Thermophiles

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 bi-stable conformation . RESULTS: We introduce a local procedure for predicting mutations, by generating and analyzing eigenvalue tables, that are capable of transforming the wild-type sequence into a bi-stable conformation . The method is independent of the folding algorithms but relies on their success . It can be used in conjunction with existing tools, as well as being incorporated into more general RNA prediction packages . We apply this procedure on three well-studied structures . First, the method is validated on the mutation leading to a conformational switch in the spliced leader RNA from Leptomonas collosoma, a mutation that has already been confirmed by an experiment . Second, the method is used to predict a mutation that can lead to a novel conformational switch in the P5abc subdomain of the group I intron ribozyme in Tetrahymena thermophila . Third, the method is applied on Hepatitis delta virus to predict mutations that transform the wild-type into a bi-stable conformation, a configuration assessed by calculating the free energies using folding prediction algorithms . The predictions in the final examples need to be verified experimentally, whereas the mutation predicted in the first example complies with the experiment . This supports the use of our proposed method on other known structures, as well as genetically engineered ones . AVAILABILITY: An eigenvalue application will be available in the near future attached to one of the existing tools.

Waste Manag Res, 2003 Dec, 21(6), 515 - 26
The optimisation of food waste addition as a co-substrate in anaerobic digestion of sewage sludge; Kim HW et al.; Food waste has been regarded as the main source of various environmental pollution in Korea due to the high volatile solids (VS) and moisture content caused by the features of dietary habits . The feasibility of food waste as a co-substrate in anaerobic digestion of sewage sludge was investigated in mesophilic and thermophilic conditions using batch tests . Cumulative methane production, dissolved organic carbon (DOC) and volatile fatty acids (VFA) were monitored to find the optimal mixing ratios of food waste and sewage sludge for the enhanced performance of co-digestion . It was observed that adequately mixed food waste led to the enhanced methane production both at mesophilic and thermophilic conditions . However, a conventional linear regression conducted for the optimisation of co-substrate mixing ratios was not accurate in describing exact methane production trends of co-digestion because of the different biodegradability of substrates . Therefore, a remodified Gompertz equation showing nonlinear relationship between variables was developed to find exact information with the same experimental data obtained at 2g VS/l generally used in biochemical methane potential (BMP) tests . Based on an influential parameter, methane production rate (MPR), the optimal mixing ratios of food waste were 39.3% and 50.1% in mesophilic and thermophilic conditions, respectively . To confirm the application of the remodified Gompertz equation, secondary batch tests were conducted with the substrate concentrations of 1-4g VS/l . In overall range tested, the confident mixing ratios of food waste was adjusted to 30-40% and 40% in mesophilic and thermophilic conditions, respectively . The most significant factor for enhanced performance was the improved organic carbon content provided by additional food waste.

Parasitol Int, 2004 Mar, 53(1), 23 - 7
Detection and significance of the potentially pathogenic amoeboflagellate Naegleria italica in Australia; Robinson BS et al.; Thermophilic amoeboflagellates in the genus Naegleria include both virulent and benign species . One of the less studied species, N . italica, has not been detected in the environment since the first reports from Italy in the 1980s; its virulence is known only from infection of laboratory mice . Two recent strains from recreational water in Western Australia (AWQC NG960, NG961) were tentatively identified as N . italica from the characteristic mobilities of seven isozymes . Sequences of the 5.8S rRNA gene and its flanking ITS aligned with a 380+bp length of the published sequence for N . italica with 98% identity . Differences from the type strain were confined to ITS2 . Shorter alignments (<320 bp) were observed with other Naegleria species, corresponding to conserved regions of the 5.8S gene and ITS . Unlike the European type strain of N . italica, the Australian isolates failed to infect laboratory mice intranasally, confirming that infectivity of this species is variable and often lower than in N . fowleri.

Cell Motil Cytoskeleton, 2004 May, 58(1), 30 - 8
Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment; Toba S et al.; An important challenge is to understand the functional specialization of dynein heavy chains . The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma . In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain . The three heavy chains remained full-length and retained MgATPase activity . The beta/gamma dimer bound microtubules in an ATP-sensitive fashion . The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP . The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein . The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility . In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions . The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer . We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized . The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track .

Protein Eng, 2003 Dec, 16(12), 871 - 4
Optimized electrostatic surfaces parallel increased thermostability: a structural bioinformatic analysis; Alsop E et al.; It has been known for some time that thermophilic proteins generally have increased numbers of non-covalent interactions (salt bridges, hydrogen bonds, etc.) compared with their mesophilic orthologs . Recently, anecdotal structural comparisons suggest that non-specific acid-base ion pairs on the protein surface can be an evolutionary efficient mechanism to increase thermostability . In this comprehensive structural analysis, we confirm this to be the case . Comparison of 127 orthologous mesophilic- thermophilic protein groups indicates a clear preference for stabilizing acid-base pairs on the surface of thermophilic proteins . Compared with positions in the core, stabilizing surface mutations are less likely to disrupt the tertiary structure, and thus more likely to be evolutionarily selected . Therefore, we believe that our results, in addition to being theoretically interesting, will facilitate identification of charge-altering mutations likely to increase the stability of a particular protein structure.

Environ Toxicol Chem, 2004 Feb, 23(2), 292 - 7
Development of a model microbial predator-prey system suitable for studies of the behavior of toxic trace metals; Patton LE et al.; Interactions between microbial predators and their prey can significantly influence the behavior of toxic trace metals . Ingested bacterial prey-bound metals can either accumulate within a predator or be excreted and potentially reintroduced into the dissolved phase . A defined predator-prey system suitable for developing a more fundamental understanding of metal behavior in simple microbial food chains was designed and tested by using lead (Pb) as a representative cationic transition metal . Desired features of this system were the ability to define the chemical speciation of dissolved metals as well as to distinguish between prey- and predator-bound metals . Pseudomonas putida and the ciliate protozoan Tetrahymena thermophila were selected as representative bacterial prey and predator species, respectively . In addition, the use of fluorescent microspheres was evaluated as an experimental surrogate for bacterial prey . Filtration techniques for size-selective separation were developed so that the distribution of Pb between cells of T . thermophila, cells of P . putida or microspheres, and the dissolved phase could be assessed . Filtration units were selected based on their ability to perform separations with minimal metal loss at circumneutral pH . Five-micron polycarbonate filter membranes successfully separated T . thermophila from P . putida with good cell retention and low metal loss . Centrifuge filters successfully separated dissolved and particle-bound metal (<5,000 nominal molecular wt limit) . Exemplary experimental results are presented and show that predation on Pb-exposed cells of P . putida or microspheres increases uptake of Pb by T . thermophila.

Biosci Biotechnol Biochem, 2004 Feb, 68(2), 286 - 92
Isolation of thermophilic ammonium-tolerant bacterium and its application to reduce ammonia emission during composting of animal wastes; Kuroda K et al.; A thermophilic bacterium, strain TAT105, was isolated from compost made of animal wastes . TAT105 had high tolerance to ammonium nitrogen up to 1200 mM, and highly assimilated nitrogen during the growth on swine feces . The strain was classified into Bacillus, close to Bacillus pallidus . To evaluate the effect of adding TAT105 to ammonia (NH3) emission during the composting process of animal wastes, laboratory scale composting was done . NH3 emission tended to be lower and nitrogen loss was smaller in the TAT105-added material than in the control material to which TAT105 was not added . Thermophilic ammonium-tolerant bacteria in the TAT105-added material increased to about 8x10(9) CFU/g of dry matter on the average during the tests, and most of them were judged to be TAT105 from morphological colony discrimination . These results suggested the possibility of reducing NH3 emission from composting of animal wastes by adding TAT105.

Biochemistry, 2004 Mar 2, 43(8), 2228 - 40
Subunit exchange by CheA histidine kinases from the mesophile Escherichia coli and the thermophile Thermotoga maritima; Park SY et al.; Dimerization of the chemotaxis histidine kinase CheA is required for intersubunit autophosphorylation {Swanson, R . V., Bourret, R . B., and Simon, M . I . (1993) Mol . Microbiol . 8, 435-441} . Here we show that CheA dimers exchange subunits by the rate-limiting dissociation of a central four-helix bundle association domain (P3), despite the high stability of P3 versus unfolding . P3 alone determines the stability and exchange properties of the CheA dimer . For CheA proteins from the mesophile Escherichia coli and the thermophile Thermotoga maritima, subunit dissociation activates at temperatures where the respective organisms live (37 and 80 degrees C) . Under destabilizing conditions, P3 dimer dissociation is cooperative with unfolding . Chemical denaturation is reversible for both EP3 and TP3 . Aggregation accompanies thermal unfolding for both proteins under most conditions, but thermal unfolding is reversible and two-state for EP3 at low protein concentrations . Residue differences within interhelical loops may account for the contrasted thermodynamic properties of structurally similar EP3 and TP3 (41% sequence identity) . Under stabilizing conditions, greater correlation between activation energy for dimer dissociation and P3 stability suggests more unfolding in the dissociation of EP3 than TP3 . Furthermore, destabilization of extended conformations by glycerol slows relative dissociation rates more for EP3 than for TP3 . Nevertheless, at physiological temperatures, neither protein likely unfolds completely during subunit exchange . EP3 and TP3 will not exchange subunits with each other . The receptor coupling protein CheW reduces the subunit dissociation rate of the T . maritima CheA dimer by interacting with the regulatory domain P5.

Orig Life Evol Biosph, 2004 Feb, 34(1-2), 243 - 56
Comparative genomics and the gene complement of a minimal cell; Islas S et al.; The concept of a minimal cell is discussed from the viewpoint of comparative genomics . Analysis of published DNA content values determined for 641 different archaeal and bacterial species by pulsed field gel electrophoresis has lead to a more precise definition of the genome size ranges of free-living and host-associated organisms . DNA content is not an indicator of phylogenetic position . However, the smallest genomes in our sample do not have a random distribution in rRNA-based evolutionary trees, and are found mostly in (a) the basal branches of the tree where thermophiles are located; and (b) in late clades, such as those of Gram positive bacteria . While the smallest-known genome size for an endosymbiont is only 450 kb, no free-living prokaryote has been described to have genomes < 1450 kb . Estimates of the size of minimal gene complement can provide important insights in the primary biological functions required for a sustainable, reproducing cell nowadays and throughout evolutionary times, but definitions of the minimum cell is dependent on specific environments.

Biol Chem, 2004 Jan, 385(1), 31 - 9
Structural destabilization of the recombinant thermophilic TthL11 ribosomal protein by a single amino acid substitution; Triantafillidou D et al.; Thermus thermophilus L11 protein has previously been reported to be resistant against tryptic and chymotryptic proteolysis under native conditions . With a single amino acid substitution, namely Trp101Arg, conformational changes were induced that resulted in the exhibition of specific amino acids that served as targets for tryptic and chymotryptic action and rendered the protein highly unstable even during purification . This unexpected process was evidenced by the isolation with size exclusion gel chromatography of the well-structured chymotryptic N-terminal domain in a high amount and its characterization both by Edman degradation and QTOF-EMS spectroscopy . On the other hand, the substitution of Val38Cys, which did not contribute to structural changes, indicates a very possible implication of this amino acid in the protein methylation process . The data reported in this work illustrate the distinctive amino acid dynamics in a thermophilic protein, which, while serving the function common to its counterparts from mesophilic organisms, has had to adapt to the extreme environmental conditions typical of thermophilic organisms.

Water Res, 2004 Mar, 38(5), 1327 - 39
Modeling of aerobic biodegradation of feces using sawdust as a matrix; Lopez Zavala MA et al.; Composting in the bio-toilet system is a continuous thermophilic-aerobic biodegradation process . Unlike to the traditional composting systems, biodegradation rates of organic matter are very important because feces are daily added into the composting reactor of the bio-toilet and an accelerated decomposition is aimed . The models developed for conventional composting processes include simple formulations of biodegradation kinetics and deal mainly with energy and water balances . Therefore, formulation of kinetics that can reasonably describe the biodegradation process in the bio-toilet system is required for better modeling predictions . In this work, a bio-kinetic model was introduced to describe the aerobic biodegradation of feces in the bio-toilet system . This model includes three processes for carbonaceous material degradation and is prepared by using the activated sludge modeling techniques and formulations . Stoichiometric parameters were adopted from literature on activated sludge processes . Kinetic parameters were estimated by conducting batch tests for several organic loadings and by using respirometry, curve-fitting techniques, and sensitivity analysis . Feasibility and applicability of these parameters were assessed by conducting feces intermittent-feeding tests and by simulating the experimental respiration rates . Model, stoichiometric and kinetic parameters proved to be affordable for describing the biodegradation of feces in the bio-toilet system.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2951 - 6 Epub 2004 Feb 18.
Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes; Paz A et al.; The mechanism of an organism's adaptation to high temperatures has been investigated intensively in recent years . It was suggested that the macromolecules of thermophilic microorganisms (especially proteins) have structural features that enhance their thermostability . We compared mRNA sequences of 72 fully sequenced prokaryotic proteomes (14 thermophilic and 58 mesophilic species) . Although the differences between the percentage of adenine plus guanine content of whole mRNAs of different prokaryotic species are much lower than those of guanine plus cytosine content, the thermophile purine-pyrimidine (R/Y) ratio within their mRNAs is significantly higher than that of the mesophiles . The first and third codon positions of both thermophiles and mesophiles are purine-biased, with the bias more pronounced by the thermophiles . Thermophile mRNAs that display the highest R/Y ratio (1.43-1.69) are those of the ribosomal proteins, histone-like proteins, DNA-dependent RNA polymerase subunits, and heat-shock proteins . Within mesophilic prokaryotes and five eukaryotic species, the R/Y ratio of the mRNAs of heat-shock proteins is higher than their average over coding part of the genome . Polypurine tracts (R)(n) (with n > or = 5) are much more abundant within the thermophile mRNAs compared with mesophiles . Between two sequential pure-purinic codons of thermophile mRNAs, there is a rather strong tendency for the occurrence of adenine but not guanine tracts . The data suggest that mixed adenine.guanine and polyadenine tracts in mRNAs increase the thermostability beyond the contribution of amino acids encoded by purine tracts, which highlights the importance of ecological stress in the evolution of genome architecture.

Klin Lab Diagn, 2003 Dec, (12), 47 - 9
{A simple biological method for the fast identification of antibiotics}; Suchkov IuG et al.; A method used to determine the quantitative and qualitative determination of antibiotics in blood, injury discharge, in human and animal urine as well as in foodstuffs (meat, milk and products made of them) is described . It is based on using obligate, thermophilic bacteria Bacillus stearothermophilus, strains KK BKM B-213OD and BKM B-718 with an optimum growth at 55-60 degrees C . Meso- and psychrophilic bacteria cannot grow at the above temperature, therefore, the studied non-sterile material does not need any preliminary thermal treatment prior to the indicator-strain, which is highly sensitive to all widely used antibiotics, is sown in the test-culture bacterial lawn.

J Food Prot, 2004 Feb, 67(2), 403 - 6
Changes in galactose and lactic acid content of sweet whey during storage; Rao RD et al.; Whey is often stored or transported for a period of time prior to processing . During this time period, galactose and lactic acid concentrations may accumulate, reducing the quality of spray-dried whey powders in regard to stickiness and agglomeration . This study surveyed industry samples of Cheddar and mozzarella cheese whey streams to determine how galactose and lactic acid concentrations changed with storage at appropriate (4 degrees C) and abuse (37.8 degrees C) temperatures . Samples stored at 4 degrees C did not exhibit significant increases in levels of lactic acid or galactose . Mozzarella whey accumulated the greatest amount of galactose and lactic acid with storage at 37.8 degrees C . Whey samples derived from cheese made from single strains of starter culture were also evaluated to determine each culture's contribution to galactose and lactic acid production . Starter cultures evaluated included Streptococcus salivarius ssp . thermophilus . Lactobacillus helveticus, Lactobacillus delbrueckii ssp . bulgaricus, Lactococcus lactis ssp . cremoris, and Lactococcus lactis ssp . lactis . Whey derived from L . helveticus accumulated a significantly greater amount of lactic acid upon storage at 37.8 degrees C as compared with the other cultures . Galactose accumulation was significantly decreased in whey from L . lactis ssp . lactis stored at 37.8 degrees C in comparison with the other cultures . Results from this study indicate that proper storage conditions (4 degrees C) for whey prevent accumulation of galactose and lactic acid while the extent of accumulation during storage at 37.8 degrees C varies depending on the culture(s) used in cheese production.

Protein Expr Purif, 2003 Dec, 32(2), 239 - 45
Cloning, purification, and characterization of thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis; Chen Q et al.; Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) from a newly characterized thermophile Thermoanaerobacter tengcongensis was expressed in Escherichia coli and purified . Analytical gel filtration suggested that the enzyme exist as a homotetramer in solution . The optimal pH for the forward reaction was found to be 8.0 and the optimal temperature 70 degrees C . The steady-state kinetic characteristics suggest that hypoxanthine is the most effective substrate . This enzyme showed a half-life of 75min at 50 degrees C and no apparent loss of activity after 3 months at 4 degrees C.

J Biol Chem, 2004 Apr 23, 279(17), 18085 - 90 Epub 2004 Feb 12.
The F subunit of Thermus thermophilus V1-ATPase promotes ATPase activity but is not necessary for rotation; Imamura H et al.; V(1)-ATPase from the thermophilic bacterium Thermus thermophilus is a molecular rotary motor with a subunit composition of A(3)B(3)DF, and its central rotor is composed of the D and F subunits . To determine the role of the F subunit, we generated an A(3)B(3)D subcomplex and compared it with A(3)B(3)DF . The ATP hydrolyzing activity of A(3)B(3)D (V(max) = 20 s(-1)) was lower than that of A(3)B(3)DF (V(max) = 31 s(-1)) and was more susceptible to MgADP inhibition during ATP hydrolysis . A(3)B(3)D was able to bind the F subunit to form A(3)B(3)DF . The C-terminally truncated F((Delta85-106)) subunit was also bound to A(3)B(3)D, but the F((Delta69-106)) subunit was not, indicating the importance of residues 69-84 of the F subunit for association with A(3)B(3)D . The ATPase activity of A(3)B(3)DF((Delta85-106)) (V(max) = 24 s(-1)) was intermediate between that of A(3)B(3)D and A(3)B(3)DF . A single molecule experiment showed the rotation of the D subunit in A(3)B(3)D, implying that the F subunit is a dispensable component for rotation itself . Thus, the F subunit binds peripherally to the D subunit, but promotes V(1)-ATPase catalysis.

J Appl Microbiol, 2004, 96(3), 481 - 90
Evaluation of bacterial communities belonging to natural whey starters for Grana Padano cheese by length heterogeneity-PCR; Lazzi C et al.; AIMS: To detect bacteria present in controlled dairy ecosystems with defined composition by length-heterogeneity (LH)-PCR . LH-PCR allows to distinguish different organisms on the basis of natural variations in the length of 16S rRNA gene sequences . METHODS AND RESULTS: LH-PCR was applied to depict population structure of the lactic acid bacteria (LAB) species recoverable from Grana Padano cheese whey starters . Typical bacterial species present in the LAB community were evidenced and well discriminated . Small differences in species composition, e.g . the frequent finding of Streptococcus thermophilus and the constant presence of thermophilic lactobacilli (Lactobacillus helveticus, Lact . delbrueckii subsp . lactis/bulgaricus and Lact . fermentum) were reliably highlighted . Specificity of LH-PCR was confirmed by species-specific PCR from total DNA of the cultures . CONCLUSIONS: LH-PCR is a useful tool to monitor microbial composition and population dynamics in dairy starter cultures . When present, non-dominant bacterial species present in the whey starters, such as Strep . thermophilus, can easily be visualized and characterized without isolating and cultivating single strains . A similar approach can be applied to more complex dairy ecosystems such as milk or cheese curd . SIGNIFICANCE AND IMPACT OF THE STUDY: Community members and differences in population structure of controlled dairy ecosystems such as whey starters for hard cheeses can be evaluated and compared in a relative easy, fast, reliable and highly reproducible way.

Gene, 2004 Feb 18, 327(1), 75 - 9
A unique ATG triplet downstream of gene start in archaea: implications for translation initiation and evolution; Xiaohui C et al.; Searching for unique features of archaeal genome may shed light on the mechanism of gene regulation in primitive life forms . Statistical analysis of ATG frequency on the complete genome sequences of 16 archaea, 20 bacteria and 2 eukaryotes revealed that most of the archaeal genomes have a remarkably high ATG frequency at the position of nine nucleotide (nt) downstream of the translation initiation site (the first nucleotide of the translation initiation codon is designated as 0) . To understand the role of this unique ATG in archaea, we further analyzed the ATG-initiated genes and non-ATG-initiated genes separately, and the results indicated that only the non-ATG-initiated genes contribute to the high ATG frequency at position +9 . This led us to speculate that the in-frame ATG at +9 may serve as a remedial initiation site for archaea in case of initiation failure at the regular site . In addition, it seems that this phenomenon does not result from the harsh environment that archaea are usually viable according to the fact that no considerably high ATG frequency at +9 was observed in all the four thermophilic bacteria that also live in harsh environment . We proposed that the high ATG frequency at position +9 might reflect the decreased efficiency of the translation initiation machinery in archaea . Since archaea evolve very slowly, this unique characteristic of high ATG frequency at position +9 may present the primitive state of the Universal Ancestor.

Eukaryot Cell, 2004 Feb, 3(1), 157 - 69
A non-long terminal repeat retrotransposon family is restricted to the germ line micronucleus of the ciliated protozoan Tetrahymena thermophila; Fillingham JS et al.; The ciliated protozoan Tetrahymena thermophila undergoes extensive programmed DNA rearrangements during the development of a somatic macronucleus from the germ line micronucleus in its sexual cycle . To investigate the relationship between programmed DNA rearrangements and transposable elements, we identified several members of a family of non-long terminal repeat (LTR) retrotransposons (retroposons) in T . thermophila, the first characterized in the ciliated protozoa . This multiple-copy retrotransposon family is restricted to the micronucleus of T . thermophila . The REP (Tetrahymena non-LTR retroposon) elements encode an ORF2 typical of non-LTR elements that contains apurinic/apyrimidinic endonuclease (APE) and reverse transcriptase (RT) domains . Phylogenetic analysis of the RT and APE domains indicates that the element forms a deep-branching clade within the non-LTR retrotransposon family . Northern analysis with a probe to the conserved RT domain indicates that transcripts from the element are small and heterogeneous in length during early macronuclear development . The presence of a repeated transposable element in the genome is consistent with the model that programmed DNA deletion in T . thermophila evolved as a method of eliminating deleterious transposons from the somatic macronucleus.

Lipids, 2003 Dec, 38(12), 1269 - 74
Two distinct pathways for the formation of hydroxy FA from linoleic acid by lactic acid bacteria; Kishimoto N et al.; Twenty-three of 86 strains of lactic acid bacteria transformed linoleic acid into hydroxy FA . Two distinct conversion pathways were in operation . Two strains of Lactobacillus acidophilus and a strain of Pediococcus pentosaceus produced 13(S)-hydroxy-9-octadecenoic acid 113(S)-OH 18:11 and 10,13dihydroxyoctadecanoic acid (10,13-OH 18:0) as main and minor products, respectively, whereas 13 strains, including L . casei subsp . casei, L . paracasei subsp . paracasei, L . rhamnosus, L . lactis subsp . cremoris, and Streptococcus salivarius subsp . thermophilus produced 10-hydroxy-12-octadecenoic acid (10-OH 18:1) . Seven strains of L . plantarum converted linoleic acid to 10-hydroxyoctadecanoic acid (10-OH 18:0) through 10-OH 18:1 . Linoleic acid at 2 g/L was converted by L . acidophilus IFO13951T to 1.3 g of 13(S)-OH 18:1 and 0.09 g of 10,13-OH 18:0 in 7 d . Lactobacillus paracasei subsp . paracasei JCM 1111 produced 10-OH 18:1 in 91% yield, and L . plantarum JCM 8341, 10-OH 18:0 in 59% yield from linoleic acid (2 g/L) under optimal conditions . To our knowledge, this is the first report on the production of 13(S)-OH 18:1 by lactic acid bacteria other than ruminal bacteria, and of 10,13-OH 18:0 by any bacteria.

