for form II . X-ray data have been collected to 1.70 and 1.75 A resolution for crystal forms I and II, respectively. Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 515 - 7 Epub 2004 Feb 25. Crystallization and preliminary X-ray crystallographic studies of the small form of glucose-inhibited division protein A from Thermus thermophilus HB8; Iwasaki W et al.; Glucose-inhibited division protein A (GidA) acts in tRNA modification . It has been suggested that GidA is involved in the biosynthesis of the hypermodified nucleotide 5-methylaminomethyl-2-thiouridine in the wobble position of bacterial tRNAs, which stabilizes codon-anticodon interactions . Thermus thermophilus HB8 has a putative small gidA gene in addition to the normal gidA gene . The crystallization and preliminary X-ray crystallographic studies of the product of this small gidA gene (GidA(small)) are reported here . The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 78.51, c = 66.10 A and one monomer per asymmetric unit . The crystals were found to diffract X-rays to beyond 1.65 A resolution. Biochemistry, 2004 Mar 9, 43(9), 2533 - 40 The ionic track in the F1-ATPase from the thermophilic Bacillus PS3; Bandyopadhyay S et al.; Only beta-beta cross-links form when the alpha(3)(betaE(395)C)(3)gammaK(36)C (MF(1) residue numbers) double mutant subcomplex of TF(1), the F(1)-ATPase from the thermophilic Bacillus PS3, is slowly inactivated with CuCl(2) in the presence or absence of MgATP . The same slow rate of inactivation and extent of beta-beta cross-linking occur upon treatment of the alpha(3)(betaE(395)C)(3)gamma single mutant subcomplex with CuCl(2) under the same conditions . In contrast, the alpha(3)(betaE(395)C)(3)gammaR(33)C and alpha(3)(betaE(395)C)(3)gammaR(75)C double mutant subcomplexes of TF(1) are rapidly inactivated by CuCl(2) under the same conditions that is accompanied by complete beta-gamma cross-linking . The ATPase activity of each mutant enzyme containing the betaE(395)C substitution is stimulated to a much greater extent by the nonionic detergent lauryldimethylamine oxide (LDAO) than wild-type enzyme, whereas the ATPase activities of the gammaR(33)C, gammaK(36)C, and gammaR(75)C single mutants are stimulated to about the same extent as wild-type enzyme by LDAO . This indicates that the E(395)C substitution in the (394)DELSEED(400) segment of beta subunits increases propensity of the enzyme to entrap inhibitory MgADP in a catalytic site during turnover . These results are discussed in perspective with (i) the ionic track predicted from molecular dynamics simulations to operate during energy-driven ATP synthesis by MF(1), the F(1)-ATPase from bovine heart mitochondria {Ma, J., Flynn, T . C., Cui, Q., Leslie, A . G . W., Walker, J . E., and Karplus, M . (2002) Structure 10, 921-931}; and (ii) the possibility that the betaE(395)C substitution might induce a global effect that alters affinity of noncatalytic sites for nucleotides or alters communication between noncatalytic sites and catalytic sites during ATP hydrolysis. Extremophiles, 2004 Jun, 8(3), 209 - 17 Epub 2004 Feb 27. Transcriptional analysis of dynamic heat-shock response by the hyperthermophilic bacterium Thermotoga maritima; Pysz MA et al.; The thermal stress response of the hyperthermophilic bacterium Thermotoga maritima was characterized using a 407-open reading frame-targeted cDNA microarray . Transient gene expression was followed for 90 min, following a shift from 80 degrees C to 90 degrees C . While some aspects of mesophilic heat-shock response were conserved in T . maritima, genome content suggested differentiating features that were borne out by transcriptional analysis . Early induction of predicted heat-shock operons hrcA-grpE-dnaJ (TM0851-TM0850-TM0849), groES-groEL (TM0505-TM0506), and dnaK-sHSP (TM0373-TM0374) was consistent with conserved CIRCE elements upstream of hrcA and groES . Induction of the T . maritima rpoE/ sigW and rpoD/ sigA homologs suggests a mechanism for global heat-shock response in the absence of an identifiable ortholog to a major heat-shock sigma factor . In contrast to heat-shock response in Escherichia coli, the majority of genes encoding ATP-dependent proteases were downregulated, including clpP (TM0695), clpQ (TM0521), clpY (TM0522), lonA (TM1633), and lonB (TM1869) . Notably, T . maritima showed indications of a late heat-shock response with the induction of a marR homolog (TM0816), several other putative transcriptional regulators (TM1023, TM1069), and two alpha-glucosidases (TM0434 and TM1068) . Taken together, the results reported here indicate that, while T . maritima shares core elements of the bacterial heat-shock response with mesophiles, the thermal stress regulatory strategies of this organism differ significantly . However, it remains to be elucidated whether these differences are related to thermophilicity or phylogenetic placement . Nucleic Acids Res, 2004 Feb 27, 32(4), 1439 - 47 Print 2004. A bipolar DNA helicase gene, herA, clusters with rad50, mre11 and nurA genes in thermophilic archaea; Constantinesco F et al.; We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon-like structure with a third gene (nurA) encoding a 5' to 3' exonuclease . Here, we show that the rad50, mre11 and nurA genes from the hyperthermophilic archaeon Sulfolobus acidocaldarius are co-transcribed with a fourth gene encoding a DNA helicase . This enzyme (HerA) is the prototype of a new class of DNA helicases able to utilize either 3' or 5' single-stranded DNA extensions for loading and subsequent DNA duplex unwinding . To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously . Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases . HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together . These results suggest that the archaeal Rad50-Mre11 complex might act in association with a 5' to 3' exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous recombination. J Biol Chem, 2004 May 28, 279(22), 22809 - 19 Epub 2004 Feb 29. Biosynthetic Ca2+/Sr2+ exchange in the photosystem II oxygen-evolving enzyme of Thermosynechococcus elongatus; Boussac A et al.; The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+ . Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures . A protocol was developed to purify a fully active Sr(2+)-containing photosystem II (PSII) . The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese . The Sr(2+)- and Ca(2+)-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography . The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII . This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves . In addition, the Sr(2+)-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD* is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ* to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr(2+)-substituted enzyme as well as in the normal enzyme . The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects. Plant Cell Physiol, 2004 Feb, 45(2), 171 - 5 Improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1; Iwai M et al.; We improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by combining electroporation with a top agar method . Transformation was also improved when a disruptant of a putative type I restriction endonuclease (tll2230) was used as recipient cells . In particular, some constructs, with which wild type has never been transformed, were successfully integrated into the tll2230-disruptant . Single-crossover recombination was detected more frequently than the double-crossover recombination . In accordance with the presence of all the homologs of pil genes in Synechocystis sp . PCC 6803, we found that T . elongatus is naturally transformable with exogenous DNA. Bioinformatics, 2004 Aug 12, 20(12), 1861 - 9 Epub 2004 Feb 26. Second eigenvalue of the Laplacian matrix for predicting RNA conformational switch by mutation; Barash D; MOTIVATION: Conformational switching in RNAs is thought to be of fundamental importance in several biological processes, including translational regulation, regulation of self-cleavage in viruses, protein biosynthesis and mRNA splicing . Current methods for detecting bi-stable RNAs that can lead to structural switching when triggered by an outside event rely on kinetics, energetics and properties of the combinatorial structure space of RNAs . Based on these properties, tools have been developed to predict whether a given sequence folds to a structure characterized by a bi-stable conformation, or to design multi-stable RNAs by an iterative algorithm . A useful addition is in developing a local procedure to prescribe, given an initial sequence, the least amount of mutations needed to drive the system into an optimal b