J Biochem (Tokyo), 2003 Dec, 134(6), 843 - 51
Glutamine:phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8; Hosono A et al.; A subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42,271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8 . Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Igamma . The protein shows around 40% identity with both T . thermophilus aspartate aminotransferase {EC 2.6.1.1} and mammalian glutamine:phenylpyruvate aminotransferase {EC 2.6.1.64} . The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42,000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids . These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase . This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells . As the presence of aromatic amino acid:2-oxoglutarate aminotransferase {EC 2.6.1.57} has not been reported in T . thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis . It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis . The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases . The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T . thermophilus and E . coli aspartate aminotransferases.

Appl Environ Microbiol, 2004 Feb, 70(2), 937 - 42
Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression; Heydari M et al.; L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps . This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase . The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18) . The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa . The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively . No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine . The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD) . The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively . When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G . stearothermophilus . The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.

Appl Environ Microbiol, 2004 Feb, 70(2), 900 - 12
Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics; Vaningelgem F et al.; Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as Streptococcus thermophilus and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium . In addition, the acidification rates of the different strains were compared with their milk clotting behaviors . The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening . EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium . Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium . An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa . A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa) . Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished . Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method . All of the EPS from the S . thermophilus strains tested were classified into six groups according to their monomer compositions . Apart from galactose and glucose, other monomers, such as (N-acetyl)galactosamine, (N-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents . Three strains were found to produce EPS containing (N-acetyl)glucosamine, which to our knowledge was never found before in an EPS from S . thermophilus . Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures . Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures.

Appl Environ Microbiol, 2004 Feb, 70(2), 837 - 44
A new alkali-thermostable azoreductase from Bacillus sp . strain SF; Maier J et al.; A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a Bacillus sp . strain isolated out of the wastewater drain of a textile finishing company . An NADH-dependent azoreductase of this strain, Bacillus sp . strain SF, was found to be responsible for the decolorization of azo dyes . This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3 . The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80 degrees C . Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction . A 26% similarity of this azoreductase to chaperonin Cpn60 from a Bacillus sp . was found by peptide mass mapping experiments . Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(R)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline . Those dyes with NO2 substituents, especially in the ortho position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.

Eur J Biochem, 2004 Feb, 271(4), 834 - 44
Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR-249 and biochemical characterization of its thermostable recombinant enzyme; Lee AY et al.; A gene encoding thermostable Lon protease from Brevibacillus thermoruber WR-249 was cloned and characterized . The Br . thermoruber Lon gene (Bt-lon) encodes an 88 kDa protein characterized by an N-terminal domain, a central ATPase domain which includes an SSD (sensor- and substrate-discrimination) domain, and a C-terminal protease domain . The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis sigmaA-dependent promoter, but in the absence of CIRCE (controlling inverted repeat of chaperone expression) . Bt-lon was expressed in Escherichia coli, and its protein product was purified . The native recombinant Br . thermoruber Lon protease (Bt-Lon) displayed a hexameric structure . The optimal temperature of ATPase activity for Bt-Lon was 70 degrees C, and the optimal temperature of peptidase and DNA-binding activities was 50 degrees C . This implies that the functions of Lon protease in thermophilic bacteria may be switched, depending on temperature, to regulate their physiological needs . The peptidase activity of Bt-Lon increases substantially in the presence of ATP . Furthermore, the substrate specificity of Bt-Lon is different from that of E . coli Lon in using fluorogenic peptides as substrates . Notably, the Bt-Lon protein shows chaperone-like activity by preventing aggregation of denatured insulin B-chain in a dose-dependent and ATP-independent manner . In thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, Tm of 71.5 degrees C . Sequence comparison with mesophilic Lon proteases shows differences in the rigidity, electrostatic interactions, and hydrogen bonding of Bt-Lon relevant to thermostability.

J Biol Chem, 2004 Apr 16, 279(16), 16518 - 25 Epub 2004 Feb 03.
Crystal structures of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8: induced fit and substrate recognition; Goto M et al.; The following three-dimensional structures of three forms of glutamine:phenylpyruvate aminotransferase from Thermus thermophilus HB8 have been determined and represent the first x-ray analysis of the enzyme: the unliganded pyridoxal 5'-phosphate form at 1.9 A resolution and two complexes with 3-phenylpropionate and alpha-keto-gamma-methylthiobutyrate at 2.35 and 2.6 A resolution, respectively . The enzyme shows high activity toward phenylalanine, tyrosine, tryptophan, kynurenine, methionine, and glutamine . The enzyme is a homodimer, and each subunit is divided into an N-terminal arm and small and large domains . Based on its folding, the enzyme belongs to fold type I, aminotransferase subclass Ib . The subclass I aminotransferases whose structures have so far been determined exhibit a large movement of the small domain region upon binding of a substrate . Similarly, the glutamine:phenylpyruvate aminotransferase undergoes a large movement in part of the small domain to close the active site . The active-site pocket has a shape and size suitable to enclose the side chain of an aromatic amino acid or that of methionine . The inner side of the pocket is mostly hydrophobic, but also has polar sites . The kynurenine complex generated by computer modeling fits the pocket of the enzyme and its hydrophilic groups interact with the polar sites of the pocket.

FEMS Microbiol Lett, 2004 Jan 30, 230(2), 251 - 8
Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops; Wu J et al.; Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp . WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region . The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%) . Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins . However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions . Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution . The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form . After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide . The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.

Biochemistry (Mosc), 2003 Dec, 68(12), 1307 - 12
Thermostable DNA polymerase from Thermus thermophilus B35: preparation and study of a modified form of the enzyme with high affinity to ddNTP; Akishev AG et al.; The hybrid protein consisting of Tte DNA polymerase fragment and mutant Taq DNA polymerase (F667Y) fragment in the ratio 20 : 1 was constructed . Affinity of the modified enzyme (substitutions F669Y, V667I, and S692Q) to ddNTP was two orders higher than that of the wild type enzyme . The modified enzyme was used for sequencing DNA fragment with total deoxyguanosine and deoxycytidine content of 68% . In the polymerase chain reaction, the modified enzyme exhibits properties typical of the wild type Tte DNA polymerase.

Chembiochem, 2004 Feb 6, 5(2), 231 - 9
Adaptation of class-13 alpha-amylases to diverse living conditions; Linden A et al.; There are currently 35 available nonredundant molecular structures of class-13 alpha-amylases (EC 3.2.1.1), mostly from microbial organisms living under a wide range of environmental conditions . One of the most recent additions has been the first alpha-amylase structure of a hyperthermophilic archaeon {Linden et al., J . Biol . Chem . 2003, 278, 9875-9884} . The structure has been used for comparative analyses with a representative set of three alpha-amylases from thermophilic, mesophilic and psychrophilic sources to identify molecular parameters for environmental adaptation . Our analysis supports generally observed trends such as an increase in structural compactness as well as an increase in salt bridges in order to cope with high-temperature conditions . The two representative thermophilic structures used in this comparative study have independently evolved di-metal centres--not present in the mesophilic and psychrophilic structures--in the vicinity of the active site . These observations may provide impetus for the design of alpha-amylases with improved molecular properties to enhance their utility in biotechnological processes.

Biotechnol Bioeng, 2004 Feb 20, 85(4), 434 - 41
Effect of cobalt on the anaerobic thermophilic conversion of methanol; Paulo PL et al.; The importance of cobalt on the anaerobic conversion of methanol under thermophilic conditions was studied in three parallel lab-scale UASB-reactors and in cobalt-limited enriched cultures . Reactors R1, R2, and R3 were fed with methanol in a bicarbonate-buffered medium, supplied with iron and macronutrients: in R1 all metals were supplied (control), R2 was cobalt deprived, and in R3 all metals were deprived . In the 136 days of continuous experiment, a drop in performance was observed over the last 30 days . Particularly in R3, both methanol removal and methane formation dropped by 7.1% and 13.7%, respectively, compared to the control reactor, R1 . When the medium was cobalt-deprived, acetate was not produced and, as a consequence, the enriched consortium lost its capacity to degrade acetate, indicating that the acetotrophic microorganisms were washed out . The addition of 0.5 microM of cobalt to a cobalt-deprived enrichment culture led to acetate accumulation . The results obtained in this study indicate that the mixed consortium requires a proper amount of cobalt, and its addition to a concentration of 0.1 microM leads to the highest methanol conversion rate, with methane as the sole end product from methanol .

Proc Natl Acad Sci U S A, 2004 Feb 10, 101(6), 1679 - 84 Epub 2004 Jan 30.
Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena; Liu Y et al.; Genome-wide DNA elimination accompanies development of the somatic macronucleus from the germ-line micronucleus during the sexual process of conjugation in the ciliated protozoan Tetrahymena thermophila . Small RNAs, referred to as "scan RNAs" (scnRNAs), that accumulate only during conjugation are highly enriched in the eliminated sequences, and mutations that prevent DNA elimination also affect the accumulation of scnRNAs, suggesting that an RNA interference (RNAi)-like mechanism is involved in DNA elimination . Histone H3 that is methylated at lysine 9 (K9) is a hallmark of heterochromatin and, in Tetrahymena, is found only in developing macronuclei (anlagen) in association with chromatin containing the sequences undergoing elimination . In this article, we demonstrate that a mutation in the TWI1 gene that eliminates the accumulation of scnRNAs also abolishes H3 methylation at K9 . We created mutant strains of Tetrahymena in which the only major H3 contained a K9Q mutation . These mutants accumulated scnRNAs normally during conjugation but showed dramatically reduced efficiency of DNA elimination . These results provide strong genetic evidence linking an RNAi-like pathway, H3 K9 methylation, and DNA elimination in Tetrahymena.

Water Sci Technol, 2003, 48(11-12), 77 - 83
A collection and treatment system for organic waste and wastewater in a sensitive rural area; Malmen L et al.; In the municipality of Sund, located in a sensitive rural area in Aland, a demonstration project is now carried out with the overall objective to move the most concentrated fractions of wastewater from the coastal area to a treatment plant situated close to arable land . Blackwater and greywater septic sludge from about twenty households and two tourist camps are treated together with energy rich organic material from a nearby potato-chip factory . The collection concept is based on the use of extremely efficient water-saving toilets, with separate systems for the blackwater and greywater in the households . The collected materials are co-treated in a batchwise aerobic thermophilic treatment process (wet composting process), where the materials reach at least 55 degrees C during a minimum of 10 hours . The dry matter content of the collected material was about 2% . After stabilisation and sanitation (by the temperature rise caused by microbial activity during the treatment process), the compost slurry is utilized as a liquid organic fertilizer on arable land.

Appl Microbiol Biotechnol, 2004 Aug, 65(2), 149 - 57 Epub 2004 Jan 30.
Metabolic selectivity and growth of Clostridium thermocellum in continuous culture under elevated hydrostatic pressure; Bothun GD et al.; The continuous culture of Clostridium thermocellum, a thermophilic bacterium capable of producing ethanol from cellulosic material, is demonstrated at elevated hydrostatic pressure (7.0 MPa, 17.3 MPa) and compared with cultures at atmospheric pressure . A commercial limitation of ethanol production by C . thermocellum is low ethanol yield due to the formation of organic acids (acetate, lactate) . At elevated hydrostatic pressure, ethanol:acetate (E/A) ratios increased >10(2) relative to atmospheric pressure . Cell growth was inhibited by approximately 40% and 60% for incubations at 7.0 MPa and 17.3 MPa, respectively, relative to continuous culture at atmospheric pressure . A decrease in the theoretical maximum growth yield and an increase in the maintenance coefficient indicated that more cellobiose and ATP are channeled towards maintaining cellular function in pressurized cultures . Shifts in product selectivity toward ethanol are consistent with previous observations of hydrostatic pressure effects in batch cultures . The results are partially attributed to the increasing concentration of dissolved product gases (H2, CO2) with increasing pressure; and they highlight the utility of continuous culture experiments for the quantification of the complex role of dissolved gas and pressure effects on metabolic activity.

Microb Ecol, 2004 Feb, 47(2), 186 - 96 Epub 2004 Feb 02.
Microbial diversity in inactive chimney structures from deep-sea hydrothermal systems; Suzuki Y et al.; Massive chimney structures, which are characteristic of many hydrothermally active zones, harbor diverse microbial communities containing both thermophilic and hyperthermophilic microbes . However, vent chimneys ultimately become hydrothermally inactive, and the changes that occur in the microbial communities upon becoming inactive have not been documented . We thus collected inactive chimneys from two geologically and geographically distinct hydrothermal fields, Iheya North in the western Pacific Ocean and the Kairei field in the Indian Ocean . The chimneys displayed easily distinguishable strata, which were analyzed with regard to both mineralogical and microbiological properties . X-ray diffraction pattern and energy-dispersive spectroscopic analyses revealed that the main mineral components of the chimney substructures from Iheya North and the Kairei field were barite (BaSO4) and chalcopyrite (CuFeS2), respectively . Microbial cell densities in the substructures determined by DAPI counting ranged from 1.7 x 10(7) cells g(-1) to 3.0 x 10(8) cells g(-1) . The proportions of archaeal rDNA in the whole microbial rDNA assemblages in all substructures were, at most, a few percent as determined by quantitative fluorogenic PCR . The microbial rDNA clone analysis and whole-cell fluorescence in situ hybridization revealed a community that was decidedly different from any communities previously reported in active chimneys . Curiously, both samples revealed the abundant presence of a group of Bacteria related to a magnetosome-bearing bacterium, " Magnetobacterium bavaricum" of the Nitrospirae division . These results suggest that inactive chimneys provide a distinct microbial habitat .

Proc Natl Acad Sci U S A, 2004 Feb 10, 101(6), 1479 - 84 Epub 2004 Jan 28.
NMR structure of the KaiC-interacting C-terminal domain of KaiA, a circadian clock protein: implications for KaiA-KaiC interaction; Vakonakis I et al.; KaiA is a two-domain circadian clock protein in cyanobacteria, acting as the positive element in a feedback loop that sustains the oscillation . The structure of the N-terminal domain of KaiA is that of a pseudo-receiver, similar to those of bacterial response regulators, which likely interacts with components of the clock-resetting pathway . The C-terminal domain of KaiA is highly conserved among cyanobacteria and enhances the autokinase activity of KaiC . Here we present the NMR structure of the C-terminal domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 . This domain adopts a novel all alpha-helical homodimeric structure . Several mutations known to affect the period of the circadian oscillator are shown to be located at an exposed groove near the dimer interface . This NMR structure and a 21-A-resolution electron microscopy structure of the hexameric KaiC particle allow us to postulate a mode of KaiA-KaiC interaction, in which KaiA binds a linker region connecting two globular KaiC domains.

Mol Cell Biol, 2004 Feb, 24(4), 1769 - 78
U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production; Atzorn V et al.; Saccharomyces cerevisiae snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) required for the processing of 18S rRNA . Here, we show that the previously characterized human, reptilian, amphibian, and fish U17 snoRNAs represent the vertebrate homologues of yeast snR30 . We also demonstrate that U17/snR30 is present in the fission yeast Schizosaccharomyces pombe and the unicellular ciliated protozoan Tetrahymena thermophila . Evolutionary comparison revealed that the 3'-terminal hairpins of U17/snR30 snoRNAs contain two highly conserved sequence motifs, the m1 (AUAUUCCUA) and m2 (AAACCAU) elements . Mutation analysis of yeast snR30 demonstrated that the m1 and m2 elements are essential for early cleavages of the 35S pre-rRNA and, consequently, for the production of mature 18S rRNA . The m1 and m2 motifs occupy the opposite strands of an internal loop structure, and they are located invariantly 7 nucleotides upstream from the ACA box of U17/snR30 snoRNAs . U17/snR30 is the first identified box H/ACA snoRNA that possesses an evolutionarily conserved role in the nucleolytic processing of eukaryotic pre-rRNA.

Am J Clin Nutr, 2004 Feb, 79(2), 261 - 7
Long-term consumption of infant formulas containing live probiotic bacteria: tolerance and safety; Saavedra JM et al.; BACKGROUND: Nonpathogenic live bacteria are consumed as food by many children, particularly in the form of yogurt . The tolerance and safety of long-term consumption of specific types and strains of probiotic bacteria are not well documented . OBJECTIVE: The goal was to evaluate tolerance to formulas containing 2 levels of probiotic supplementation and effects on growth, general clinical status, and intestinal health in free-living healthy infants . DESIGN: This was a prospective, double-blind, randomized, placebo-controlled study of healthy infants aged 3-24 mo . Infants were assigned to receive a standard milk-based formula containing 1 x 10(7) colony-forming units (CFU)/g each of Bifidobacterium lactis and Streptococcus thermophilus, formula containing 1 x 10(6) CFU/g each of B . lactis and S . thermophilus, or unsupplemented formula . Clinical outcomes included formula intake, gastrointestinal tolerance, anthropometric measures, daycare attendance, and history of illness . RESULTS: One hundred eighteen infants aged ( +/- SD) 7.0 +/- 2.9 mo at enrollment consumed formula for 210 +/- 127 d . There were no significant differences in age, sex, formula consumption, or length of study between groups . The supplemented formulas were well accepted and were associated with a lower frequency of reported colic or irritability (P < 0.001) and a lower frequency of antibiotic use (P < 0.001) than was the unsupplemented formula . There were no significant differences between groups in growth, health care attention seeking, daycare absenteeism, or other health variables . CONCLUSION: Long-term consumption of formulas supplemented with B . lactis and S . thermophilus was well tolerated and safe and resulted in adequate growth, reduced reporting of colic or irritability, and a lower frequency of antibiotic use.

Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 357 - 8 Epub 2004 Jan 23.
Expression, purification, crystallization and preliminary crystallographic analysis of osmotically inducible protein C; Rehse PH et al.; Selenium-incorporated osmotically inducible protein C from the thermophilic bacterium Thermus thermophilus was overexpressed, purified and crystallized . The crystals belong to space group P1, with unit-cell parameters a = 37.58, b = 40.95, c = 48.14 A, alpha = 76.93, beta = 74.04, gamma = 64.05 degrees . Five data sets were collected from a single crystal to 1.6 A using synchrotron radiation for MAD phasing . Self-rotation functions and the Matthews coefficient are consistent with two molecules in the asymmetric unit.

J Biol Chem, 2004 Apr 16, 279(16), 16272 - 7 Epub 2004 Jan 26.
Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase; Ueoka-Nakanishi H et al.; In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis . We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T . (2001) J . Biol . Chem . 276, 39505-39507) . The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma . In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted . ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation . We successfully observed inverse redox switching of rotation of gamma using this mutant complex . The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered . Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level . These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.

Biophys J, 2004 Feb, 86(2), 1089 - 104
Adenylation-dependent conformation and unfolding pathways of the NAD+-dependent DNA ligase from the thermophile Thermus scotoductus; Georlette D et al.; In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases . This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium . However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood . From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation . Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme . These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA . These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA . In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability . Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states . Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase . These data provide further insights into the properties of partially folded intermediates.

Dtsch Tierarztl Wochenschr, 2003 Dec, 110(12), 487 - 93
{Airborne microorganisms in a rearing henhouse for layers during vaccination}; Albrecht A et al.; Airborne microorganisms are proved regularly in livestock houses as a part of stable dust and its amount depends on housing conditions, the flow of air and the movement of material . Health of animals and farmers can be influenced in a negative way by these bioaerosols . In a rearing house for layers concentrations of various groups of airborne microorganisms were measured during vaccination by a veterinary and his three assistants . During the vaccination activities the concentrations of some airborne microorganisms increased by a factor of ten to the following medians of colony forming units (cfu) on used selective agars (cfu/m3): 10(3) on MacConkey (36 degrees C), 10(3) on Dichloran-Glycerol (25 degrees C), 10(7) on Tryptone Soy (CaSo, 36 degrees C), 10(3) on Salmonella-Shigella (36 degrees C), 10(2) yeasts on Sabouraud (36 degrees C), and 10(2) on Campylobacter (36 degrees C) . Thermophilic fungi were only grown on some of the used Maltextract agar dishes (45 degrees C) in concentrations near of the limit of detection . Some aerial samples were analysed for Chlamydia . Chlamydophila psittaci was not detected . Concentrations of airborne microorganisms in livestock houses depends not only on housing conditions but also on specific work procedures of farmers or on the activity of the animals.

J Mol Evol, 2003 Dec, 57(6), 721 - 30
The universal ancestor and the ancestor of bacteria were hyperthermophiles; Di Giulio M; The definition of the node of the last universal common ancestor (LUCA) is justified in a topology of the unrooted universal tree . This definition allows previous analyses based on paralogous proteins to be extended to orthologous ones . In particular, the use of a thermophily index (based on the amino acids' propensity to enter the {hyper} thermophile proteins more frequently) and its correlation with the optimal growth temperature of the various organisms allow inferences to be made on the habitat in which the LUCA lived . The reconstruction of ancestral sequences by means of the maximum likelihood method and their attribution to the set of mesophilic or hyperthermophilic sequences have led to the following conclusions: the LUCA was a hyperthermophile "organism," as were the ancestors of the Archaea and Bacteria domains, while the ancestor of the Eukarya domain was a mesophile . These conclusions are independent of the presence of hyperthermophile bacteria in the sample of sequences used in the analysis and are therefore independent of whether or not these are the first lines of divergence in the Bacteria domain, as observed in the topology of the universal tree of ribosomal RNA . These conclusions are thus more easily understood under the hypothesis that the origin of life took place at a high temperature.

Arch Microbiol, 2004 Apr, 181(4), 269 - 77 Epub 2004 Jan 27.
Thermobaculum terrenum gen . nov., sp . nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia; Botero LM et al.; A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C . This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies . Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia . This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure . Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane . The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella . Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0 . This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light . A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen . nov., sp . nov . (16S rDNA gene GenBank accession AF391972) . This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).

Bioorg Khim, 2003 Nov-Dec, 29(6), 597 - 604
{Catechol siderophore, produced by thermoresistent strain of Bacillus licheniformis VK21}; Temirov IuV et al.; Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for producers of siderophores . It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe3+, in response to addition of manganese(II) salts . The growth of the producer on a minimum medium containing MnSO4 under the conditions of iron deficiency is accompanied by the accumulation of a catechol product, the content of which reaches a maximum at the beginning of the stationary growth phase of culture . In the presence of FeCl3, the amount of the catechol product in the medium considerably decreases . The siderophore, called SVK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA . The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm . According to amino acid analysis and NMR spectrometry, the metabolite SVK21 is 2,3-dihydroxybenzoyl-glycyl-threonine . The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol . 29, no . 6; see also http://www.maik.ru.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 227 - 33
Thermodesulfatator indicus gen . nov., sp . nov., a novel thermophilic chemolithoautotrophic sulfate-reducing bacterium isolated from the Central Indian Ridge; Moussard H et al.; A thermophilic, marine, anaerobic, chemolithoautotrophic, sulfate-reducing bacterium, strain CIR29812T, was isolated from a deep-sea hydrothermal vent site at the Kairei vent field on the Central Indian Ridge . Cells were Gram-negative motile rods that did not form spores . The temperature range for growth was 55-80 degrees C, with an optimum at 70 degrees C . The NaCl concentration range for growth was 10-35 g l(-1), with an optimum at 25 g l(-1) . The pH range for growth was 6-6.7, with an optimum at approximately pH 6.25 . H2 and CO2 were the only electron donor and carbon source found to support growth of the strain . However, several organic compounds were stimulatory for growth . Sulfate was used as electron acceptor, whereas elemental sulfur, thiosulfate, sulfite, cystine, nitrate and fumarate were not . No fermentative growth was observed with malate, pyruvate or lactate . The phenotypic characteristics of strain CIR29812T were similar to those of Thermodesulfobacterium hydrogeniphilum, a recently described thermophilic, chemolithoautotrophic sulfate-reducer . However, phylogenetic analyses of the 16S rRNA gene sequences showed that the new isolate was distantly related to members of the family Thermodesulfobacteriaceae (similarity values of less than 90%) . The chemotaxonomic data (fatty acids and polar lipids composition) also indicated that strain CIR29812T could be distinguished from Thermodesulfobacterium commune, the type species of the type genus of the family Thermodesulfobacteriaceae . Finally, the G+C content of the genomic DNA of strain CIR29812T (46.0 mol%) was not in the range of values obtained for members of this family . On the basis of phenotypic, chemotaxonomic and genomic features, it is proposed that strain CIR29812T represents a novel species of a new genus, Thermodesulfatator, of which Thermodesulfatator indicus is the type species . The type strain is CIR29812T (=DSM 15286T=JCM 11887T).

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 175 - 81
Thermovibrio ammonificans sp . nov., a thermophilic, chemolithotrophic, nitrate-ammonifying bacterium from deep-sea hydrothermal vents; Vetriani C et al.; A thermophilic, anaerobic, chemolithoautotrophic bacterium was isolated from the walls of an active deep-sea hydrothermal vent chimney on the East Pacific Rise at 9 degrees 50' N . Cells of the organism were Gram-negative, motile rods that were about 1.0 microm in length and 0.6 microm in width . Growth occurred between 60 and 80 degrees C (optimum at 75 degrees C), 0.5 and 4.5% (w/v) NaCl (optimum at 2%) and pH 5 and 7 (optimum at 5.5) . Generation time under optimal conditions was 1.57 h . Growth occurred under chemolithoautotrophic conditions in the presence of H2 and CO2, with nitrate or sulfur as the electron acceptor and with concomitant formation of ammonium or hydrogen sulfide, respectively . Thiosulfate, sulfite and oxygen were not used as electron acceptors . Acetate, formate, lactate and yeast extract inhibited growth . No chemoorganoheterotrophic growth was observed on peptone, tryptone or Casamino acids . The genomic DNA G+C content was 54.6 mol% . Phylogenetic analyses of the 16S rRNA gene sequence indicated that the organism was a member of the domain Bacteria and formed a deep branch within the phylum Aquificae, with Thermovibrio ruber as its closest relative (94.4% sequence similarity) . On the basis of phylogenetic, physiological and genetic considerations, it is proposed that the organism represents a novel species within the newly described genus Thermovibrio . The type strain is Thermovibrio ammonificans HB-1T (=DSM 15698T=JCM 12110T).

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 169 - 74
Petrotoga mexicana sp . nov., a novel thermophilic, anaerobic and xylanolytic bacterium isolated from an oil-producing well in the Gulf of Mexico; Miranda-Tello E et al.; A novel anaerobic, thermophilic, xylanolytic, motile rod-shaped bacterium with a sheath-like outer structure (toga) was isolated from a Mexican oil well in the Gulf of Mexico . Strain MET12T was a Gram-negative bacterium, reducing elemental sulfur, thiosulfate and sulfite to hydrogen sulfide . Its optimum growth conditions were 55 degrees C, pH 6.6, 3% NaCl and 0.15% MgCl2.6H2O . The DNA G+C content was 36.1 mol% . Phylogenetically, strain MET12T was related to members of genus Petrotoga, with similarities to Petrotoga mobilis, Petrotoga sibirica, Petrotoga miotherma and Petrotoga olearia varying from 97.6 to 98.8% . However DNA-DNA relatedness values between these species and strain MET12T were lower than 70% . As strain MET12T (=DSM 14811T=CIP 107371T) was genomically and phenotypically different from existing Petrotoga species, it is proposed as the type strain of a novel species, Petrotoga mexicana sp . nov.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 41 - 5
Caminibacter profundus sp . nov., a novel thermophile of Nautiliales ord . nov . within the class 'Epsilonproteobacteria', isolated from a deep-sea hydrothermal vent; Miroshnichenko ML et al.; A novel moderately thermophilic, microaerobic to anaerobic, chemolithoautotrophic bacterium, designated strain CRT, was isolated from a deep-sea hydrothermal vent site at 36 degrees N on the Mid-Atlantic Ridge . Cells were Gram-negative, non-motile rods . The organism grew at 45-65 degrees C and pH 6.5-7.4, with optimum growth at 55 degrees C and pH 6.9-7.1 . The NaCl range for growth was 5-50 g l(-1) (optimum 30 g l(-1)) . Strain CRT was an obligate chemolithoautotroph, growing with H2 as energy source, sulfur, nitrate or oxygen as electron acceptors and CO2 as carbon source . Hydrogen sulfide and ammonium were the respective products of sulfur and nitrate reduction . The G+C content of the genomic DNA was 32.1 mol% . Based on 16S rRNA gene sequence analysis, this organism was most closely related to Caminibacter hydrogeniphilus (94.9% similarity) . On the basis of phenotypic and phylogenetic data, it is proposed that the isolate represents a novel species, Caminibacter profundus sp . nov . The type strain is CRT (=DSM 15016T=JCM 11957T) . The phylogenetic data also correlate well with the significant phenotypic differences between the lineage encompassing the genera Nautilia and Caminibacter and other members of the class 'Epsilonproteobacteria' . The lineage encompassing the genera Nautilia and Caminibacter is therefore proposed as a new order, Nautiliales ord . nov., represented by a single family, Nautiliaceae fam . nov.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 33 - 9
Sulfurihydrogenibium azorense, sp . nov., a thermophilic hydrogen-oxidizing microaerophile from terrestrial hot springs in the Azores; Aguiar P et al.; Five hydrogen-oxidizing, thermophilic, strictly chemolithoautotrophic, microaerophilic strains, with similar (99-100%) 16S rRNA gene sequences were isolated from terrestrial hot springs at Furnas, Sao Miguel Island, Azores, Portugal . The strain, designated Az-Fu1T, was characterized . The motile, 0.9-2.0 microm rods were Gram-negative and non-sporulating . The temperature growth range was from 50 to 73 degrees C (optimum at 68 degrees C) . The strains grew fastest in 0.1% (w/v) NaCl and at pH 6, although growth was observed from pH 5.5 to 7.0 . Az-Fu1T can use elemental sulfur, sulfite, thiosulfate, ferrous iron or hydrogen as electron donors, and oxygen (0.2-9.0%, v/v) as electron acceptor . Az-Fu1T is also able to grow anaerobically, with elemental sulfur, arsenate and ferric iron as electron acceptors . The Az-Fu1T G+C content was 33.6 mol% . Maximum-likelihood analysis of the 16S rRNA phylogeny placed the isolate in a distinct lineage within the Aquificales, closely related to Sulfurihydrogenibium subterraneum (2.0% distant) . The 16S rRNA gene of Az-Fu1T is 7.7% different from that of Persephonella marina and 6.8% different from Hydrogenothermus marinus . Based on the phenotypic and phylogenetic characteristics presented here, it is proposed that Az-Fu1T belongs to the recently described genus Sulfurihydrogenibium . It is further proposed that Az-Fu1T represents a new species, Sulfurihydrogenibium azorense.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 25 - 32
Hydrogenimonas thermophila gen . nov., sp . nov., a novel thermophilic, hydrogen-oxidizing chemolithoautotroph within the epsilon-Proteobacteria, isolated from a black smoker in a Central Indian Ridge hydrothermal field; Takai K et al.; A novel thermophilic bacterium, strain EP1-55-1%T, was isolated from an in-situ colonization system deployed in a superheated, deep-sea, hydrothermal vent emission at the Kairei Field on the Central Indian Ridge in the Indian Ocean . The cells were highly motile rods, each possessing a single polar flagellum . Growth was observed between 35 and 65 degrees C (optimum temperature, 55 degrees C; 70 min doubling time) and between pH 4.9 and 7.2 (optimum, pH 5.9) . The isolate was a microaerobic-to-anaerobic chemolithoautotroph capable of using molecular hydrogen as the sole energy source and carbon dioxide as the sole carbon source . Molecular oxygen, nitrate or elemental sulfur (S0) could serve as electron acceptors to support growth . The G+C content of the genomic DNA was 34.6 mol% . Phylogenetic analysis based on 16S rDNA sequences indicated that strain EP1-55-1%T represents the first strain for which taxonomic properties have been characterized within the previously uncultivated phylogroup classified as belonging to the uncultivated epsilon-Proteobacteria group A; the name Hydrogenimonas thermophila gen . nov., sp . nov . is proposed, with strain EP1-55-1%T (=JCM 11971T=ATCC BAA-737T) as the type strain.

J Mol Biol, 2004 Feb 6, 336(1), 275 - 85
A chaperone network for the resolubilization of protein aggregates: direct interaction of ClpB and DnaK; Schlee S et al.; The molecular chaperones ClpB (Hsp104) and DnaK (Hsp70) co-operate in the ATP-dependent resolubilization of aggregated proteins . A sequential mechanism has been proposed for this reaction; however, the mechanism and the functional interplay between both chaperones remain poorly defined . Here, we show for the first time that complex formation of ClpB and DnaK can be detected by using various types of affinity chromatography methods . The finding that the DnaK chaperone of Escherichia coli is not co-operating with ClpB from Thermus thermophilus further strengthens the specificity of this complex . The affinity of the complex is weak and interaction between both chaperones is nucleotide-dependent . The presence of ADP, which is shown to cause dissociation of ClpB(Tth), as well as ClpB deletion mutants incapable of oligomer formation prevent ClpB-DnaK complex formation . The experiments presented indicate a correlation between the oligomeric state of ClpB and its ability to interact with DnaK . The chaperone complex described here might facilitate transfer of intermediates between ClpB and DnaK during refolding of substrates from aggregates.

J Mol Biol, 2004 Feb 6, 336(1), 241 - 51
Heterogeneous folding of the trpzip hairpin: full atom simulation and experiment; Yang WY et al.; The beta-hairpin trpzip2 can be tuned continuously from a two-state folder to folding on a rough energy landscape without a dominant refolding barrier . At high denaturant concentration, this extremely stable peptide exhibits a single apparent "two-state" transition temperature when monitored by different spectroscopic techniques . However, under optimal folding conditions the hairpin undergoes an unusual folding process with three clusters of melting transitions ranging from 15 degrees C to 160 degrees C, as monitored by 12 different experimental and computational observables . We explain this behavior in terms of a rough free energy landscape of the unfolded peptide caused by multiple tryptophan interactions and alternative backbone conformations . The landscape is mapped out by potentials of mean force derived from replica-exchange molecular dynamics simulations . Implications for deducing cooperativity from denaturant titrations, for the origin of folding cooperativity, and for the folding of thermophilic proteins are pointed out . trpzip is an excellent small tunable model system for the glass-like folding transitions predicted by landscape theory.

J Dairy Sci, 2003 Dec, 86(12), 3841 - 8
Use of dry milk protein concentrate in pizza cheese manufactured by culture or direct acidification; Shakeel-Ur-Rehman et al.; Milk protein concentrate (MPC) contains high concentrations of casein and calcium and low concentrations of lactose . Enrichment of cheese milk with MPC should, therefore, enhance yields and improve quality . The objectives of this study were: 1) to compare pizza cheese made by culture acidification using standardized whole milk (WM) plus skim milk (SM) versus WM plus MPC; and 2) compare cheese made using WM + MPC by culture acidification to that made by direct acidification . The experimental design is as follows: vat 1 = WM + SM + culture (commercial thermophilic lactic acid bacteria), vat 2 = WM + MPC + culture, and vat 3 = WM + MPC + direct acid (2% citric acid) . Each cheese milk was standardized to a protein-to-fat ratio of approximately 1.4 . The experiment was repeated three times . Yield and composition of cheeses were determined by standard methods, whereas the proteolysis was assessed by urea polyacrylamide gel electrophoresis (PAGE) and water-soluble N contents . Meltability of the cheeses was determined during 1 mo of storage, in addition to pizza making . The addition of MPC improved the yields from 10.34 +/- 0.57% in vat 1 cheese to 14.50 +/- 0.84% and 16.65 +/- 2.23%, respectively, in vats 2 and 3 and cheeses . The percentage of fat and protein recoveries showed insignificant differences between the treatments, but TS recoveries were in the order, vat 2 > vat 3 > vat 1 . Most of the compositional parameters were significantly affected by the different treatments . Vat 2 cheese had the highest calcium and lowest lactose contencentrations . Vat 3 cheese had the best meltability . Vat 1 cheese initially had better meltability than vat 2 cheese; however, the difference became insignificant after 28 d of storage at 4 degrees C . Vat 3 cheese had the softest texture and produced large-sized blisters when baked on pizza . The lowest and highest levels of proteolysis were found in vats 2 and 3 cheeses, respectively . The study demonstrates the use of MPC in pizza cheese manufacture with improved yield both by culture acidification as well as direct acidification.

J Dairy Sci, 2003 Dec, 86(12), 3831 - 40
Proteolysis on Reggianito Argentino cheeses manufactured with natural whey cultures and selected strains of Lactobacillus helveticus; Hynes ER et al.; Reggianito Argentino cheese is traditionally manufactured with whey starter cultures that provide typical and intense flavor but can cause poor quality standardization . In this study, the influence of natural and selected starters on Reggianito Argentino cheese proteolysis was investigated . Cheeses were manufactured with three strains of Lactobacillus helveticus (SF133, SF138 and SF209) cultured individually in sterile whey and used as single or mixed starters . Control cheeses were made with natural whey starter culture . Cheeses were analyzed to determine gross composition, as well as total thermophilic lactic flora . Proteolysis was assessed by N fractions, electrophoresis and liquid chromatography . Gross composition of the cheeses did not significantly differ, while viable starter cell counts were lower for cheeses made with strain SF209 alone or combined with other strains . Soluble N at pH 4.6 was the same for cheeses made with natural or selected starters, but soluble N in 12% trichloroacetic acid and 2.5% phosphotungstic acid was significantly higher in cheeses made with starters containing strain SF209 . Nitrogen fractions results indicated that natural whey starter cultures could be replaced by several starters composed of the selected strains without significant changes to proteolysis patterns . Starter cultures prepared only with SF209 or with the three selected L . helveticus strains produced cheese products with significantly more proteolysis than control cheeses . Chromatographic profiles analyzed by principal components showed that three main peaks on chromatograms, presumptively identified as Tyr, Phe, and Trp, explained most of variability . Principal component scores indicated that cheese samples were grouped by ripening time, which was confirmed by linear discriminant analysis . On the contrary, samples did not cluster by Lactobacillus strain or type of starter.

J Biol Chem, 2004 Mar 12, 279(11), 9685 - 8 Epub 2004 Jan 22.
Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme; Pavlova P et al.; During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits . Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species . At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution . At higher concentrations, however, ATP hydrolysis recovers . Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D . (2002) J . Biol . Chem . 277, 20117-20119) . We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations . Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.

Nucleic Acids Res, 2004 Jan 22, 32(2), 465 - 76 Print 2004.
A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase; Roovers M et al.; The modified nucleoside 1-methyladenosine (m(1)A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea) . In the T-loop of eukaryotic and bacterial tRNAs, m(1)A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m(1)A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m(1)I57) . In yeast, the formation of m(1)A58 is catalysed by the essential tRNA (m(1)A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide . Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer . However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m(1)A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs . The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences . The origin and evolution of m(1)A MTases is discussed in the context of different hypotheses of the tree of life.

J Biol Chem, 2004 Apr 16, 279(16), 16471 - 8 Epub 2004 Jan 20.
Distant structural homology leads to the functional characterization of an archaeal PIN domain as an exonuclease; Arcus VL et al.; Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized . One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding . The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here . Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site . The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro . The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures . These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis . They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation.

J Eukaryot Microbiol, 2003 Nov-Dec, 50(6), 427 - 9
Caspase-like activity is required for programmed nuclear elimination during conjugation in Tetrahymena; Ejercito M et al.; During conjugation in the binucleate ciliate, Tetrahymena thermophila, the old macronucleus is eliminated as new macronuclei and micronuclei are ontogenetically derived from the zygote nucleus . The mechanism of programmed nuclear elimination in ciliates may be related to the mechanism of apoptosis in higher organisms since its chromatin undergoes major condensation, its DNA is digested into nucleosome-sized fragments, and it stains positively for TUNEL . The present study explores whether caspases are involved in programmed macronuclear degradation in Tetrahymena . We show here that caspase-like activity is detectable using two specific colorimetric substrates, and that the activity is reduced with specific caspase inhibitors . In addition, using the fluorigenic substrate PhiPhiLux, active caspase-like activity is detected in living cells, localized to cytoplasmic vesicles; activity is not detected in pre- or post-condensed macronuclei . Finally, three different inhibitors of caspase activity cause a block to macronuclear chromatin condensation and elimination . Therefore, a caspase-like enzyme activity is necessary for regulating macronuclear elimination in Tetrahymena . These data support the possibility that macronuclear elimination is related, evolutionarily, to regulated cell death in multicellular organisms.

J Eukaryot Microbiol, 2003 Nov-Dec, 50(6), 403 - 8
Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila; Hosein RE et al.; Green fluorescent protein (GFP)-tagged actin was used to investigate the distribution and function of actin in Tetrahymena . A strain that expresses both GFP-actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA-based replicative vector . Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP-actin to basal bodies and the contractile ring . Incorporation of the fusion protein into these and other actin-related structures correlated with severe impairment of macronuclear elongation and cytokinesis . At 30 degrees C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division . At 20 degrees C macronuclear elongation failed to occur in 2% of the population . Arrest of cytokinesis coincided with failure of macronuclear elongation . Arrested cells developed into homopolar doublets with two sets of oral structures . This study indicates a requirement for actin in nuclear elongation and cytokinesis . Although GFP-actin can interfere with the functioning of actin-containing structures, the GFP-actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.

Mol Microbiol, 2004 Feb, 51(3), 791 - 8
A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6; Aoshima M et al.; Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced . The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters . Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate . The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s) . Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides . N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation . Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase . Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate . These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 644 - 50 Epub 2004 Jan 17.
Arabinoxylan and mono- and dimeric ferulic acid release from brewer's grain and wheat bran by feruloyl esterases and glycosyl hydrolases from Humicola insolens; Faulds CB et al.; An enzyme preparation from the thermophilic fungus Humicola insolens, Ultraflo L, was able to solubilise more than half of the biomass of brewer's grain and wheat bran, two agro-industrial co-products . While almost all of the ferulic acid was released in the free form, the majority of diferulates were released still attached to soluble feruloylated oligosaccharides, except for the 8,5' benzofuran form, which remained mostly in the residue . H . insolens also produced an esterase capable of releasing over 50% of p-coumaric acid present in wheat bran, but only 9% from the brewer's grain . The polysaccharide content in the residues after enzyme treatment comprised mostly cellulose and arabinoxylan, which suggests that part of the arabinoxylan in these residues is inaccessible to the xylanases of H . insolens . Differences in the solubilised arabinose-to-xylose ratio coupled to high free ferulate release suggest that the structure of feruloylated arabinoxylan in barley and wheat may differ.

J Biol Chem, 2004 Apr 16, 279(16), 15723 - 7 Epub 2004 Jan 16.
Trigonal DnaK-DnaJ complex versus free DnaK and DnaJ: heat stress converts the former to the latter, and only the latter can do disaggregation in cooperation with ClpB; Watanabe YH et al.; DnaK from Thermus thermophilus (TDnaK) is unique because significant fractions of cellular TDnaK exist as a trigonal K.J complex that consists of three copies each of TDnaK, TDnaJ, and an assembly factor TDafA . Here, chaperone functions of the K.J complex and free TDnaK plus free TDnaJ (K+J) were compared . Substrate proteins were completely denatured at 72-73 degrees C or 89 degrees C in the absence or the presence of K.J complex or K+J and were subsequently incubated at a moderate temperature of 55 degrees C . TGrpE and ATP were always included in the K.J complex and K+J, and TClpB was supplemented at 55 degrees C . At 72-73 degrees C, both the K.J complex and K+J suppressed heat aggregation of substrate proteins . During the next incubation at 55 degrees C, K+J, assisted by TClpB, was able to disaggregate the heat aggregates and efficiently reactivate activities of the proteins, whereas the K.J complex was not; it reactivated only the soluble inactivated proteins . When substrate proteins were heated to 89 degrees C, both the K.J complex and K+J were no longer able to prevent heat aggregation, and because of selective, irreversible denaturation of TDafA the K.J complex dissociated into K+J, which then exhibited disaggregation activity during the next incubation at 55 degrees C . Thus, TClpB-assisted disaggregation activity belongs only to K+J, and TDafA is a potential thermosensor for converting the K.J complex to K+J in response to heat stress.

Gene, 2004 Feb 4, 326, 97 - 105
Identification of a developmentally regulated translation elongation factor 2 in Tetrahymena thermophila; Malave TM et al.; Protein synthesis elongation factor 2 (eEF2) catalyzes the translocation of the peptidyl-tRNA from the A site to the P site of the ribosome . Most organisms encode a single EF2 protein and its activity is regulated by phosphorylation . We have identified a family of genes in Tetrahymena thermophila that encode proteins homologous to eEF2, yet are expressed only during sexual reproduction . These genes have been designated EFR for Elongation Factor 2 Related . EFR transcripts were not detected in vegetative cell cultures but rapidly increased about 6 h after the start of conjugation (mating) . For comparison, we cloned, sequenced and analyzed the expression of the standard eEF2 gene from T . thermophila . Unlike EFR, transcripts from eEF2 were detected in vegetative cells but were present at lower concentrations during conjugation . Despite the high sequence identity between EFR and eEF2 from other organisms (about 42% at the amino acid level), key regulatory sequences that are involved in the regulation of eEF2 are altered in EFR . The sequence and expression data suggest that EFR is an eEF2 variant involved in a major translation regulatory mechanism that occurs during the formation of the macronuclear genome in conjugating cells.

Eur J Biochem, 2004 Feb, 271(3), 517 - 23
Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis; Huang SH et al.; This study describes a modified colorimetric assay for uricase activity in flexible 96-well microtiter plates using the uricase/uric acid/horseradish peroxidase/4-aminoantipyrine/3,5-dichloro-2-hydroxybenzene sulfonate colorimetric reaction . The utility of this assay was demonstrated in a screen for mutant uricase enzymes derived from the uricase gene of the thermophilic bacterium Bacillus subtilis by a modified staggered extension process (StEP) mutagenesis . An Escherichia coli library of StEP-derived uricase mutant clones was screened yielding two identical active mutant uricase genes . Two motifs conserved in eukaryotic and prokaryotic uricases are highly conserved in the mutant uricase . The mutant uricase protein was found to exhibit high uricase activity (13.1 U.mg(-1)) . Finally, the modified colorimetric method is much more efficient than the conventional ones and greatly reduces assay time from 4 days to less than 20 h.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 671 - 4 Epub 2004 Jan 16.
CYP175A1 from Thermus thermophilus HB27, the first beta-carotene hydroxylase of the P450 superfamily; Blasco F et al.; The biological function of thermostable P450 monooxygenase CYP175A1 from Thermus thermophilus HB27 was studied by functional complementation in Escherichia coli . The gene product of CYP175A1 added hydroxyl groups to both beta rings of beta-carotene to form zeaxanthin (beta,beta-carotene-3,3'-diol) in E . coli, which produces beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes . In addition, spectroscopic methods revealed that E . coli carrying CYP175A1 and the cDNA of the Haematococcus pluvialis carotene ketolase was able to synthesise hydroxyechinenone . The predicted amino acid sequence of the enzyme from T . thermophilus does not show substantial similarity with other known beta-carotene hydroxylases, but 41% with the cytochrome P450 monooxygenase from Bacillus megaterium (CYP102A1, P450 BM3) . It is concluded that CYP175 A1 represents a new type of beta-carotene hydroxylase of the P450 superfamily.

Arch Microbiol, 2004 Mar, 181(3), 195 - 203 Epub 2004 Jan 15.
Chorismate mutase of Thermus thermophilus is a monofunctional AroH class enzyme inhibited by tyrosine; Helmstaedt K et al.; aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized . TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa . The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/ Clostridia group and have recently also been described for gram-negative organisms . The native molecular mass is consistent with a pseudo-alpha/beta barrel enzyme that is organized as a trimer . Comparison of the enzyme's structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the protein's surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase . The kinetic data show Michaelis-Menten substrate saturation with a Km of 290 microM, and a kcat/ Km value of 180 s(-1) mM(-1) . TtCM was inhibited by tyrosine with a Ki =34 microM, possibly in a competitive manner.

J Biol Chem, 2004 Apr 9, 279(15), 15177 - 82 Epub 2004 Jan 15.
NMR analysis shows that a b-type variant of Hydrogenobacter thermophilus cytochrome c552 retains its native structure; Wain R et al.; Conversion of Hydrogenobacter thermophilus cytochrome c(552) into a b-type cytochrome by mutagenesis of both heme-binding cysteines to alanines significantly reduces the stability of the protein (Tomlinson, E . J., and Ferguson, S . J . (2000) Proc . Natl . Acad . Sci . U . S . A . 97, 5156-5160) . To understand the effects of this change on the structure and dynamics of the protein, hetero-nuclear (15)N-edited NMR techniques have been used to characterize this b-type variant . The backbone (15)N, (1)H(N), and (1)H(alpha), and (1)H(beta) resonances of the protein have been assigned . Analysis of (3)J(HN)alpha coupling constants, nuclear Overhauser enhancement intensities, and chemical shift index data demonstrates that the four alpha-helices present in the wild-type protein are retained in the b-type variant . Comparison of the chemical shifts for the b-type and wild-type proteins indicates that the tertiary structures of the two proteins are closely similar . Some subtle differences are, however, observed for residues in the N-terminal region and in the vicinity of the heme-binding pocket . Hydrogen exchange studies show that there are 25 backbone amide protons that exchange very slowly in the b-type variant and confirm that the fluctuations within the b-type protein are of a similar extent to those in the wild-type protein . These data demonstrate the notable retention of the native secondary structure and tertiary fold despite the absence of covalent linkages between the heme group and the protein.

Gut, 2004 Feb, 53(2), 241 - 5
Randomised clinical trial of synbiotic therapy in elective surgical patients; Anderson AD et al.; BACKGROUND: It is possible to manipulate the composition of the gastrointestinal microflora by administration of pre- and probiotics . This may help to preserve gut barrier function and reduce the incidence of septic morbidity . AIMS: To assess the effects of a combination of pre- and probiotics (synbiotic) on bacterial translocation, gastric colonisation, systemic inflammation, and septic morbidity in elective surgical patients . PATIENTS: Patients were enrolled two weeks prior to elective abdominal surgery . Seventy two patients were randomised to the synbiotic group and 65 to the placebo group . Patients were well matched regarding age and sex distribution, diagnoses, and POSSUM scores . METHODS: Patients in the synbiotic group received a two week preoperative course of Lactobacillus acidophilus La5, Bifidobacterium lactis Bb-12, Streptococcus thermophilus, and Lactobacillus bulgaricus, together with the prebiotic oligofructose . Patients in the placebo group received placebo capsules and sucrose powder . At surgery, a nasogastric aspirate, mesenteric lymph node, and scrapings of the terminal ileum were harvested for microbiological analysis . Serum was collected preoperatively and on postoperative days 1 and 7 for measurement of C reactive protein, interleukin 6, and antiendotoxin antibodies . Septic morbidity and mortality were recorded . RESULTS: There were no significant differences between the synbiotic and control groups in bacterial translocation (12.1% v 10.7%; p = 0.808, chi(2)), gastric colonisation (41% v 44%; p = 0.719), systemic inflammation, or septic complications (32% v 31%; p = 0.882) . CONCLUSIONS: In this study, synbiotics had no measurable effect on gut barrier function in elective surgical patients . Further studies investigating the place of pre- and probiotics in clinical practice are required.

Water Res, 2004 Feb, 38(3), 619 - 30
Long-term effects of operating temperature and sulphate addition on the methanogenic community structure of anaerobic hybrid reactors; Pender S et al.; The diversity, population dynamics, and activity profiles of methanogens in anaerobic granular sludges from two anaerobic hybrid reactors treating a molasses wastewater both mesophilically (37 degrees C) and thermophilically (55 degrees C) during a 1081 day trial were determined . The influent to one of the reactors was supplemented with sulphate, after an acclimation period of 112 days, to determine the effect of competition with sulphate-reducing bacteria on the methanogenic community structure . Sludge samples were removed from the reactors at intervals throughout the operational period and examined by amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) and partial sequencing of 16S rRNA genes . In total, 18 operational taxonomic units (OTUs) were identified, 12 of which were sequenced . The methanogenic communities in both reactors changed during the operational period . The seed sludge and the reactor biomass sampled during mesophilic operation, both in the presence and absence of sulphate, was characterised by a predominance of Methanosaeta spp . Following temperature elevation, the dominant methanogenic sequences detected in the non-sulphate supplemented reactor were closely related to Methanocorpusculum parvum . By contrast, the dominant OTUs detected in the sulphate-supplemented reactor upon temperature increase were related to the hydrogen-utilising methanogen, Methanobacterium thermoautotrophicum . The observed methanogenic community structure in the reactors correlated with the operational performance of the reactors during the trial and with physiological measurements of the reactor biomass . Both reactors achieved chemical oxygen demand (COD) removal efficiencies of over 90% during mesophilic operation, with or without sulphate supplementation . During thermophilic operation, the presence of sulphate resulted in decreased reactor performance (effluent acetate concentrations of >3000 mg/l and biogas methane content of <25%) . It was demonstrated that methanogenic conversion of acetate at 55 degrees C was extremely sensitive to inhibition by sulphide (50% inhibition at 8-17 mg/l unionised sulphide at pH 7.6-8.0), while the conversion of H(2)/CO(2) methanogenically was favoured . The combination of experiments carried out demonstrated the presence of specific methanogenic populations during periods of successful operational performance.

J Appl Microbiol, 2004, 96(2), 340 - 51
Characterization of three Lactobacillus delbrueckii subsp . bulgaricus phages and the physicochemical analysis of phage adsorption; Quiberoni A et al.; AIMS: Three indigenous Lactobacillus delbrueckii subsp . bulgaricus bacteriophages and their adsorption process were characterized . METHODS AND RESULTS: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1) . They showed low burst size and short latent periods . A remarkably high sensitivity to pH was also demonstrated . Indigenous phage genomes were linear and double-stranded DNA molecules of approx . 31-34 kbp, with distinctive restriction patterns . Only one phage genome appeared to contain cohesive ends . Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation . The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50 degrees C . SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably . No significant inhibitory effect on phage adsorption was observed for the saccharides tested . This study also revealed the irreversibility of phage adsorption to their hosts . CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on phages of thermophilic lactic acid bacteria.

J Appl Microbiol, 2004, 96(2), 263 - 70
PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese; Ercolini D et al.; AIMS: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product . METHODS AND RESULTS: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture . Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments . The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC . Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms . LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far . Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture . CONCLUSIONS: The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development . SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.

J Appl Microbiol, 2004, 96(2), 223 - 9
Peptidase activity in various species of dairy thermophilic lactobacilli; Gatti M et al.; AIMS: The aim of the present work was to evaluate the enzymatic potential manifested by aminopeptidase activity of different thermophilic Lactobacillus biotypes and to measure the influence of cell growth phase on enzyme expression . METHODS AND RESULTS: The activities were evaluated by the hydrolysis of beta-naphthylamide substrates for both whole and mechanically disrupted cells of L . helveticus, L . delbrueckii subsp . bulgaricus and L . delbrueckii subsp . lactis strains, collected from both the exponential and the stationary growth phase . In general, activities were higher for cells in the exponential rather than in the stationary phase and the disrupted cells showed higher activities than the whole cells . The highest activity expressed by all strains corresponded to X-prolyl-dipeptidyl aminopeptidase while a moderate activity was observed towards Arg-betaNa, Lys-betaNa and Leu-betaNa . The lowest activity was observed for Pro-betaNa . CONCLUSIONS: It may be inferred that the cell structure and the cell physiology are crucial to define the level of efficiency of expression for aminopeptidase activity . The two species may be characterized by a different enzymatic system that hydrolyses N-terminal leucine . SIGNIFICANCE AND IMPACT OF THE STUDY: The differences of peptidase activities in L . helveticus and L . delbrueckii species acquires an importance to comprehend their role in the biochemical events occurring in cheese ripening.

Parasitol Res, 2004 Mar, 92(4), 289 - 98 Epub 2004 Jan 13.
Characterisation and differentiation of pathogenic and non-pathogenic Acanthamoeba strains by their protein and antigen profiles; Walochnik J et al.; Free-living amoebae of the genus Acanthamoeba are the causative agents of Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis . Acanthamoebae occur ubiquitously in the environment and are thus a constant cause of antigenic stimulation . In a previous study we have shown that compared to control sera, AK patients exhibit markedly lower immunoreactivities to whole cell antigen of Acanthamoeba spp . As the pathogenicity of acanthamoebae primarily relies on the excretion of proteins, it was the aim of the present study to investigate the immunoreactivity of metabolic antigen from different Acanthamoeba strains of varying pathogenicity . Three Acanthamoeba strains, one highly pathogenic, one non-pathogenic but thermophilic and one non-thermophilic non-pathogenic, were used for antigen extraction . The antigen was harvested before and after contact with human cells and all strains were tested with AK sera and with sera from healthy individuals . It was shown that the somatic protein profiles of the Acanthamoeba strains correlated to the morphological groups, and that within morphological group II-the group associated with AK-the profiles of the metabolic antigens correlated to strain pathogenicity . Moreover, it was shown that the control sera showed markedly higher immunoreactivities than the sera of the AK patients and that this immunoreactivity was generally higher to the non-pathogenic strains than to the pathogenic strain . Altogether our results once again raise the question of whether there is an immunological predisposition in AK . To our knowledge this is the first study on the immunoreactivity of metabolic antigen of acanthamoebae.

Am J Vet Res, 2004 Jan, 65(1), 45 - 52
Comparison of the type and number of microorganisms and concentration of endotoxin in the air of feedyards in the Southern High Plains; Purdy CW et al.; OBJECTIVES: To determine the bacterial, fungal, and endotoxin concentrations in aerosolized ambient air during the winter and summer in feedyards located in the Southern High Plains, identify aerosolized microbial pathogens, and determine the size of microbial and dust components . SAMPLE POPULATION: Aerosol samples were obtained from 7 feedyards . PROCEDURE: Aerosol samples were collected upwind, on-site, and downwind from each feedyard at a point 1 m above the ground by use of biological 2- and 6-stage cascade impactors . RESULTS: Significantly more microbes were cultured from on-site and downwind samples than upwind samples . There were significantly more microbes during the summer than during the winter . However, mean endotoxin concentration was significantly higher during the winter (8.37 ng/m3) than the summer (2.63 ng/m3) . Among 7 feedyards, mean +/- SE number of mesophilic bacteria (1,441 +/- 195 colony-forming units {CFUs}/m3) was significantly higher than mean number of anaerobic bacteria (751 +/- 133 CFUs/m3) or thermophilic bacteria (54 +/- 10 CFUs/m3) in feedyard air . Feedyard aerosol samples contained more mesophilic fungi (78 +/- 7 CFUs/m3) than thermophilic fungi (2 +/- 0.2 CFUs/m3) . Eighteen genera of bacteria were identified by use of an automated identification system . CONCLUSIONS AND CLINICAL RELEVANCE: It appeared that gram-negative enteric pathogens offered little risk to remote calves or humans via ambient aerosols and that gram-positive pathogens of the Bacillus, Corynebacterium, and Staphylococcus spp can be spread by aerosols in and around feedyards . It was common to detect concentrations of endotoxin in the ambient air of 7 feedyards.

J Exp Biol, 2004 Feb, 207(Pt 4), 579 - 85
The thermogenesis of digestion in rattlesnakes; Tattersall GJ et al.; Some snakes have a feeding regime characterized by the infrequent ingestion of relatively large meals, causing impressive increments in post-prandial metabolism . Metabolism remains elevated for many days, while digestion proceeds, resulting in considerable investment of time and energy . Snakes actively adjust thermoregulatory behavior to raise their body temperature during digestion, exhibiting a post-prandial thermophilic response that accelerates digestion at the expense of higher metabolic rates . In the present study, we investigated the possibility that endogenously derived heat, originating as a byproduct of the post-prandial increase in metabolism, could itself contribute to the elevated body temperature during digestion in the South American rattlesnake Crotalus durissus . We assessed heat production, at a constant environmental temperature, by taking infrared (IR) images of snakes during fasting and after being fed meals varying from 10% to 50% of their own body masses . Our results show clearly that digesting rattlesnakes have significantly increased body temperatures, even when precluded from adjusting their thermoregulatory behavior . The feeding-derived thermogenesis caused the surface body temperature of rattlesnakes to increase by 0.9-1.2 degrees C, a temperature change that will significantly affect digestive performance . The alterations in body temperature following feeding correlated closely with the temporal profile of changes in post-prandial metabolism . Moreover, the magnitude of the thermogenesis was greater for snakes fed large meals, as was the corresponding metabolic response . Since IR imaging only assesses surface temperatures, the magnitude of the thermogenesis and the changes in deep core temperature could be even more pronounced than is reported here.

BMC Microbiol . 2004 Jan 12;4(1):2.
The single-stranded DNA-binding protein of Deinococcus radiodurans; Eggington JM et al.; BACKGROUND: Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation . Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair . The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein . The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms . Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored . RESULTS: A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented . The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF) . The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds . The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins . Like the Thermus SSB proteins, the SSBDr functions as a homodimer . The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer . CONCLUSIONS: The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date . The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer . The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions . The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

Eur J Biochem, 2004 Jan, 271(2), 429 - 38
Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding; Miyanaga A et al.; Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated . As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones . In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center . The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value . The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions . It showed no detectable activity . A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure . In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases . Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one . The alphaT109S mutant showed similar characteristics to the wild-type enzyme . However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed . The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P . thermophila.

Eur J Biochem, 2004 Jan, 271(2), 272 - 80
Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus; Bugaytsova Z et al.; The moderately thermophilic bacterium Acidithiobacillus caldus is found in bacterial populations in many bioleaching operations throughout the world . This bacterium oxidizes elemental sulfur and other reduced inorganic sulfur compounds as the sole source of energy . The purpose of this study was to purify and characterize the tetrathionate hydrolase of A . caldus . The enzyme was purified 16.7-fold by one step chromatography using a SP Sepharose column . The purified enzyme resolved into a single band in 10% polyacrylamide gel, both under denaturing and native conditions . Its homogeneity was confirmed by N-terminal amino acid sequencing . Tetrathionate hydrolase was shown to be a homodimer with a molecular mass of 103 kDa (composed from two 52 kDa monomers) . The purified enzyme had optimum activity at pH 3.0 and 40 degrees C and an isoelectric point of 9.8 . The periplasmic localization of the enzyme was determined by differential fractionation of A . caldus cells . Detected products of the tetrathionate hydrolase reaction were thiosulfate and pentathionate as confirmed by RP-HPLC analysis . The activity of the purified enzyme was drastically enhanced by divalent metal ions.

Cell Biochem Biophys, 2003, 39(3), 175 - 81
Association of alpha-subunits with nucleotide-modified beta-subunits induces asymmetry in the catalytic sites of the F1-ATPase alpha3beta3-hexamer; Burgard S et al.; The photoaffinity spin-labeled ATP analog, 2-N3-SL-adenosine triphosphate (ATP), was used to covalently modify isolated beta-subunits from F1-ATPase of the thermophilic bacterium PS3 . Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark . Irradiation leads to covalent incorporation of the nucleotide into the binding site . ESR spectra of the complex show a signal that is typical for protein-immobilized radicals . Addition of isolated alpha-subunits to the modified beta-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites . The relative ratio of the signals is approx 2:1 in favor of the more closed conformation . The data show for the first time that when nucleotides are bound to isolated beta-subunits, binding of alpha-subunits induces asymmetry in the catalytic sites even in the absence of the gamma-subunit.

Appl Environ Microbiol, 2004 Jan, 70(1), 182 - 90
Biodiversity among Lactobacillus helveticus strains isolated from different natural whey starter cultures as revealed by classification trees; Gatti M et al.; Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium used extensively for manufacturing Swiss type and aged Italian cheese . In this study, the phenotypic and genotypic diversity of strains isolated from different natural dairy starter cultures used for Grana Padano, Parmigiano Reggiano, and Provolone cheeses was investigated by a classification tree technique . A data set was used that consists of 119 L . helveticus strains, each of which was studied for its physiological characters, as well as surface protein profiles and hybridization with a species-specific DNA probe . The methodology employed in this work allowed the strains to be grouped into terminal nodes without difficult and subjective interpretation . In particular, good discrimination was obtained between L . helveticus strains isolated, respectively, from Grana Padano and from Provolone natural whey starter cultures . The method used in this work allowed identification of the main characteristics that permit discrimination of biotypes . In order to understand what kind of genes could code for phenotypes of technological relevance, evidence that specific DNA sequences are present only in particular biotypes may be of great interest.

Appl Environ Microbiol, 2004 Jan, 70(1), 137 - 44
Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melanocarpus albomyces; Kiiskinen LL et al.; The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces . This gene has five introns, and it encodes a protein consisting of 623 amino acids . The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes . In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension . M . albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter . Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences . The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S . cerevisiae alpha-factor gene . The role of the C-terminal extension in laccase production in S . cerevisiae was also studied . Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus.

Appl Environ Microbiol, 2004 Jan, 70(1), 96 - 103
Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303; Deutsch SM et al.; phi-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction . In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of phi-0303 in Escherichia coli DH5alpha . The lytic activity was detected by its expression, using whole cells of the sensitive strain L . helveticus CNRZ 892 as the substrate . The lysin gene was within a 4.1-kb DNA fragment of phi-0303 containing six open reading frames (ORFs) and two truncated ORFs . No sequence homology with holin genes was found within the cloned fragment . An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene . The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned . The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da . Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages . Mur-LH was expressed in E . coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L . helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa . The N-terminal sequence of the protein confirmed the start codon . Hydrolysis of cell walls of L . helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity . Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.

Appl Environ Microbiol, 2004 Jan, 70(1), 18 - 24
Identification of distinct Campylobacter lari genogroups by amplified fragment length polymorphism and protein electrophoretic profiles; Duim B et al.; Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters (NARTC) and the biochemical C . lari variants, including the urease-positive campylobacters (UPTC), the nalidixic acid-susceptible campylobacters (NASC), and the urease-producing nalidixic acid-susceptible campylobacters . To study the taxonomic and epidemiological relationships among strains of the C . lari variants, amplified fragment length polymorphism (AFLP) profiling and whole-cell protein profile analysis were performed with 55 C . lari strains . Great genetic heterogeneity in AFLP and protein profiles was observed . Numerical analysis of AFLP profiles and of partial protein profiles allowed discrimination of four distinct genogroups . AFLP cluster I included nearly homogeneous patterns for C . lari NARTC strains (genogroup I) . UPTC strains together with non-urease-producing NASC strains produced highly diverse patterns and were placed in genogroup II . The genogroup III strains had the NASC phenotype and produced more homogeneous patterns . Finally, genogroup IV strains had the classical NARTC phenotype and produced AFLP patterns that were very distinct from those of other genogroups . One UPTC strain had aberrant patterns and clustered separately, which may indicate that there is an additional genogroup . Preliminary DNA-DNA hybridization experiments suggested that genogroups I and III represent a single genomic species and that genogroup IV represents a distinct species . The detection of moderate levels of DNA-DNA hybridization between a genogroup II reference strain and genogroup I and III reference strains highlights the need for further DNA-DNA hybridization experiments to clarify the taxonomic status of the former group . No correlation of genogroups with different sources of strains was identified . These data show that UPTC strains are genetically diverse and distinct from NARTC strains . In addition, they indicate that the classical NARTC phenotype encompasses at least two genogroups.

Plasmid, 2004 Jan, 51(1), 24 - 36
Characterization of a theta-replicating plasmid from Streptococcus thermophilus; Turgeon N et al.; Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-D) . Here, we report the characterization of plasmids of group B and D . The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family . The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs . Two ORFs likely encode a primase/helicase and an integrase . Northern blot experiments demonstrate that these two orfs are transcribed within the three strains containing plasmids of group B . Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta mechanism . To our knowledge, this is the first report of a plasmid replicating via a theta mode in S . thermophilus . Finally, a classification of 20 sequenced S . thermophilus plasmids into six groups based on their mode of replication is proposed.

EMBO Rep, 2004 Jan, 5(1), 66 - 70
Adaptation to extreme environments: macromolecular dynamics in bacteria compared in vivo by neutron scattering; Tehei M et al.; Mean macromolecular dynamics was quantified in vivo by neutron scattering in psychrophile, mesophile, thermophile and hyperthermophile bacteria . Root mean square atomic fluctuation amplitudes determining macromolecular flexibility were found to be similar for each organism at its physiological temperature ( approximately 1 A in the 0.1 ns timescale) . Effective force constants determining the mean macromolecular resilience were found to increase with physiological temperature from 0.2 N/m for the psychrophiles, which grow at 4 degrees C, to 0.6 N/m for the hyperthermophiles (85 degrees C), indicating that the increase in stabilization free energy is dominated by enthalpic rather than entropic terms . Larger resilience allows macromolecular stability at high temperatures, while maintaining flexibility within acceptable limits for biological activity.

J Am Chem Soc, 2004 Jan 14, 126(1), 88 - 95
Chemically distinct Ni sites in the A-cluster in subunit beta of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila: Ni L-edge absorption and X-ray magnetic circular dichroism analyses; Funk T et al.; The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of acetate C-C bond cleavage in which acetyl-CoA serves as the acetyl donor substrate reacting at the ACDS beta subunit active site . The properties of Ni in the active site A-cluster in the ACDS beta subunit from Methanosarcina thermophila were investigated . A recombinant, C-terminally truncated form of the beta subunit was employed, which mimics the native subunit previously isolated from the ACDS complex, and contains an A-cluster composed of an {Fe(4)S(4)} center bridged to a binuclear Ni-Ni site . The electronic structures of these two Ni were studied using L-edge absorption and X-ray magnetic circular dichroism (XMCD) spectroscopy . The L-edge absorption data provided evidence for two distinct Ni species in the as-isolated enzyme, one with low-spin Ni(II) and the other with high-spin Ni(II) . XMCD spectroscopy confirmed that the species producing the high-spin signal was paramagnetic . Upon treatment with Ti(3+) citrate, an additional Ni species emerged, which was assigned to Ni(I) . By contrast, CO treatment of the reduced enzyme converted nearly all of the Ni in the sample to low-spin Ni(II) . The results implicate reaction of a high-spin tetrahedral Ni site with CO to form an enzyme-CO adduct transformed to a low-spin Ni(II) state . These findings are discussed in relation to the mechanism of C-C bond activation, in connection with the model of the beta subunit A-cluster developed from companion Ni and Fe K edge, XANES, and EXAFS studies.

J Agric Food Chem, 2004 Jan 14, 52(1), 8 - 14
Development of a new method, based on a bioreactor coupled with an L-lactate biosensor, toward the determination of a nonspecific inhibition of L-lactic acid production during milk fermentation; Zaydan R et al.; The development and characteristics of a bioreactor employing bacteria (Streptococcus thermophilus) encapsulated in Ca-alginate beads coupled with an L-lactate biosensor are reported . The biosensor comprises a carbon paste electrode modified with enzymes HRP (horseradish peroxidase), LOD (lactate oxidase), and FcH (ferrocene) as redox mediator . The measurement of L-lactate is based on the signal produced by H(2)O(2), the product of the enzymatic oxidation of L-lactate by LOD . The detection of H(2)O(2) is performed at the electrode surface via HRP/FcH at low operating potential (-100mV vs Ag/AgCl) . Optimization studies were performed using the bioreactor in conjunction with an L-lactate electrode operating in a flow injection system to assess the ability of encapsulated bacteria to ferment carbohydrate solutions . The possibility of using the developed method to assess the fermentation capability of milk samples was evaluated . Bronopol (2-bromo-2-nitro propane-1,3-diol) was chosen to simulate the effect of an inhibitory agent of milk fermentation . The obtained results indicated that the evaluation of the amount of L-lactate amount produced through the bioreactor could be used as a measure of inhibition of lactic acid production in milk samples.

Genome Res, 2004 Jan, 14(1), 109 - 15
A cross-genomic approach for systematic mapping of phenotypic traits to genes; Jim K et al.; We present a computational method for de novo identification of gene function using only cross-organismal distribution of phenotypic traits . Our approach assumes that proteins necessary for a set of phenotypic traits are preferentially conserved among organisms that share those traits . This method combines organism-to-phenotype associations,along with phylogenetic profiles,to identify proteins that have high propensities for the query phenotype; it does not require the use of any functional annotations for any proteins . We first present the statistical foundations of this approach and then apply it to a range of phenotypes to assess how its performance depends on the frequency and specificity of the phenotype . Our analysis shows that statistically significant associations are possible as long as the phenotype is neither extremely rare nor extremely common; results on the flagella,pili, thermophily,and respiratory tract tropism phenotypes suggest that reliable associations can be inferred when the phenotype does not arise from many alternate mechanisms.

DNA Repair (Amst), 2004 Feb 3, 3(2), 163 - 9
Multiple uracil-DNA glycosylase activities in Deinococcus radiodurans; Sandigursky M et al.; The extremely radiation resistant bacterium, Deinococcus radiodurans, contains a spectrum of genes that encode for multiple activities that repair DNA damage . We have cloned and expressed the product of three predicted uracil-DNA glycosylases to determine their biochemical function . DR0689 is a homologue of the Escherichia coli uracil-DNA glycosylase, the product of the ung gene; this activity is able to remove uracil from a U : G and U : A base pair in double-stranded DNA and uracil from single-stranded DNA and is inhibited by the Ugi peptide . DR1751 is a member of the class 4 family of uracil-DNA glycosylases such as those found in the thermophiles Thermotoga maritima and Archaeoglobus fulgidus . DR1751 is also able to remove uracil from a U : G and U : A base pair; however, it is considerably more active on single-stranded DNA . Unlike its thermophilic relatives, the enzyme is not heat stable . Another putative enzyme, DR0022, did not demonstrate any appreciable uracil-DNA glycosylase activity . DR0689 appears to be the major activity in the organism based on inhibition studies with D . radiodurans crude cell extracts utilizing the Ugi peptide . The implications for D . radiodurans having multiple uracil-DNA glycosylase activities and other possible roles for these enzymes are discussed.

Exp Dermatol, 2003 Oct, 12(5), 615 - 20
Effect of the lactic acid bacterium Streptococcus thermophilus on stratum corneum ceramide levels and signs and symptoms of atopic dermatitis patients; Di Marzio L et al.; A reduced amount of total ceramides could be responsible for functional abnormalities of the skin of atopic dermatitis (AD) patients . The ability of an experimental cream containing sonicated Streptococcus thermophilus to increase skin ceramide levels in healthy subjects has been previously reported . The aim of the present work was to investigate the effects of the topical administration of a S . thermophilus-containing cream on ceramide levels of stratum corneum from AD patients . A 2-week application of the cream, containing a sonicated preparation of the lactic acid bacterium S . thermophilus, in the forearm skin of 11 patients led to a significant and relevant increase of skin ceramide amounts, which could have resulted from the sphingomyelin hydrolysis through the bacterial sphingomyelinase . Moreover, in all patients the topical application of our experimental cream also resulted in the improvement of the signs and symptoms characteristic of AD skin (i.e . erythema, scaling, pruritus).

Water Environ Res, 2003 Nov-Dec, 75(6), 539 - 48
Effect of solids retention time on the performance of thermophilic and mesophilic digestion of combined municipal wastewater sludges; Moen G et al.; The steady-state performance of thermophilic (55 degrees C) and mesophilic (35 degrees C) anaerobic digestion as a function of solids retention time (SRT) was evaluated in laboratory digesters at SRTs ranging from 4 to 15 days, and in pilot-plant digesters at a 20-day SRT . All of the digesters were fed the same source of municipal combined primary and secondary waste sludge . The destruction efficiency of volatile solids increased from 53% to 66% as the SRT was increased from 6 days to 20 days . The average destruction efficiency of volatile solids was 3 percentage points higher for the thermophilic digester at the 6-day SRT and approximately 1 percentage point higher for the higher SRTs, but the difference was only statistically significant at the 15-day SRT . Based on volatile suspended solids measurements, the thermophilic solids destruction efficiency was approximately 4 percentage points higher at the 10- and 15-day SRTs . At a 4-day SRT, methanogenic activity could only be maintained in the thermophilic digester . The pH, alkalinity, ammonia, volatile fatty acid, and soluble chemical oxygen demand concentrations were higher for the thermophilic digester at each SRT . At SRTs of 10 days and less, the thermophilic digester had a much higher propionate and slightly higher butyrate concentration . Carbohydrates were readily degraded by both digesters, protein was the major component in the sludge at the long SRTs, and lipid degradation increased with increasing SRT.

J Biol Chem, 2004 Mar 26, 279(13), 12860 - 7 Epub 2003 Dec 31.
Structural studies on flavin reductase PheA2 reveal binding of NAD in an unusual folded conformation and support novel mechanism of action; van den Heuvel RH et al.; The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system . The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism . The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols . We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 A), which is the first structure of a member of this flavin reductase family . We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 A) . PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix . The tightly bound FAD prosthetic group (K(d) = 10 nm) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent . The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking . This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide . The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product . Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate . In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis.

J Comput Aided Mol Des, 2003 Aug, 17(8), 537 - 49
Representation, searching and discovery of patterns of bases in complex RNA structures; Harrison AM et al.; We describe a graph theoretic method designed to perform efficient searches for substructural patterns in nucleic acid structural coordinate databases using a simplified vectorial representation . Two vectors represent each nucleic acid base and the relative positions of bases with respect to one another are described in terms of distances between the defined start and end points of the vectors on each base . These points comprise the nodes and the distances the edges of a graph, and a pattern search can then be performed using a subgraph isomorphism algorithm . The minimal representation was designed to facilitate searches for complex patterns but was first tested on simple, well-characterised arrangements of bases such as base pairs and GNRA-tetraloop receptor interactions . The method performed very well for these interaction types . A survey of side-by-side base interactions, of which the adenosine platform is the best known example, also locates examples of similar base rearrangements that we consider to be important in structural regulation . A number of examples were found, with GU platforms being particularly prevalent . A GC platform in the RNA of the Thermus thermophilus small ribosomal subunit is in an analogous position to an adenosine platform in other species . An unusual GG platform is also observed close to one of the substrate binding sites in Haloarcula marismortui large ribosomal subunit RNA.

J Bacteriol, 2004 Jan, 186(2), 570 - 4
Cloning and characterization of acetohydroxyacid synthase from Bacillus stearothermophilus; Porat I et al.; Five genes from the ilv-leu operon from Bacillus stearothermophilus have been sequenced . Acetohydroxyacid synthase (AHAS) and its subunits were separately cloned, purified, and characterized . This thermophilic enzyme resembles AHAS III of Escherichia coli, and regulatory subunits of AHAS III complement the catalytic subunit of the AHAS of B . stearothermophilus, suggesting that AHAS III is functionally and evolutionally related to the single AHAS of gram-positive bacteria.

J Bacteriol, 2004 Jan, 186(2), 351 - 5
Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus; Wilquet V et al.; A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T . thermophilus dehydrogenase {DH(Tt)} gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate) . DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A . Henne, personal communication) . The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family . The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group . We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.

J Biol Chem, 2004 Mar 19, 279(12), 11825 - 33 Epub 2003 Dec 29.
Induced fit movements and metal cofactor selectivity of class II aldolases: structure of Thermus aquaticus fructose-1,6-bisphosphate aldolase; Izard T et al.; Fructose-1,6-bisphosphate (FBP) aldolase is an essential glycolytic enzyme that reversibly cleaves its ketohexose substrate into triose phosphates . Here we report the crystal structure of a metallo-dependent or class II FBP aldolase from an extreme thermophile, Thermus aquaticus (Taq) . The quaternary structure reveals a tetramer composed of two dimers related by a 2-fold axis . Taq FBP aldolase subunits exhibit two distinct conformational states corresponding to loop regions that are in either open or closed position with respect to the active site . Loop closure remodels the disposition of chelating active site histidine residues . In subunits corresponding to the open conformation, the metal cofactor, Co(2+), is sequestered in the active site, whereas for subunits in the closed conformation, the metal cation exchanges between two mutually exclusive binding loci, corresponding to a site at the active site surface and an interior site vicinal to the metal-binding site in the open conformation . Cofactor site exchange is mediated by rotations of the chelating histidine side chains that are coupled to the prior conformational change of loop closure . Sulfate anions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not only the previously unknown second phosphate-binding site but also provide a mechanism that regulates loop closure during catalysis . Modeling of FBP substrate into the active site is consistent with binding by the acyclic keto form, a minor solution species, and with the metal cofactor mediating keto bond polarization . The Taq FBP aldolase structure suggests a structural basis for different metal cofactor specificity than in Escherichia coli FBP aldolase structures, and we discuss its potential role during catalysis . Comparison with the E . coli structure also indicates a structural basis for thermostability by Taq FBP aldolase.

Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 570 - 5
Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication; Furuya T et al.; The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50 degrees C . The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases . The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B . subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase . Thus, to clone a gene encoding flavin reductase from B . subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E . coli was used as a selection . Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity . The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens . In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B . subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55 degrees C . This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.

Proteins, 2004 Feb 1, 54(2), 323 - 32
Phenomenological similarities between protein denaturation and small-molecule dissolution: Insights into the mechanism driving the thermal resistance of globular proteins; Ragone R; This article shows that the stability profiles of thermophilic proteins are significantly displaced toward higher temperatures as compared to those of mesophilic proteins . A similar trend characterizes the aqueous transfer of N-alkyl amides . In fact, as a general feature of transfer processes, liquid dissolution profiles are centered at temperatures higher than those of solid ones . This behavior is governed by packing contributions . A partition of the unfolding thermodynamics based on the analysis of phenomenological temperatures common to dissolution and unfolding phenomena provides a clue to understanding the mechanism of thermal stabilization . In fact, the position of stability profiles along the temperature axis does not appear to depend on solvation of internal residues . Instead, it is notably affected by solidlike components, whose progressive decrease appears to drive the heat denaturation temperature increase of most thermostable proteins . As a corollary, it is shown that there are actually two limiting mechanisms of thermal stabilization .

Biophys J, 2004 Jan, 86(1 Pt 1), 420 - 7
Crystal structure of the hyperthermophilic inorganic pyrophosphatase from the archaeon Pyrococcus horikoshii; Liu B et al.; A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii . This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88 degrees C . To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 A resolution . The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry . The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius . A detailed comparison of the crystal structure of Pho-PPase with related structures from S . acidocaldarius, Thermus thermophilus, and Escherichia coli shows significant differences that may account for the difference in their thermostabilities . A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase . In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus . The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.

Cell Motil Cytoskeleton, 2004 Feb, 57(2), 73 - 83
Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila; Deckman CM et al.; In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming . The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known . Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli . We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+ . In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming . These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal . The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal . An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus . Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+ .

Protein Sci, 2004 Jan, 13(1), 134 - 44
Expression and biochemical characterization of two small heat shock proteins from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7; Usui K et al.; We expressed and characterized two sHsps, StHsp19.7 and StHsp14.0, from a thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 . StHsp19.7 forms a filamentous structure consisting of spherical particles and lacks molecular chaperone activity . Fractionation of Sulfolobus extracts by size exclusion chromatography with immunoblotting indicates that StHsp19.7 exists as a filamentous structure in vivo . On the other hand, StHsp14.0 exists as a spherical oligomer like other sHsps . It showed molecular chaperone activity to protect thermophilic 3-isopropylmalate dehydrogenase (IPMDH) from thermal aggregation at 87 degrees C . StHsp14.0 formed variable-sized complexes with denatured IPMDH at 90 degrees C . Using StHsp14.0 labeled with fluorescence or biotin probe and magnetic separation, subunit exchanges between complexes were demonstrated . This is the first report on the filament formation of sHsp and also the high molecular chaperone activity of thermophilic archaeal sHsps.

Protein Sci, 2004 Jan, 13(1), 89 - 99
Thermostability of multidomain proteins: elongation factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms; Sanderova H et al.; Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF-Tus, their isolated G-domains, and six chimeric EF-Tus composed of domains of either EF-Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized . BstEF-Tu and BstG-domain bound GDP and GTP with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of EcEF-Tu . In contrast, the EcG-domain bound the nucleotides with much lower, micromolar affinities . The exchange of domains 2 and 3 had essentially no effect on the GDP-binding activity; all complexes of chimeric EF-Tus with GDP retained K(d) values in the nanomolar range . The final thermostability level of either EF-Tu was the result of a cooperative interaction between the G-domains and domains 2 + 3 . The G-domains set up a "basic" level of the thermostability, which was approximately 20 degrees C higher with the BstG-domain than with the EcG-domain . This correlated with the growth temperature optimum difference of both bacteria and two distinct thermostabilization features of the BstG-domain: an increase of charged residues at the expense of polar uncharged residues (CvP bias), and a decrease in the nonpolar solvent-accessible surface area . Domains 2 + 3 contributed by further stabilization of alpha-helical regions and, in turn, the functions of the G-domains to the level of the respective growth temperature optima . Their contributions were similar irrespective of their origin but, with Ecdomains 2 + 3, dependent on the guanine nucleotide binding state . It was lower in the GTP conformation, and the mechanism involved the destabilization of the alpha-helical regions of the G-domain by Ecdomain 2.

Traffic, 2004 Feb, 5(2), 63 - 8
Out with a bang! Tetrahymena as a model system to study secretory granule biogenesis; Turkewitz AP; The release of polypeptides in response to extracellular cues is a notable feature of endocrine, exocrine and neuronal cells, and is based on regulated exocytosis via dense-core secretory granules . There is interest in this mode of secretion because of its importance in human physiology and also because regulated exocytosis reflects a complex pathway of membrane traffic that includes compartment-specific reversible macromolecular assembly, coat-independent vesicle budding, maturation/remodeling of both lumenal and membrane constituents, and stimulus-dependent membrane fusion . Secretory granules are absent in most unicellular model organisms but are highly developed in the Ciliates, which therefore offer attractive systems to study these phenomena . In Tetrahymena thermophila, biochemical and genetic approaches have begun yielding insights into issues ranging from control of granule core assembly, based on reverse genetic analysis of granule cargo, to questions about factors involved in granule biogenesis, based on random mutational approaches.

Eur J Biochem, 2004 Jan, 271(1), 48 - 57
Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions; Zoldak G et al.; The conformational dynamics of NADH oxidase from Thermus thermophilus was modulated by the Hofmeister series of anions (H2PO4-, SO42-, CH3COO-, Cl-, Br-, I-, ClO4-, SCN-) in the concentration range 0-3 M . Both chaotropic and kosmotropic anions, at high concentration, inhibit the enzyme by different mechanisms . Chaotropic anions increase the apparent Michaelis constant and decrease the activation barrier of the reaction . Kosmotropic anions have the opposite effect . Anions from the middle of the Hofmeister series do not significantly affect the enzyme activity even at high concentration . We detected no significant changes in ellipticity of the aromatic region in the presence of the anions studied . There is a decreased Stern-Volmer quenching constant for FAD fluorescence quenching in the presence of kosmotropic anions and an increased quenching constant in the presence of chaotropic anions . All of this indicates that active site flexibility is important in the function of the enzyme . The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions, and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity . The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain, with potential importance in modulating extremozyme activity at room temperature.

Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 178 - 80 Epub 2003 Dec 18.
Expression, purification, crystallization and preliminary X-ray studies of geranylgeranyl diphosphate synthase from Thermus thermophilus HB8; Nishio K et al.; Geranylgeranyl diphosphate (GGPP) synthase from Thermus thermophilus HB8 was expressed in Escherichia coli, purified to homogeneity and crystallized both as the recombinant native protein and its selenomethionine (SeMet) derivative . Well diffracting crystals of these proteins were obtained belonging to the tetragonal space group P4(1) or P4(3), with unit-cell parameters a = b = 139.88, c = 73.37 A . There were two homodimers in the asymmetric unit . A native data set was collected to 1.55 A resolution and a data set suitable for MAD phasing was collected to 2.40 A resolution on beamline BL40B2 at SPring-8.

Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 97 - 104 Epub 2003 Dec 18.
Structure and implications for the thermal stability of phosphopantetheine adenylyltransferase from Thermus thermophilus; Takahashi H et al.; Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyzes the rate-limiting step in coenzyme A (CoA) biosynthesis by transferring an adenylyl group from ATP to 4'-phosphopantetheine (Ppant), yielding 3'-dephospho-CoA (dPCoA) . The crystal structure of PPAT from Thermus thermophilus HB8 (Tt PPAT) complexed with Ppant has been determined by the molecular-replacement method at 1.5 A resolution . The overall fold of the enzyme is almost the same as that of Escherichia coli PPAT, a hexamer having point group 32 . The asymmetric unit of Tt PPAT contains a monomer and the crystallographic triad and dyad coincide with the threefold and twofold axes of the hexamer, respectively . Most of the important atoms surrounding the active site in E . coli PPAT are conserved in Tt PPAT, indicating similarities in their substrate binding and enzymatic reaction . The notable difference between E . coli PPAT and Tt PPAT is the simultaneous substrate recognition by all six subunits of Tt PPAT compared with substrate recognition by only three subunits in E . coli PPAT . Comparative analysis also revealed that the higher stability of Tt PPAT arises from stabilization of each subunit by hydrophobic effects, hydrogen bonds and entropic effects.

Proc Natl Acad Sci U S A, 2004 Jan 6, 101(1), 59 - 64 Epub 2003 Dec 18.
Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase; Iwata M et al.; The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment . The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-A resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments . The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V(1) (soluble) and the V(o) (membrane bound) domains . Subunit C, attached to the V(o) domain, seems to have a socket like function in attaching the central-stalk subunits of the V(1) domain . This architecture seems essential for the reversible association/dissociation of the V(1) and the V(o) domains, unique for V-ATPase activity regulation.

Water Sci Technol, 2003, 48(8), 69 - 77
Airlift-reactor design and test for aerobic environmental bioprocesses with extremely high solid contents at high temperatures; Feitkenhauer H et al.; Bioprocesses at high temperatures gained considerably in importance within the last years and several new applications for aerobic, extreme thermophilic environmental bioprocesses are emerging . However, this development is not yet matched by adequate bioreactor designs, especially if it comes to the treatment of solids . In this communication we propose the use of airlift reactors to bridge this gap . The design of an internal draught tube bioreactor (Area(Riser)/Area(Downcomer) . 1; Height/Diameter . 8) is described in detail . The influence of the temperature on gas hold-up, liquid velocity and mixing characteristics was investigated . It was shown that this reactor could hold up to 1 t quartz sand per m3 in suspension at moderate aeration rates . Despite the decreasing oxygen solubility, the oxygen transfer rate increased with rising temperature due to the improved mass transfer parameters . With rising solid content, the oxygen transfer rate increased and reached a maximum at a solid content of about 140 kg m(-3) before it decreased again . However, it is only slightly reduced at the highest solid contents . The results demonstrate that aerobic bioprocesses at high temperatures are not only feasible, but can be very efficient if carried out in proper bioreactors.

RNA, 2004 Jan, 10(1), 28 - 33
Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model; Rodriguez-Correa D et al.; A mechanistic understanding of ribosome function demands knowledge of the conformational changes that occur during protein synthesis . One current model proposes a conformational switch in Helix 27 (H27) of 16S rRNA involved in the decoding of mRNA . This model was based on the behavior of mutations in the 912 region of H27 of Escherichia coli 16S rRNA, which were predicted to stabilize the helix in either of two alternative conformations . This interpretation was supported by evidence from both genetics and structural biochemistry . However, recently published X-ray crystallographic structures of the Thermus thermophilus 30S subunit at different stages of tRNA selection have raised doubts regarding the validity of this model . We have therefore revisited the model genetically by constructing a H27 quadruple mutation (C912G, C910G, G885C, and G887C), which would create multiple mismatches in the proposed alternative conformation without perturbing the native H27 conformation seen in the crystal structures . Inconsistent with the H27 switch model, cells containing pure populations of quadruple mutant ribosomes grow at essentially wild-type rates . The mutants used to construct the H27 switch model all carried A2058G in 23S rRNA and C1192U in 16S rRNA as selectable markers . The quadruple mutant carrying these additional marker mutations is deleterious, and we conclude that they have a synergistic effect when combined with other mutations and are not phenotypically silent . Their presence confounded the interpretation of the original mutant phenotypes and, in light of the viability of the quadruple mutant, we conclude that the genetic evidence no longer supports the model.

Mikrobiologiia, 2003 Sep-Oct, 72(5), 681 - 8
{Sulfobacillus sibiricus sp . nov., a new moderately thermophilic bacterium}; Melamud VS et al.; In the course of pilot industrial testing of a biohydrometallurgical technology for processing goldarsenic concentrate obtained from the Nezhdaninskoe ore deposit (East Siberia, Sakha, Yakutiya), a new gram-positive rod-shaped spore-forming moderately thermophilic bacterium (designated as strain N1) oxidizing Fe2+, S0, and sulfide minerals in the presence of yeast extract (0.02%) was isolated from a dense pulp . Physiologically, strain N1 differs from previously described species of the genus Sulfobacillus in having a somewhat higher optimal growth temperature (55 degrees C) . Unlike the type strain of S . thermosulfidooxidans, strain N1 could grow on medium with 1 mM thiosulfate or sodium tetrathionate as a source of energy only within several passages and failed to grow, in the absence of an inorganic energy source, on media with sucrose, fructose, glucose, reduced glutathione, alanine, cysteine, sorbitol, sodium acetate, or pyruvate . The G + C content of the DNA of strain N1 was 48.2 mol % . The strain showed 42% homology after DNA-DNA hybridization with the type strain of S . thermosulfidooxidans and 10% homology with the type strain of S . acidophilus . The isolate differed from previously studied strains of S . thermosulfidooxidans in the structure of its chromosomal DNA (determined by the method of pulsed-field gel electrophoresis) which remained stable as growth conditions were changed . According to the results of the 16S rRNA gene analysis, the new strain forms a single cluster with the bacteria of the species Sulfobacillus thermosulfidooxidans (sequence similarity of 97.9-98.6%) . Based on these genetic and physiological features, strain N1 is described as a new species Sulfobacillus sibiricus sp . nov.

J Appl Microbiol, 2004, 96(1), 209 - 19
Characterization of urease genes cluster of Streptococcus thermophilus; Mora D et al.; AIMS: The milk acidification rate of Streptococcus thermophilus strains can be affected by several factors, one of which is the hydrolysis of urea by the urease complex . To evaluate the technological suitability of S . thermophilus strains deprived of urease activity in milk fermentation, the genetic cluster related to urease enzymatic activity has been characterized in the type strain DSM 20167T . METHODS AND RESULTS: Amplification of the urease genes of S . thermophilus DSM 20167T was developed on the basis of the urease gene cluster of the phylogenetically related S . salivarius . Nucleotide sequencing revealed the presence of eight open reading frames, which were most homologous to ureABC (structural genes) and ureI, ureEFGD (accessory genes) of S . salivarius and other ureolytic bacteria . Reverse transcriptase PCR experiments were in agreement with an operon organization for the eight genes (ureIABCEFGD) . A food grade mutant A16 (DeltaureC3) with a 693 bp in-frame deletion in ureC gene exhibited a urease negative (Ure-) phenotype . Unlike the wild-type strain, the acidification rate of the mutant in reconstituted skimmed milk was not affected by the presence of urea or nickel ions . A small-scale yoghurt fermentation trials were carried out using the wild-type or the Ure- mutant A16 (DeltaureC3) in co-culture with Lactobacillus delbrueckii subsp . bulgaricus ATCC 11842 in presence of urea . The result obtained underlines that when the Ure- mutant was used as a co-starter the acidification rate was higher than that obtained using the wild-type strain . CONCLUSIONS: The study provides the first genetic characterization and the technological implication of S . thermophilus DSM 20617T urease activity . SIGNIFICANCE AND IMPACT OF THE STUDY: The detrimental effect of ureolytic activity on the rate of milk acidification was evaluated and superseded using a food-grade Ure- recombinant strain . Small-scale yoghurt production trials highlighted the positive role of a Ure-S . thermophilus mutant as a co-starter in milk fermentations . Moreover, the vector pMI108 developed for the construction of the Ure- strain, should be considered as a potential tool for the generation of Ure- dairy S . thermophilus strains selected for other relevant technological properties but characterized by the undesirable ureolytic phenotype.

J Appl Microbiol, 2004, 96(1), 110 - 6
Evaluation of the effect of cleaning regimes on biofilms of thermophilic bacilli on stainless steel; Parkar SG et al.; AIMS: To determine the mechanism for both the removal and inactivation of 18-h biofilms of a thermophilic Bacillus species that optimally grows at 55 degrees C on stainless steel . METHODS AND RESULTS: The cleaning strategies tested were based on biofilm biochemistry and physiology, and focused on the chemistry of the cleaners, the duration and temperature of the cleaning process and a combination of various cleaners . The success of the cleaning regimes was determined based on the removal of cells and organic debris and the elimination of viable cells . The results confirmed that a caustic (75 degrees C for 30 min) and acid (75 degrees C for 30 min) wash, relied upon heavily in most food processing industries for cleaning-in-place systems, was successful in removing these biofilms . However, any changes in the concentrations of these cleaners or the temperature of cleaning drastically affected the overall outcome . Alternative cleaning agents based on enzymatic or nonenzymatic breakdown of cellular proteins or polysaccharides, surfactant action, use of oxidative attack and free radicals varied in degrees of their success . Combining proteolytic action with surfactants increased wetability and therefore enhanced the cleaning efficiency . CONCLUSIONS: Several procedures, including caustic/acid and enzyme based cleaners, will be satisfactory, provided that the correct process parameters are observed i.e . concentration, time, temperature and kinetic energy (flow) . Confirmation of these results should be carried out in a pilot plant through several use/clean cycles . SIGNIFICANCE AND IMPACT OF THE STUDY: Confidence in standard and alternative cleaning procedures for food manufacturing plant to prevent contamination with thermophilic bacilli that threaten product quality.

Ann Agric Environ Med, 2003, 10(2), 241 - 8
Exposure to bioaerosols in a municipal sewage treatment plant; Prazmo Z et al.; Microbiological air sampling was performed in a medium-size sewage treatment plant processing municipal wastewater from a city located in eastern Poland . Air samples for determination of the concentrations of viable mesophilic bacteria, Gram-negative bacteria, thermophilic actinomycetes, fungi and endotoxin were collected at 12 sites associated with various phases of sewage treatment process . The concentrations of total mesophilic bacteria (both Gram-positive and Gram-negative) were within a range of 2.4-70.7 x 10(2) cfu/m(3) . Gram-positive coryneform bacteria and cocci were dominant, forming respectively 56.6 % and 24.0 % of the total count . The concentrations of Gram-negative bacteria, thermophilic actinomycetes, and fungi were respectively within ranges of 0.2-5.7 x 10(2) cfu/m(3), 0-0.5 x 10(2) cfu/m(3), and 0.24-1.4 x 10(2) cfu/m(3) . Among Gram-negative bacteria, commonly occurred Enterobacter cloacae (17.3 % of the total count), followed by Acinetobacter calcoaceticus (16.2 %), Pseudomonas spp . (14.0 %) and Stenotrophomonas maltophilia (11.1 %) . Among thermophilic actinomycetes prevailed Thermoactinomyces thalpophilus (47.2 %) and Thermoactinomyces vulgaris (22.2 %), while among fungi, Geotrichum candidum (32.2 %), Penicillium spp . (20 %), Cladosporium lignicola (12.2 %), and Alternaria alternata (10.4 %) . Altogether, 20 potentially pathogenic species or genera of bacteria and fungi were identified in the air samples taken in the examined plant . The values of the respirable fraction of airborne microflora varied within a fairly wide range and were between 24.1-100 % . The concentrations of airborne endotoxin were in the range of 0.104-5.2 ng/m(3) . In conclusion, the concentrations of microorganisms and endotoxin in the examined municipal sewage treatment plant were low and did not exceed proposed occupational exposure limit values . A moderate risk for the workers may be associated with the presence of potentially pathogenic microbial species having allergenic and/or immunotoxic properties.

Biochemistry, 2003 Dec 23, 42(50), 14968 - 76
Distinct domain functions regulating de novo DNA synthesis of thermostable DNA primase from hyperthermophile Pyrococcus horikoshii; Matsui E et al.; DNA primases are essential components of the DNA replication apparatus in every organism . Reported here are the biochemical characteristics of a thermostable DNA primase from the thermophilic archaeon Pyrococcus horikoshii, which formed the oligomeric unit L(1)S(1) and synthesized long DNA primers 10 times more effectively than RNA primers . The N-terminal (25KL) and C-terminal halves (20KL) of the large subunit (L) play distinct roles in regulating de novo DNA synthesis of the small catalytic subunit (S) . The 25KL domain has a dual function . One function is to depress the large affinity of the intrasubunit domain 20KL for the template DNA until complex (L(1)S(1) unit) formation . The other function is to tether the L subunit tightly to the S subunit, probably to promote effective interaction between the intrasubunit domain 20KL and the active center of the S subunit . The 20KL domain is a central factor to enhance the de novo DNA synthesis activity of the catalytic S subunit since the total affinity of the L(1)S(1) unit is mainly derived from the affinity of 20KL, which is elevated more than 10 times by the heterodimer formation, presumably due to the cancellation of the inhibitory activity of 25KL through tight binding to the S subunit.

Mol Cell Biochem, 2003 Dec, 254(1-2), 173 - 83
Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: purification and biochemical properties of PHA synthase; Pantazaki AA et al.; The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus . Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively . Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (Mw: 480,000 g mol(-1)) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64% . In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents . In contrast, the polyester (Mw: 391,000 g mol(-1)) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD) . Activities of PHA synthase, a beta-ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T . thermophilus . The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a Km of 0.25 mM for 3HB-CoA . The optimal temperature of PHA synthase activity was about 70 degrees C and acts optimally at pH near 7.3 . PHA synthase activity was inhibited 50% with 25 microM CoA and lost all of its activity when it was treated with alkaline phosphatase . T . thermophilus PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used . Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme . Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme . A major protein (55 kDa) was detected by SDS-PAGE . When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.

J Gen Appl Microbiol, 2003 Oct, 49(5), 287 - 93
Cellular polyamines of the acidophilic, thermophilic and thermoacidophilic archaebacteria, Acidilobus, Ferroplasma, Pyrobaculum, Pyrococcus, Staphylothermus, Thermococcus, Thermodiscus and Vulcanisaeta; Hamana K et al.; Cellular polyamines of newly isolated acidophilic, thermophilic and thermoacidophilic archaebacteria were investigated for the chemotaxonomic significance of polyamine distribution profiles . In addition to spermidine, spermine and agmatine, a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, was found in thermophilic Thermococcus waiotapuensis, Thermococcus aegaeus and Pyrococcus glycovorans belonging to the order Thermococcales . An acidophilic euryarchaeon, Ferroplasma acidiphilum located in the order Thermoplasmatales, contained spermidine and agmatine . Norspermidine, spermidine, norspermine and spermine were found in thermoacidophilic Acidilobus aceticus and thermophilic Thermodiscus maritimus located in the order Desulfurococcales, and in thermophilic Pyrobaculum arsenaticum, Pyrobaculum oguniense, Vulcanisaeta distributa and Vulcanisaeta souniana belonging to the order Thermoproteales; however, the four genera differ on their tetra- and penta-amine levels . Thermophilic Staphylothermus hellenicus belonging to Desulfurococcales contained caldopentamine, caldohexamine and N1-acetylcaldopentamine in addition to norspermidine, spermidine and norspermine . This is the first report on the occurrence of acetylated penta-amine in nature.

J Biol Chem, 2004 Feb 27, 279(9), 8396 - 402 Epub 2003 Dec 12.
Crystal structures of the CP1 domain from Thermus thermophilus isoleucyl-tRNA synthetase and its complex with L-valine; Fukunaga R et al.; Isoleucyl-tRNA synthetase (IleRS) links tRNA(Ile) with not only its cognate isoleucine but also the nearly cognate valine . The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNA(Ile) . We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-A resolutions, respectively . In the complex structure, the Asp(328) residue, which was shown to be critical for the editing reaction against Val-tRNA(Ile) by a previous mutational analysis, recognizes the valine NH(3)(+) group . The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His(319) . The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNA(Ile) in addition to Val-tRNA(Ile), indicating that the valine-binding mode found in this study represents that in the Val-tRNA(Ile) editing reaction . Analyses of the Val-tRNA(Ile) editing activities of T . thermophilus IleRS mutants revealed the importance of Thr(228), Thr(229), Thr(230), and Asp(328), which are coordinated with water molecules in the present structure . The structural model for the Val-adenosine moiety of Val-tRNA(Ile) bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T . thermophilus leucyl-tRNA synthetase . For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized.

J Dairy Sci, 2003 Nov, 86(11), 3405 - 15
Effects of pH, temperature, supplementation with whey protein concentrate, and adjunct cultures on the production of exopolysaccharides by Streptococcus thermophilus 1275; Zisu B et al.; Effects of pH, temperature, supplementation with whey protein concentrate (WPC), and non-EPS culture on the exopolysaccharide (EPS) production by Streptococcus thermophilus 1275 were studied . The organism was grown in 10% reconstituted skim milk (RSM) in a Biostat B fermenter for 24 h at various pH (4.5, 5.5 and 6.5) and temperatures (30, 37, 40, and 42 degrees C), and supplementation with WPC 392, and non-EPS producing S . thermophilus 1303 and the amount of EPS produced were determined . Bacterial counts were enumerated and the concentrations of lactic acid, lactose, glucose, and galactose were also determined . A maximum of 406 mg/L of EPS was produced in RSM at 37 degrees C after 24 h of fermentation at pH 4.08 when the pH was not controlled . A pH of 5.5 and temperature of 40 degrees C were found to be optimal for EPS production by S . thermophilus 1275, yielding 458 mg/L . The EPS production increased when RSM was supplemented with WPC 392 . At optimum pH and at 37 degrees C with WPC supplementation, the level of EPS increased to 1029 mg/L . Co-culturing S . thermophilus 1275 with non-EPS S . thermophilus 1303 increased EPS production at 37 degrees C and pH 5.5 to 832 mg/L . High temperature (42 degrees C) reduced the amount of EPS production, and EPS production ceased at pH 4.5 when maintained constantly at this pH . The level of lactose utilization and lactic acid production depended on growth conditions of the organism . No glucose was detected, while galactose was found to accumulate in the medium.

Biodegradation, 2003 Dec, 14(6), 367 - 72
Degradation of polycyclic aromatic hydrocarbons and long chain alkanes at 60-70 degrees C by Thermus and Bacillus spp {corrected}; Feitkenhauer H et al.; Although polycyclic aromatic hydrocarbons (PAH) and alkanes are biodegradable at ambient temperature, in some cases low bioavailabilities are the reason for slow biodegradation . Considerably higher mass transfer rates and PAH solubilities and hence bioavailabilities can be obtained at higher temperatures . Mixed and pure cultures of aerobic, extreme thermophilic microorganisms (Bacillus spp., Thermus sp.) were used to degrade PAH compounds and PAH/alkane mixtures at 65 degrees C . The microorganisms used grew on hydrocarbons as sole carbon and energy source . Optimal growth temperatures were in the range of 60-70 degrees C at pH values of 6-7 . The conversion of PAH with 3-5 rings (acenaphthene, fluoranthene, pyrene, benzo{e}pyrene) was demonstrated . Efficient PAH biodegradation required a second, degradable liquid phase . Thermus brockii Hamburg metabolized up to 40 mg (1 h)(-1) pyrene and 1000 mg (1 h)(-1) hexadecane at 70 degrees C . Specific growth rates of 0.43 h(-1) were measured for this strain with hexadecane/pyrene mixtures as the sole carbon and energy source in a 2-liter stirred bioreactor . About 0.7 g cell dry weight were formed from 1 g hydrocarbon . The experiments demonstrate the feasibility and efficiency of extreme thermophilic PAH and alkane biodegradation.

Parasitology, 2003, 126 Suppl, S87 - 93
Cadmium effects on Ichthyophthirius: evidence for metal-sequestration in fish tissues following administration of recombinant vaccines; Bisharyan Y et al.; We are developing Tetrahymena thermophila as a delivery system for recombinant vaccines against parasitic protozoa, including the common fish parasite, Ichthyophthirius multifiliis . T . thermophila cell lines expressing I . multifiliis genes under the control of a cadmium-inducible metallothionein gene promoter conferred strong protection against a lethal parasite challenge when administered parenterally to naive fish . Nevertheless, given that heavy metals can be toxic to parasites, a question arose as to whether protection resulted from Cd residues carried over with the vaccine, rather than acquired immunity per se . To address this issue, we examined the sensitivity of I . multifiliis to Cd in vitro and determined Cd concentrations in different host tissues following i.p . injection of juvenile channel catfish with the recombinant vaccine . We found that CdCl2 at concentrations > or = 50 ppb were lethal to I . multifiliis theronts in vitro . Furthermore, Cd concentrations were clearly elevated in fish tissues and reached levels equivalent to 74 ng/g wet weight (74 ppb) in the skin within 14 days of injection with recombinant T . thermophila . Nevertheless, fish injected with non-transformed Tetrahymena grown in the presence or absence of CdCl2 showed no significant difference in either relative survival or parasite load following direct challenge with I . multifiliis.

Appl Biochem Biotechnol, 2003 Dec, 111(3), 153 - 66
Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase; Rey MW et al.; Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/ cellular biology is poorly understood . Only a few genes have been cloned from the Thielavia genus . We detected an extracellular glucoamylase in culture filtrates of T . terrestris and cloned the corresponding glaA gene . The coding region contains five introns . Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase . Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15 . The T . terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A . oryzae alpha-amylase promoter and an Aspergillus niger glucoamylase terminator . The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 micromol/(min x mg) with maltose as substrate . With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60 degrees C . The enzyme was stable at 60 degrees C for 30 min . The Km and kcat of the enzyme for maltotriose were determined at various pHs and temperatures . At 20 degrees C and pH 4.0, the enzyme had a Km of 0.33 +/- 0.07 mM and a kcat of (5.5 +/- 0.5) x 103 min(-1) for maltotriose . The temperature dependence of kcat/Km indicated an activation free energy of 2.8 kJ/mol across the range of 20-70 degrees C . Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi.

J Am Chem Soc, 2003 Dec 17, 125(50), 15343 - 51
The A-cluster in subunit beta of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila: Ni and Fe K-edge XANES and EXAFS analyses; Gu W et al.; The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the cleavage of acetyl-CoA in methanogens that metabolize acetate to CO(2) and CH(4), and also carries out acetyl-CoA synthesis during growth on one-carbon substrates . The ACDS complex contains five subunits, among which beta possesses an Ni-Fe-S active-site metal cluster, the A-cluster, at which reaction with acetyl-CoA takes place, generating an acetyl-enzyme species poised for C-C bond cleavage . We have used Ni and Fe K fluorescence XANES and EXAFS analyses to characterize these metals in the ACDS beta subunit, expressed as a C-terminally shortened form . Fe XANES and EXAFS confirmed the presence of an {Fe(4)S(4)} cluster, with typical Fe-S and Fe-Fe distances of 2.3 and 2.7 A respectively . An Fe:Ni ratio of approximately 2:1 was found by Kalphabeta fluorescence analysis, indicating 2 Ni per {Fe(4)S(4)} . Ni XANES simulations were consistent with two distinct Ni sites in cluster A, and the observed spectrum could be modeled as the sum of separate square planar and tetrahedral Ni sites . Treatment of the beta subunit with Ti(3+) citrate resulted in shifts to lower energy, implying significant reduction of the {Fe(4)S(4)} center, along with conversion of a smaller fraction of Ni(II) to Ni(I) . Reaction with CO in the presence of Ti(3+) citrate generated a unique Ni XANES spectrum, while effects on the Fe-edge were not very different from the reaction with Ti(3+) alone . Ni EXAFS revealed an average Ni coordination of 2.5 S at 2.19 A and 1.5 N/O at 1.89 A . A distinct feature at approximately 2.95 A most likely results from Ni-Ni interaction . The methanogen beta subunit A-cluster is proposed to consist of an {Fe(4)S(4)} cluster bridged to an Ni-Ni center with one Ni in square planar geometry coordinated by 2 S + 2 N and the other approximately tetrahedral with 3 S + 1 N/O ligands . The electronic consequences of two distinct Ni geometries are discussed.

J Biol Chem, 2004 Feb 20, 279(8), 6683 - 7 Epub 2003 Dec 07.
A role for iron in an ancient carbonic anhydrase; Tripp BC et al.; Since 1933, carbonic anhydrase research has focused on enzymes from mammals (alpha class) and plants (beta class); however, two additional classes (gamma and delta) were discovered recently . Cam, from the procaryote Methanosarcina thermophila, is the prototype of the gamma class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain . All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc . Herein, we report the anaerobic reconstitution of apo-Cam with Fe2+, which yielded Cam with an effective kcat that exceeded that for the Zn2+-reconstituted enzyme . Mossbauer spectroscopy showed that the Fe2+-reconstituted enzyme contained high spin Fe2+ that, when oxidized to Fe3+, inactivated the enzyme . Reconstitution with Fe3+ was unsuccessful . Reconstitution with Cu2+, Mn2+, Ni2+, or Cd2+ yielded enzymes with effective kcat values that were 10% or less than the value for the Zn2+-reconstituted Cam . Cam produced in Escherichia coli and purified anaerobically contained iron with effective kcat and kcat/Km values exceeding the values for Zn2+-reconstituted Cam . The results identify a previously unrecognized biological function for iron.

BMC Public Health . 2003 Dec 09;3(1):39.
Risk factors for antibiotic resistance in Campylobacter spp . isolated from raw poultry meat in Switzerland; Ledergerber U et al.; BACKGROUND: The world-wide increase of foodborne infections with antibiotic resistant pathogens is of growing concern and is designated by the World Health Organization as an emerging public health problem . Thermophilic Campylobacter have been recognised as a major cause of foodborne bacterial gastrointestinal human infections in Switzerland and in many other countries throughout the world . Poultry meat is the most common source for foodborne cases caused by Campylobacter . Because all classes of antibiotics recommended for treatment of human campylobacteriosis are also used in veterinary medicine, in view of food safety, the resistance status of Campylobacter isolated from poultry meat is of special interest . METHODS: Raw poultry meat samples were collected throughout Switzerland and Liechtenstein at retail level and examined for Campylobacter spp . One strain from each Campylobacter-positive sample was selected for susceptibility testing with the disc diffusion and the E-test method . Risk factors associated with resistance to the tested antibiotics were analysed by multiple logistic regression . RESULTS: In total, 91 Campylobacter spp . strains were isolated from 415 raw poultry meat samples . Fifty-one strains (59%) were sensitive to all tested antibiotics . Nineteen strains (22%) were resistant to a single, nine strains to two antibiotics, and eight strains showed at least three antibiotic resistances . Resistance was observed most frequently to ciprofloxacin (28.7%), tetracycline (12.6%), sulphonamide (11.8%), and ampicillin (10.3%) . One multiple resistant strain exhibited resistance to five antibiotics including ciprofloxacin, tetracycline, and erythromycin . These are the most important antibiotics for treatment of human campylobacteriosis . A significant risk factor associated with multiple resistance in Campylobacter was foreign meat production compared to Swiss meat production (odds ratio = 5.7) . CONCLUSION: Compared to the situation in other countries, the data of this study show a favourable resistance situation for Campylobacter strains isolated from raw poultry meat produced in Switzerland . Nevertheless, the prevalence of 19% ciprofloxacin resistant strains is of concern and has to be monitored . "Foreign production vs . Swiss production" was a significant risk factor for multiple resistance in the logistic regression model . Therefore, an adequate resistance-monitoring programme should include meat produced in Switzerland as well as imported meat samples.

Waste Manag Res, 2003 Oct, 21(5), 405 - 15
Intensive aerobic bioconversion of sewage sludge and food waste into fertiliser; Wang JY et al.; The aim of this research was to verify the possibility of recovering the nutrients present in sewage sludge and vegetable food waste as fertiliser after aerobic thermophilic intensive bioconversion . The process was performed in a closed reactor under controlled conditions of aeration, stirring and pH, at a temperature of 60 degrees C, after addition of a starter bacterial culture of Bacillus thermoamylovorans SW25 . End product with the best fertilising properties was obtained when sewage sludge, mixed with food waste, CaCO3 and an artificial bulking agent was thermally pretreated . The content of volatile solids and organic carbon decreased from 82.8% to 62.3% and from 37.7% to 32.5% of total solids (TS) respectively, during 12 days of bioconversion . The stable organic fertiliser produced was a powder with moisture content of 5% . Furthermore, 3.4% of nitrogen, 0.4% of phosphorus and 2.9% of potassium were also present . Addition of 10-15g of this fertiliser to 1 kg of poor fertility soil increased the growth of different plants by 113-164%.

FEMS Microbiol Lett, 2003 Dec 5, 229(1), 133 - 40
Microbiological characterization of artisanal Montasio cheese: analysis of its indigenous lactic acid bacteria; Marino M et al.; The aim of this study was to investigate the dynamics of the microflora during Montasio cheese ripening, with specific reference to some characteristics of biotechnological interest . Nine batches of Montasio cheese produced in different plants were analyzed . Streptococcus thermophilus was the predominant species throughout the whole ripening period of Montasio cheese . Enterococci were also frequently present . This microbial group resulted probably from milk, and its proportion decreased rapidly during ripening . The most acidifying microbial species was S . thermophilus, while the most proteolytic strains belonged to the genera Enterococcus . A high degree of phenotypic diversity occurred within the microbial species.

Protist, 2003 Oct, 154(3-4), 431 - 42
Cloning and sequencing of four new metallothionein genes from Tetrahymena thermophila and T . pigmentosa: evolutionary relationships in Tetrahymena MT family; Boldrin F et al.; The structure of four new MT (metallothionein) genes of Tetrahymena thermophila and T . pigmentosa were characterized . The MT-2 genes from the two species are very similar, differing by 10 out of 2259 sequenced nucleotides, and the deduced amino acid sequences are identical . The MT-1 genes from T . pigmentosa and T thermophila are also very similar, differing only by 3 nucleotides in the 5'-UT region . The promoter regions contain a TATA box and many stretches partially matching some regulatory elements such as metal-responsive (MREs), antioxidant-responsive (AREs), a CAAT box, a G-box, and AP1 and ACE-1 binding sites . The related coding and amino acid sequences were compared with those previously sequenced in Tetrahymena . This analysis revealed two independent events of duplication occurring in Cd- (MT-1 and MTT1) and Cu- (MT-2) induced MTs . This evolutionary pathway also explains the unusual length of these proteins, which are much longer than many MTs studied so far . Additionally, the orthology and paralogy relationships of the various MTs are presented . Finally, on the basis of phylogenetic analyses of Tetrahymena MTs, two evolutionary hypotheses are proposed.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14731 - 6 Epub 2003 Dec 01.
Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40 degree substep rotation; Shimabukuro K et al.; F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor . The central gamma-subunit rotates in the alpha 3 beta 3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, interim dwell, and rapid 40 degrees substep rotation . At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events . To discover which steps, we analyzed rotations of F1 subcomplex (alpha 3 beta 3 gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATP gamma S (adenosine 5'-{gamma-thio}triphosphate) by wild-type F1 . In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell . Furthermore, the results of ATP gamma S hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event . Thus, cleavage of ATP on F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1947 - 54
Balnearium lithotrophicum gen . nov., sp . nov., a novel thermophilic, strictly anaerobic, hydrogen-oxidizing chemolithoautotroph isolated from a black smoker chimney in the Suiyo Seamount hydrothermal system; Takai K et al.; A novel, extremely thermophilic bacterium, designated strain 17S(T), was isolated from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc, Japan . The cells were rods with no apparent motility, most of which were narrow in the middle in the exponential-growth phase and had several polar flagella at both ends . Growth was observed between 45 and 80 degrees C (optimum temperature, 70-75 degrees C; doubling time, 80 min) and between pH 5.0 and 7.0 (optimum pH, 5.4) . The isolate was a strictly anaerobic chemolithoautotroph that was capable of using molecular hydrogen as its sole energy source and carbon dioxide as its sole carbon source . Elemental sulfur (S(0)) was required for growth as an electron acceptor . The G+C content of the genomic DNA was 34.6 mol% . Phylogenetic analysis based on 16S rDNA sequences indicated that the isolate was related to Thermovibrio ruber ED11/3LLK(T) and Desulfurobacterium thermolithotrophum BSA(T), whilst it appeared to be a novel lineage prior to the divergence of these genera . This isolate could also be differentiated from both T . ruber ED11/3LLK(T) and D . thermolithotrophum BSA(T) on the basis of physiological properties . The name Balnearium lithotrophicum gen . nov., sp . nov . is proposed for this isolate (type strain, 17S(T)=JCM 11970(T)=ATCC BAA-736(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1843 - 51
Anaerolinea thermophila gen . nov., sp . nov . and Caldilinea aerophila gen . nov., sp . nov., novel filamentous thermophiles that represent a previously uncultured lineage of the domain Bacteria at the subphylum level; Sekiguchi Y et al.; Two thermophilic, Gram-negative, non-spore-forming, multicellular filamentous micro-organisms were isolated from thermophilic granular sludge in an upflow anaerobic sludge blanket reactor treating fried soybean-curd manufacturing waste water (strain UNI-1(T)) and from a hot spring sulfur-turf in Japan (strain STL-6-O1(T)) . The filaments were longer than 100 microm and of 0.2-0.3 microm (strain UNI-1(T)) or 0.7-0.8 microm (strain STL-6-O1(T)) in width . Strain UNI-1(T) was a strictly anaerobic organism . The optimum temperature for growth was around 55 degrees C; growth occurred in the range 50-60 degrees C . The optimum pH for growth was around 7.0; growth occurred in the range pH 6.0-8.0 . Strain STL-6-O1(T) was a facultatively aerobic bacterium . The optimum temperature for growth was around 55 degrees C; growth occurred in the range 37-65 degrees C . The optimum pH for growth was around 7.5-8.0; growth occurred in the range pH 7.0-9.0 . The two organisms grew chemo-organotrophically on a number of carbohydrates and amino acids in the presence of yeast extract . The G+C content of the DNA of strains UNI-1(T) and STL-6-O1(T) was 54.5 and 59.0 mol%, respectively . Major cellular fatty acids for strain UNI-1(T) were C(16 : 0), C(15 : 0), C(14 : 0) and C(18 : 0), whereas those for strain STL-6-O1(T) were C(18 : 0), C(16 : 0), C(17 : 0) and iso-C(17 : 0) . MK-10 was the major quinone from aerobically grown STL-6-O1(T) cells . Phylogenetic analyses based on 16S rDNA sequences revealed that both strains belong to an uncultured, previously recognized clone lineage of the phylum Chloroflexi (formerly known as green non-sulfur bacteria) . These phenotypic and genetic properties suggested that each strain should be classified into a new independent genus; hence, the names Anaerolinea thermophila and Caldilinea aerophila are proposed for strains UNI-1(T) (=JCM 11387(T)=DSM 14523(T)) and STL-6-O1(T)(=JCM 11388(T)=DSM 14525(T)), respectively . These strains represent the type and sole species of the genera Anaerolinea and Caldilinea, respectively.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1791 - 9
Soehngenia saccharolytica gen . nov., sp . nov . and Clostridium amygdalinum sp . nov., two novel anaerobic, benzaldehyde-converting bacteria; Parshina SN et al.; Two anaerobic, benzaldehyde-converting bacteria were isolated from an anaerobic upflow anaerobic sludge bed (UASB)-reactor treating potato starch waste water . Strain BOR-Y(T) converted benzaldehyde to benzoate and benzylalcohol in approximately equimolar concentrations . Benzaldehyde conversion did not support growth . Strain BOR-Y(T) was Gram-positive and rod-shaped, and its cells were slightly thickened in the middle . The strain was a mesophilic spore-former that grew between 15 and 40 degrees C, with optimum growth at 30-37 degrees C . The optimum pH for growth was pH 7.0 . Strain BOR-Y(T) grew on a wide range of carbohydrates and some other carbon sources including yeast extract, cysteine and serine . The G+C content of its DNA was 42 mol% . According to physiological characteristics and 16S rRNA gene sequence analysis, confirmed by DNA-DNA hybridization with its phylogenetic neighbours, strain BOR-Y(T) belongs to a novel genus of cluster XII of the clostridia, namely Soehngenia; the name Soehngenia saccharolytica is proposed for the type species (type strain BOR-Y(T)=DSM 12858(T)=ATCC BAA-502(T)) . Strain BR-10(T) reduced benzaldehyde to benzylalcohol . This conversion was coupled to growth . In a medium containing yeast extract, the presence of benzaldehyde resulted in the accumulation of more than twofold more cells . Strain BR-10(T) was a Gram-positive organism that was characterized by oval- or rod-shaped cells with oval ends, which occurred singly, in pairs or sometimes in chains . The strain was moderately thermophilic and grew between 20 and 60 degrees C, with optimum growth at 45 degrees C . The optimum pH for growth was between pH 7.0 and 7.5 . Strain BR-10(T) grew on a wide range of carbon sources including carbohydrates, yeast extract, casein and some amino acids . The G+C content of its DNA was 32 mol% . As determined by 16S rRNA gene sequence analysis, strain BR-10(T) represents a novel species of cluster XIVa of the clostridia; the name Clostridium amygdalinum is proposed for this novel species (type strain BR-10(T)=DSM 12857(T)=ATCC BAA-501(T)).

Nucleic Acids Res, 2003 Dec 15, 31(24), 7247 - 54
Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1; Blondal T et al.; Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology . We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology . We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes . Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus . The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA . Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.

Eur J Biochem, 2003 Dec, 270(24), 4887 - 97
Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus; Zoldak G et al.; NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function . As is typical for an enzyme isolated from a thermophile, the catalytic rate, kcat, is low at low temperatures and increases with temperature, achieving an optimum at the physiological temperature of the organism, i.e . at approximately 70 degrees C for T . thermophilus . At low temperatures, the kcat of several enzymes from thermophilic and mesophilic organisms can be increased by chaotropic agents . The catalytic rate of NADH oxidase increases in the presence of urea . At concentrations of 1.0-1.3 m urea it reaches 250% of the activity in the absence of urea, at 20 degrees C . At higher urea concentrations the enzyme activity is inhibited . The urea-dependent activity changes correlate with changes in the fluorescence intensity of Trp47, which is located in the active site of the enzyme . Both fluorescence and circular dichroism measurements indicate that the activation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme . This is not related to the global structure of NADH oxidase . The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin-dependent oxidases . A comparison of the crystal structure with the activation thermodynamic parameters, deltaH* and TdeltaS*, obtained from the temperature dependence of kcat, suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring . The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase.

Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 1297 - 302
Characterization of two different 2-oxoglutarate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus TK-6; Yamamoto M et al.; A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive TCA cycle . 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes of this cycle . Strain TK-6 has two distinct OGOR enzymes termed For and Kor . These enzymes were purified and characterized following heterologous expression in Escherichia coli . The specific activity of For was approximately one-tenth of that of Kor . Additionally, For showed higher thermo-stability than Kor under both aerobic and anaerobic conditions . Western blot analysis showed that both of For and Kor were expressed when strain TK-6 was grown under aerobic conditions . In contrast, only Kor was expressed when the strain was grown under anaerobic conditions using nitrate as a terminal electron acceptor . These results indicate that For supports the optimal growth of strain TK-6 in the presence of oxygen.

Mol Microbiol, 2003 Dec, 50(5), 1605 - 15
Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo; Guo R et al.; Proteins of the Sac10b family, which is highly conserved among hyperthermophilic archaea, have been regarded as DNA-binding proteins . Based on their in vitro DNA-binding properties, these proteins are thought to be involved in chromosomal organization or DNA repair/recombination . We show that Ssh10b, a member of the Sac10b family from Sulfolobus shibatae, bound with similar affinities to double-stranded DNA, single-stranded DNA and RNA in vitro . However, the protein was exclusively bound to RNA in S . shibatae cells, as revealed by in vivo UV cross-linking and co-immunoprecipitation . Ribosomal RNAs were among the RNA species co-immunoprecipitated with Ssh10b . Consistent with this observation, Ssh10b was co-purified with ribosomes under low salt conditions . Furthermore, we demonstrate by UV-cross-linking hybridization that, when the cells were irradiated with UV, Ssh10b became cross-linked to 16S, 23S rRNAs and mRNAs . Our data indicate that RNA is the physiological binding target of the Sac10b family.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2334 - 6 Epub 2003 Nov 27.
Crystallization and preliminary X-ray crystallographic analysis of the Hsp100 chaperone ClpB from Thermus thermophilus; Lee S et al.; ClpB from Thermus thermophilus (TClpB) has been crystallized by the vapour-diffusion method in the presence of adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP) and adenosine 5'-(gamma-thio)triphosphate (ATPgammaS), respectively . Complete native data sets have been collected from frozen crystals, which belonged to the primitive orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 109.2, b = 139.6, c = 213.0 A, alpha = beta = gamma = 90 degrees.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2294 - 6 Epub 2003 Nov 27.
Crystallization and preliminary X-ray crystallographic studies of NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8; Lokanath NK et al.; 3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific NADP-dependent dehydrogenase (HIBADH) . HIBADH from Thermus thermophilus HB8 has been overexpressed in Escherichia coli and crystallized by the microbatch method using lithium chloride as a precipitant at 296 K . X-ray diffraction data have been collected to 1.80 A resolution at 100 K using synchrotron radiation . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 85.878, b = 106.367, c = 168.639 A . A homotetramer of HIBADH is likely to be present in the asymmetric unit, giving a V(M) of 3.0 A(3) Da(-1) and a solvent content of 59.3%.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2275 - 8 Epub 2003 Nov 27.
Crystallization and preliminary X-ray analysis of the small component of 4-hydroxyphenylacetate 3-monooxygenase (HpaC) and its cofactor complex from Thermus thermophilus HB8; Kim SH et al.; The small component of 4-hydroxyphenylacetate 3-monooxygenase (HpaC) is an NADH oxidoreductase containing a flavin molecule as a cofactor . HpaC reduces a flavin molecule and reduced flavin is subsequently supplied to the large component of 4-hydroxyphenylacetate 3-monooxygenase (HpaB) . The HpaC protein from Thermus thermophilus HB8 has been overexpressed in Escherichia coli and crystallized . During purification, the eluted HpaC protein solutions were separated into colourless and yellow-coloured fractions (i.e . apo-HpaC and HpaC-flavin complex, respectively) . Crystals of apo-HpaC grown in 5%(v/v) isopropyl alcohol, 0.1 M HEPES-NaOH pH 7.0, 40%(w/v) polyethylene glycol (PEG) 4000 and 10%(v/v) glycerol diffracted X-rays to a resolution of 1.85 A, whereas crystals of the HpaC-flavin complex grown in 20%(w/v) PEG 1000, 10%(w/v) PEG 8000 and 10%(v/v) glycerol diffracted X-rays to a resolution of 1.3 A . Both crystals belong to the monoclinic system, space group P2(1), with similar unit-cell parameters . Selenomethionyl protein crystals of the HpaC-flavin complex grown under similar conditions to the native crystals diffracted X-rays to a resolution of 1.8 A . They also belong to the monoclinic space group P2(1), but are not isomorphous to crystals of the HpaC-flavin complex of the native protein . MAD data for structure determination were successfully collected using these crystals.

Appl Biochem Biotechnol, 2003 Nov, 111(2), 93 - 112
Conversion of municipal solid wastes to carboxylic acids by thermophilic fermentation; Chan WN et al.; The purpose of this research is to generate carboxylic acids from the biodegradable fraction of municipal solid wastes (MSW) and municipal sewage sludge (MSS) by using a thermophilic (55 degrees C), anaerobic, high-solid fermentation . With terrestrial inocula, the highest total carboxylic acid concentration achieved was 20.5 g/L, the highest conversion obtained was 69%, and the highest acetic acid selectivity was 86.4% . Marine inocula were also used to compare against terrestrial sources . Continuum particle distribution modeling (CPDM) was used to predict the final acid product concentrations and substrate conversions at a wide range of liquid residence times (LRT) and volatile solid loading rates (VSLR) . "Maps" showing the product concentration and conversion for various LRT and VSLR were generated from CPDM . The predictions were compared to the experimental results . On average, the difference between the predicted and experimental values were 13% for acid concentration and 10% for conversion . CPDM "maps" show that marine inocula produce higher concentrations than terrestrial inocula.

J Photochem Photobiol B, 2003 Dec 5, 72(1-3), 45 - 53
Allophycocyanin complexes from the phycobilisome of a thermophilic blue-green alga Myxosarcina concinna Printz; Sun L et al.; The core polypeptide components of the intact phycobilisomes (PBSs) prepared by the sucrose gradients in 0.9 M phosphate buffer from a thermophilic cyanobacterium Myxosarcina concinna Printz were investigated . Three allophycocyanins, designated AP1, AP2, and AP3, of the PBS cores were successfully prepared by using the gradient polyacrylamide gel electrophoresis (PAGE) performed in neutral, instead of alkaline, buffer system . The spectral properties of AP2 and AP3 demonstrated that they both had fluorescence emission maxima at 684/685 nm at 77 K, which was identical to those of the intact PBSs, and showed the absorption of allophycocyanin B (AP-B) subunit . Sodium dodecyl sulfate-PAGE revealed that the three biliprotein complexes were all composed of heterogeneous subunits and two more linker polypeptides (Ls), AP1 alpha(22.3)alpha(19.5)beta(17.4)beta(15.7)L(13.8)L(11.3)L(9.5), AP2 alpha(22.3)alpha(19.5)beta(17.4)beta(15.7)beta(15.1)L(11.3)L(9.5), and AP3 alpha(22.3)alpha(19.5)beta(17.4)beta(15.7)beta(15.1)L(11.3)L(9.5)L(8.3) . Compared with the characteristics of AP1, beta(15.1), which belonged to the beta subunit group, was the AP-B subunit of AP2 and AP3 . Because AP2 was only obtained together with the PBS by the aid of 2% (v/v) Triton X-100, but not AP3, it was closely related to anchoring the PBS core on thylakoid membranes though the polypeptide analysis showed that AP2 had no core-membrane linker (LCM) . Aggregates of the three AP biliproteins were proposed based on the present results, and their functions in the PBS core construction and the energy transfer to PS II and PS I were discussed.

Biochim Biophys Acta, 2003 Nov 15, 1634(3), 107 - 15
A novel lipopeptide, an inhibitor of bacterial adhesion, from the thermophilic and halotolerant subsurface Bacillus licheniformis strain 603; Batrakov SG et al.; A new Bacillus licheniformis strain, 603, isolated from a mixture of drilling fluid and subsurface thermal water, has been found to produce a cyclic lipopeptide which is released into cultural medium as well as present in cells as the major lipid constituent (57% of the total cell lipids extractable with 2:1 chloroform-methanol) . The quantitative ratio of the extracellular and intracellular lipopeptide has been estimated as 23:10 . The metabolite represents a heptapeptide, L-Asp-->L-Leu-->L-Leu-->L-Val-->L-Val-->L-Glu-->L-Leu, N-acylated to the N-terminal amino acid, L-Asp, by a 3-hydroxy fatty acid (from 13:0 to 17:0 with n-, iso-, and anteiso-chains), the 3-OH group of which is esterified by the C-terminal amino acid, L-Leu . The chemical structure of the lipopeptide has been established by means of infrared (IR), 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionisation (ESI) mass spectrometry (MS), including secondary ion mass spectrometry, along with chemical and enzymatic degradation . Although a diversity of similar metabolites synthesised by various B . licheniformis strains are presently known, such a structure has not been reported thus far . Added to the growth medium of strain 603 at the concentration of 1.6 microg/ml, the lipopeptide prevents adhesion of cells to a glass surface . Also, it exhibits a considerable growth-inhibiting activity against Corynebacterium variabilis and a much lower activity against Acinetobacter sp.

Biochem Soc Trans, 2003 Dec, 31(Pt 6), 1318 - 21
The irony of manganese superoxide dismutase; Whittaker JW; The manganese and iron SODs (superoxide dismutases) form a superfamily of closely related antioxidant defence metalloenzymes . MnSOD requires Mn (not Fe) for activity . However, when MnSOD is expressed in Escherichia coli grown in medium supplemented with ferrous salts, Fe substitutes for Mn in the active site, reflecting relatively indiscriminate uptake of either Mn or Fe and a surprisingly low selectivity for the identity of the bound metal ion . X-ray crystallographic studies on Fe-substituted MnSOD show that the substrate access channel is blocked by solvent (hydroxide), providing a structural explanation for the observed metal specificity of the catalytic activity . The mechanism of metal binding has been investigated in vitro using recombinant thermophilic SODs . The thermophilic Thermus thermophilus MnSOD expressed in E . coli was isolated as the metal-free apoprotein when heat treatment was eliminated from the purification procedure . While incubation of the purified MnSOD apoprotein with metal salts at ambient temperatures did not restore SOD activity, re-activation could be achieved by heating the protein with Mn salts at elevated temperatures . This in vitro thermally triggered metal uptake is non-specific for the metal ion; both Mn and Fe bind, but only Mn restores catalytic activity . Formation of the metal complex is essentially irreversible under these conditions . The metallation process is strongly temperature-dependent, suggesting that there are substantial activation barriers to metal uptake at ambient temperatures that are overcome by a transition in the apoprotein structure under physiological conditions . Two mechanisms may be proposed for SOD metallation: one involving subunit dissociation and another involving domain separation . Thermally triggered metal binding by thermophilic SODs is providing new insight into the metallation mechanism of the SOD apoprotein, which is likely to be conserved over this family of enzymes.

J Am Chem Soc, 2003 Dec 3, 125(48), 14728 - 32
Ligand binding in a docking site of cytochrome C oxidase: a time-resolved step-scan Fourier transform infrared study; Koutsoupakis C et al.; The description of reaction regulation in enzymes responsible for activating and catalyzing small molecules (O(2), NO) requires identification of ligand movement into the binding site and out of the enzyme through specific channels and docking sites . We have used time-resolved step-scan Fourier transform infrared spectroscopy on CO-photolyzed cytochrome c oxidase ba(3) from T . thermophilus, which is responsible for the activation and reduction of both O(2) and NO, to gain insight into the structure of ligand-binding intermediates at ambient temperature . We show that, upon dissociation, the photolyzed CO becomes trapped within a ligand docking site located near the ring A propionate of heme a(3) . The 2131 cm(-1) mode of the "docked" CO we have detected corresponds to the B(1) state of Mb and persists for 35 micros . The release of CO from the docking site is not followed by recombination to the heme a(3) Fe . Our analysis indicates that this behavior reflects a mechanism in which the protein near ring A of heme a(3) propionate reorganizes about the released CO from the docking site, and establishes a transient barrier that inhibits the recombination process to the heme a(3) Fe for a few milliseconds . Rebinding to heme a(3) occurs with k(2) = 29.5 s(-1) . These results have implications for understanding the role of ligand binding/escape through docking sites and channels in heme-copper oxidases and, thus, in respiration.

J Dairy Res, 2003 Nov, 70(4), 395 - 401
A case of sporadic ovine mastitis caused by Listeria monocytogenes and its effect on contamination of raw milk and raw-milk cheeses produced in the on-farm dairy; Schoder D et al.; We describe a case of listerial mastitis in a flock of 130 sheep . The animals were housed at a farm where the bulk raw ewe milk was processed to produce raw milk soft cheese . List . monocytogenes was shed from the right mammary complex . Shedding was observed over a period of 99 d . A mean level of 4-56 x 10(4) cfu (colony forming units) Listeria monocytogenes/ml was recovered from the raw milk originating from the infected udder . The numbers ranged from 9 x10(1) to 2.95 x 10(5) . The bulk milk was contaminated by approx . 5.7 x 10(3) cfu/ml . In the cheese product, 2.0 x 10(2) cfu List . monocytogenes/g were constantly detectable for a period of 7 d post manufacture . The starter culture used for coagulation had a pivotal influence on the behaviour of List . monocytogenes during cheesemaking . Using the same mesophilic buttermilk culture as used by the farmer allowed numbers of Listeria to increase 60-fold within 12 h owing to a delayed acidification of the bulk milk . Addition of a thermophilic yogurt culture reduced the numbers of Listeria within 8 h of incubation.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14892 - 6 Epub 2003 Nov 25.
Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells; Hasegawa S et al.; Tetrahymena ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins . Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high in vivo activity, but no such strategies have been reported . Here we present a study using a new reporter enzyme, beta-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry . The reporter ribozyme constructs consist of the self-splicing Tetrahymena thermophila group I intron ribozyme that is inserted into the ORF of the mRNA of beta-lactamase . The splicing activity in single living cells can be readily detected quantitatively and visualized . Individual cells have shown considerable heterogeneity in ribozyme activity . Screening of Tetrahymena ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final in vivo activity than the native sequence . Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile in vivo selection of desired ribozyme variants.

Environ Microbiol, 2003 Nov, 5(11), 1168 - 91
Indigenous and contaminant microbes in ultradeep mines; Onstott TC et al.; Rock, air and service water samples were collected for microbial analyses from 3.2 kilometres depth in a working Au mine in the Witwatersrand basin, South Africa . The approximately metre-wide mined zone was comprised of a carbonaceous, quartz, sulphide, uraninite and Au bearing layer, called the Carbon Leader, sandwiched by quartzite and conglomerate . The microbial community in the service water was dominated by mesophilic aerobic and anaerobic, alpha-, beta- and gamma-Proteobacteria with a total biomass concentration approximately 10(4) cells ml(-1), whereas, that of the mine air was dominated by members of the Chlorobi and Bacteroidetes groups and a fungal component . The microorganisms in the Carbon Leader were predominantly mesophilic, aerobic heterotrophic, nitrate reducing and methylotrophic, beta- and gamma-Proteobacteria that were more closely related to service water microorganisms than to air microbes . Rhodamine WT dye and fluorescent microspheres employed as contaminant tracers, however, indicated that service water contamination of most of the rock samples was < 0.01% during acquisition . The microbial contaminants most likely originated from the service water, infiltrated the low permeability rock through and accumulated within mining-induced fractures where they survived for several days before being mined . Combined PLFA and terminal restriction fragment length profile (T-RFLP) analyses suggest that the maximum concentration of indigenous microorganisms in the Carbon Leader was < 10(2) cells g(-1) . PLFA, 35S autoradiography and enrichments suggest that the adjacent quartzite was less contaminated and contained approximately 10(3) cells gram(-1) of thermophilic, sulphate reducing bacteria, SRB, some of which are delta-Proteobacteria . Pore water and rock geochemical analyses suggest that these SRB's may have been sustained by sulphate diffusing from the adjacent U-rich, Carbon Leader where it was formed by radiolysis of sulphide.

Water Sci Technol, 2003, 48(6), 195 - 202
Assessment of compatible solutes to overcome salinity stress in thermophilic (55 degrees C) methanol-fed sulfate reducing granular sludges; Vallero MV et al.; High NaCl concentrations (25 g x L(-1)) considerably decreased the methanol depletion rates for sludges harvested from two lab-scale sulfate reducing UASB reactors . In addition, 25 gNaCl x L(-1) strongly affected the fate of methanol degradation, with clear increase in the acetate production at the expense of sulfide and methane production . The addition of different osmoprotectants, viz . glutamate, betaine, ectoine, choline, a mixture of compatible solutes and K+ and Mg2+, slightly increased methanol depletion rates for UASB reactors sludges . However, the acceleration in the methanol uptake rate favored the homoacetogenic bacteria, as the methanol breakdown was steered to the formation of acetate without increasing sulfate reduction and methane production rates . Thus, the compatible solutes used in this work were not effective as osmoprotectants to alleviate the acute NaCl toxicity on sulfate reducing granular sludges developed in methanol degrading thermophilic (55 degrees C) UASB reactors.

Water Sci Technol, 2003, 48(6), 95 - 101
Bicarbonate dosing: a tool to performance recovery of a thermophilic methanol-fed UASB reactor; Paulo PL et al.; The thermophilic-anaerobic treatment of methanol-containing wastewater in an upflow anaerobic sludge blanket (UASB) reactor, was found to be quite sensitive to pH shocks, both acid and alkaline . The results of the recovery experiments of sludge exposed to an alkaline shock, indicated that the addition or deprivation of sodium bicarbonate (NaHCO3) in the medium, plays an important role in the competition of methanogens and (homo)acetogens for methanol . In addition, caution has to be taken when using NaHCO3 for buffering methanol-containing wastewaters, since its introduction in the system will favour (homo)acetogenesis when proper conditions are not established . Based on these results, a recovery strategy for methanogenesis was proposed where bicarbonate is supplied stepwise, and the reactor is operated in a batch mode . This strategy was found to be appropriate, i.e . the results revealed that the recovery of methanogenesis on methanol from a reactor upset or complete failure caused by pH shock is possible, even in systems where (homo)acetogens are outcompeting methanogens . The time and the number of feedings required will depend on the degree of deterioration of the sludge.

Water Sci Technol, 2003, 48(6), 81 - 6
Acclimation of mesophilic anaerobic sludge to thermophilic conditions: PCR genera detection methodology; Cabirol N et al.; In Mexico, sludge from wastewater treatment plants is a public health problem, especially for the infant population due to the presence of pathogens and parasite eggs . Therefore, it is of great interest to apply proper treatment methods for those wastes . An option proposed for the removal of possible infectious sources is thermophilic anaerobic digestion, even though a limitation for its acceptance is the slow start-up of the reactors . The transformation of a mesophilic anaerobic sludge for its utilization as a thermophilic inoculum, by means of direct temperature increase up to 55 degrees C and feeding of the inoculum with synthetic medium was possible in two months . As a main part of the work and with the objective of evaluating the changes in the methanogenic microbial population during the sludge treatment, a specific detection technique for these bacteria has been developed and validated, based on the use of the polymerase chain reaction (PCR).

Mol Genet Genomics, 2004 Feb, 271(1), 50 - 9 Epub 2003 Nov 25.
Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: a simple and efficient method for gene transfer; Onai K et al.; Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical and X-ray crystallographic studies . We found that T . elongatus displays natural transformation, and we established a simple and efficient protocol for transferring exogenous DNAs into the organism's genome . We obtained transformants directly on selective agar plates without having to amplify them prior to plating . We constructed several targeting vectors that enabled us to insert exogenous DNAs into specific sites without disrupting endogenous genes and operons . We also developed a new selectable marker gene for T . elongatus by optimizing the codons of the gene encoding a kanamycin nucleotidyltransferase derived from the thermophilic bacterium Bacillus stearothermophilus . This synthetic gene enabled us to select transformants as kanamycin-resistant colonies on agar plates at 52 degrees C . Optimization of the conditions for natural transformation resulted in a transformation efficiency of up to 1.7 x 10(3) transformants per microg of DNA . The exogenous DNAs were integrated stably into the targeted sites of the T . elongatus genome via homologous recombination by double crossovers.

FEMS Microbiol Rev, 2003 Dec, 27(5), 593 - 616
Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria; Radianingtyas H et al.; Many studies have been undertaken to characterise alcohol dehydrogenases (ADHs) from thermophiles and hyperthermophiles, mainly to better understand their activities and thermostability . To date, there are 20 thermophilic archaeal and 17 thermophilic bacterial strains known to have ADHs or similar enzymes, including the hypothetical proteins . Some of these thermophiles are found to have multiple ADHs, sometimes of different types . A rigid delineation of amino acid sequences amongst currently elucidated thermophilic ADHs and similar proteins is phylogenetically apparent . All are NAD(P)-dependent, with one exception that utilises the cofactor F(420) instead . Within the NAD(P)-dependent group, the thermophilic ADHs are orderly clustered as zinc-dependent ADHs, short-chain ADHs, and iron-containing/activated ADHs . Distance matrix calculations reveal that thermophilic ADHs within one type are homologous, with those derived from a single genus often showing high similarities . Elucidation of the enzyme activity and stability, coupled with structure analysis, provides excellent information to explain the relationship between them, and thermophilic ADHs diversity.

Biochem Biophys Res Commun, 2003 Dec 12, 312(2), 467 - 72
A novel cold-adapted imidase from fish Oreochromis niloticus that catalyzes hydrolysis of maleimide; Huang CY et al.; In this paper we report the first comparative study of cold-adapted imidase (EC 3.5.2.2) from the fish (Oreochromis niloticus) liver and its thermophilic counterparts taken from pig liver and Escherichia coli (overexpressed recombinant hydantoinase from Agrobacterium radiobacter NRRL B1) . Approximately 6000-fold purification and a 40% yield of fish imidase activity were obtained through ammonium sulfate precipitation, octyl, chelating, DEAE, and hydroxyapatite chromatography . This cold-adapted imidase was characterized by a specific activity 10- to a 100-fold higher than those of its thermophilic counterparts below room temperature (25 degrees C or lower) conditions but less stable at elevated temperatures (40 degrees C or higher) . A less organized helical structure (compared to those of pig liver and bacterial imidases) was observed by circular dichroism . Furthermore, maleimide was first identified as a novel substrate of all imidases examined, and confirmed by HPLC and NMR analysis . These results constituted a first study to discover a novel cold-adapted imidase with surprising high activity . These findings might be also helpful for industrial application of imidase.

Biochem Biophys Res Commun, 2003 Dec 12, 312(2), 340 - 5
Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii; Hourai S et al.; The crystal structure of the nitrile hydratase (NHase) from Bacillus smithii SC-J05-1 was determined . Our analysis of the structure shows that some residues that seem to be responsible for substrate recognition are different from those of other NHases . In particular, the Phe52 in the beta subunit of NHase from B . smithii covers the metal center partially like a small lid and narrows the active site cleft . It is well known that the NHase from B . smithii especially prefers aliphatic nitriles for its substrate rather than aromatic ones, and we can now infer that the Phe52 residue may play a key role in the substrate specificity for this enzyme . This finding leads us to suggest that substitution of these residues may alter the substrate specificity of the enzyme.

J Mol Biol, 2003 Dec 5, 334(4), 811 - 21
Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface; Bjork A et al.; Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers . To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions . The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg . C in apparent melting temperature compared to the wild-type . The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact . Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity . The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.

Appl Microbiol Biotechnol, 2004 May, 64(4), 473 - 80 Epub 2003 Nov 22.
Temperature optima of enzyme-catalysed reactions in microemulsion systems; Mlejnek K et al.; Ternary phase systems (water/surfactant/organic solvent) were utilised to increase and broaden the temperature optima of enzyme-catalysed reactions . Alcohol dehydrogenases from yeast and Thermoanaerobium brockii (EC 1.1.1.1 and EC 1.1.1.2), lactate dehydrogenase from Lactobacillus delbrueckii (EC 1.1.1.28) and the particulate hydrogenase from Ralstonia eutropha (EC 1.18.99.1) were used as model enzymes in microemulsions, consisting of the surfactant Aerosol OT, and various alkane solvent and aqueous phases . All enzymes exhibited, besides an increase in specific activity, an upshift of the temperature optimum of the catalysed reaction . The temperature optimum could be further shifted by variation of the chain length of the solvent used and/or the addition of compatible solutes to the aqueous phase . Under optimised conditions, catalytic reactions of enzymes from mesophilic microorganisms had temperature optima in the range generally obtained with enzymes from thermophilic organisms .

Biotechnol Lett, 2003 Oct, 25(20), 1743 - 6
Isolation and characterization of a thermophilic bacterium, Geobacillus thermocatenulatus, degrading nylon 12 and nylon 66; Tomita K et al.; A thermophilic bacterium, identified as a neighboring species to Geobacillus thermocatenulatus, having a growth optimum at 55 degrees C and, capable of degrading nylon 12, was isolated from soil by enrichment culture technique at 60 degrees C . At this temperature, the strain grew on 5 g nylon 12 l(-1) with a decrease in its molecular weight from 41000 to 11000 over 20 d . The degradation was assumed to be due to endogenous hydrolysis of amide bond in nylon 12 . The strain degraded also nylon 66 with a decrease in its molecular weight from 43000 to 17000 in 20 d at 60 degrees C . Nylon 6 was not degraded.

Int J Food Microbiol, 2003 Dec 31, 89(2-3), 223 - 31
Rapid selection of phage-resistant mutants in Streptococcus thermophilus by immunoselection and cell sorting; Viscardi M et al.; Immunoselection and flow cytometry allowed the isolation from Streptococcus thermophilus strain Str31 of double mutants displaying resistance to the phage phi31 and good acid production . Strain Str31 is very sensitive to phage phi31 . This phage-host system seemed therefore particularly suitable to test the validity of the selection method adopted in this study . Mutants were stable with respect to both characters . The isolation of the double mutants required 4 to 5 days . The approach does not involve genetic manipulations and can therefore be an alternative to genetic engineering when this technology cannot be applied.

Arch Biochem Biophys, 2003 Dec 1, 420(1), 103 - 13
Molecular characterization and functional analysis of the manganese-containing superoxide dismutase gene (sodA) from Streptococcus thermophilus AO54; Andrus JM et al.; This report describes the isolation, sequencing, and functional analysis of the sodA gene, encoding Mn-superoxide dismutase, from Streptococcus thermophilus AO54 . The gene was found to encode a 201 amino acid polypeptide with 88 and 83% identity to SodA from Streptococcus mutans and Streptococcus agalacticae, respectively . Primer extension analysis revealed a transcriptional start site 27 nucleotides upstream of initiation codon . The gene was expressed in Escherichia coli and was able to rescue the growth of a sodAsodB mutant in a minimal-medium containing 10(-6)M paraquat . A sodA mutant of S . thermophilus was constructed and found to be more sensitive to aerobic growth than its parent strain . Supplementing the medium with MnCl(2) improved the growth of the mutant, only under microaerophilic conditions . The results suggest that sodA is essential for the aerobic growth of S . thermophilus . In the absence of functional SodA, manganese ions may provide partial protection against oxygen toxicity.

Eur J Biochem, 2003 Dec, 270(23), 4736 - 43
High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin . Linear clusters form at high pH on polypeptide unfolding; Griffin S et al.; To probe the stability of the seven-iron ferredoxin from Thermus thermophilus (FdTt), we investigated its chemical and thermal denaturation processes in solution . As predicted from the crystal structure, FdTt is extremely resistant to perturbation . The guanidine hydrochloride-induced unfolding transition shows a midpoint at 6.5 m (pH 7, 20 degrees C), and the thermal midpoint is above boiling, at 114 degrees C . The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high stability at higher pH . On FdTt unfolding at alkaline pH, new absorption bands at 520 nm and 610 nm appear transiently, resulting from rearrangement of the cubic clusters into linear three-iron species . A range of iron-sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned.

Biochemistry, 2003 Nov 25, 42(46), 13438 - 48
Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit; Poznanski J et al.; Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied . Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznanski, J., Zhukov, I., Bolewska, K., and Wierzchowski, K . L . (2001) J . Biomol . NMR 20, 181-2) . The protein proved to be sufficiently soluble and did not aggregate only in the protonated state . In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy . Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein . The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments . 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure . Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E . A., Muzzin, O., Chlenov, M., Sun, J . L., Olson, A., Weinman, O., Trester-Zedlitz, M . L., and Darst, S . A . (2002) Mol . Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D . G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M . N., Borukhov, S., and Yokoyama, S . (2002) Nature 417, 712-9.) . Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and {(1)H}-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments . Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.

J Bacteriol, 2003 Dec, 185(23), 6773 - 9
Identification and characterization of the nickel uptake system for urease biogenesis in Streptococcus salivarius 57.I; Chen YY et al.; Ureases are multisubunit enzymes requiring Ni(2+) for activity . The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes) . Urease biogenesis also requires a high-affinity Ni(2+) uptake system . By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3' to the ure operon . To determine whether these genes were involved in urease biogenesis by catalyzing Ni(2+) uptake in S . salivarius, regions 3' to ureD were amplified by PCRs from S . salivarius by using primers identical to the S . thermophilus sequences . Sequence analysis of the products revealed three ORFs . Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon . Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate (63)Ni(2+) during growth . Supplementation of the growth medium with NiCl(2) at concentrations as low as 2.5 micro M partially restored urease activity in the mutant . Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni(2+) was provided at neutral pH . Enhancement of urease activity by adding nickel was regulated at the posttranslational level . Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni(2+)-specific ATP-binding cassette transporter.

J Bacteriol, 2003 Dec, 185(23), 6764 - 72
Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth; Lessard C et al.; The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism . S . thermophilus releases galactose into the medium during growth on lactose . Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS) . Unlike S . thermophilus, S . salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars . Analysis of the C-terminal end of S . salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S . thermophilus LacS . Experiments performed with purified proteins showed that S . salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S . salivarius cells . Two other major S . salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS) . The effect of LacS phosphorylation on growth was studied with strain G71, an S . salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P) . These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.

J Biol Chem, 2004 Feb 20, 279(8), 6815 - 23 Epub 2003 Nov 15.
A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis; De Simone G et al.; The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L . In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme . The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated . The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis . Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism . In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 686 - 90 Epub 2003 Nov 13.
Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile; Topakas E et al.; A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity . The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0+/-1.5 kDa, with a mass of 33+/-1 kDa on SDS-PAGE . The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1 . The enzyme activity was optimal at pH 6.0 and 55-60 degrees C . The purified esterase was stable at the pH range 5.0-7.0 . The enzyme retained 70% of activity after 7 h at 50 degrees C and lost 50% of its activity after 45 min at 55 degrees C and after 12 min at 60 degrees C . Determination of k(cat)/ K(m) revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively . No activity on methyl sinapinate was detected . The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5- O- trans-feruloyl-alpha- l-arabinofuranoside (NPh-5-Fe-Ara f) 2-fold more efficiently than NPh-2-Fe-Ara f . Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S . thermophile (a maximum of 34% total ferulic acid released after 1 h incubation) . StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase . The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.

Nahrung, 2003 Oct, 47(5), 349 - 53
Influence of heat impact in reconstituted skim milk on the properties of yoghurt fermented by ropy or non-ropy starter cultures; Lorenzen PC et al.; The paper describes studies on the influence of heat impact in reconstituted skim milk on chemical and functional properties of yoghurt products . Reconstituted skim milk was heated for 20 min at 85 degrees C, 90 degrees C, or 95 degrees C . Ropy (producing exopolysaccharides, EPS) or non-ropy strains of S . thermophilus and L . delbrueckii subsp . bulgaricus were used as starter culture for yoghurt manufacture . The studies have shown that the fermentation times decreased with increasing heat impact when the ropy starter culture was used, while they remained to a far extent unchanged if the non-ropy starter culture was applied . The lactic acid contents of the yoghurt products were in the same range when the milk was heated at 85 degrees C or 90 degrees C, while they were different when milk was heated at 95 degrees C . There was a tendency visible that an increase in preheating leads to increased L(+)- and decreased D(-)-lactic acid contents if the non-ropy culture was applied . Using the ropy culture, it was vice versa . A slightly decrease in proteolysis with increasing heat impact was to be noted with both starter cultures . Concerning the relation of proteolysis to acidification, the fermentation process could be subdivided into three sections with different slopes if the non-ropy starter culture was used, while a linear relation was found if the ropy starter culture was applied . Regarding final product characteristics it was found that the functional properties of yoghurt decreased with increasing heat impact when the ropy starter culture was applied, while they remained to a far extent unchanged when the non-ropy starter culture was used . It can be concluded from these studies that a preheating of milk at a temperature of 85 degrees C (20 min) is optimal in regard to final yoghurt product characteristics.

Gene, 2003 Oct 23, 317(1-2), 39 - 47
Thermophilic prokaryotes have characteristic patterns of codon usage, amino acid composition and nucleotide content; Singer GA et al.; A number of recent studies have shown that thermophilic prokaryotes have distinguishable patterns of both synonymous codon usage and amino acid composition, indicating the action of natural selection related to thermophily . On the other hand, several other studies of whole genomes have illustrated that nucleotide bias can have dramatic effects on synonymous codon usage and also on the amino acid composition of the encoded proteins . This raises the possibility that the thermophile-specific patterns observed at both the codon and protein levels are merely reflections of a single underlying effect at the level of nucleotide composition . Moreover, such an effect at the nucleotide level might be due entirely to mutational bias . In this study, we have compared the genomes of thermophiles and mesophiles at three levels: nucleotide content, codon usage and amino acid composition . Our results indicate that the genomes of thermophiles are distinguishable from mesophiles at all three levels and that the codon and amino acid frequency differences cannot be explained simply by the patterns of nucleotide composition . At the nucleotide level, we see a consistent tendency for the frequency of adenine to increase at all codon positions within the thermophiles . Thermophiles are also distinguished by their pattern of synonymous codon usage for several amino acids, particularly arginine and isoleucine . At the protein level, the most dramatic effect is a two-fold decrease in the frequency of glutamine residues among thermophiles . These results indicate that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting (i) mRNA thermostability, (ii) stability of codon-anticodon interactions and (iii) increased thermostability of the protein products . We conclude that elevated growth temperature imposes selective constraints at all three molecular levels: nucleotide content, codon usage and amino acid composition . In addition to these multiple selective effects, however, the genomes of both thermophiles and mesophiles are often subject to superimposed large changes in composition due to mutational bias.

Nucleic Acids Res, 2003 Nov 15, 31(22), 6473 - 80
Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein; Perales C et al.; Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair . We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB) . The three proteins were found as dimers in solution . They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80 degrees C, and the wild-type protein could withstand incubation at 94 degrees C for 2 min . Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs . The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus . TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.

J Biol Chem, 2004 Feb 20, 279(8), 6380 - 4 Epub 2003 Nov 05.
Trigger factor from Thermus thermophilus is a Zn2+-dependent chaperone; Suno R et al.; The ribosome-associated chaperone trigger factor (TF) of Escherichia coli interacts with a variety of newly synthesized polypeptides to assist their correct folding . Here, we report that the TF of thermophilic eubacterium, Thermus thermophilus, arrested spontaneous folding of green fluorescent protein by forming a 1:1 binary complex . The complex was isolable by gel-filtration but was shown to be dynamic because green fluorescent protein was released by alpha-casein in large excess . Unexpectedly, EDTA completely abolished the folding-arrest activity of TF, and analysis revealed that the TF from our preparation contained approximately 0.5 mol Zn2+/mol TF . The folding-arrest activity of TF that was saturated with Zn2+ (approximately 1 mol/mol TF) was twice as efficient as that of untreated TF . Thus, chaperone activity of thermophilic TF is Zn2+-dependent.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Nov, 136(3), 487 - 94
Solubilization of dynein from Tetrahymena ssp . axonemes using phosphate analogues; Nakamura K et al.; One major protein was selectively solubilized when phosphate analogues, such as inorganic vanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlFx), were added to ciliary axonemes of Tetrahymena ssp . (T . pyriformis or T . thermophila) in the presence of ATP . This protein contains three high molecular weight polypeptides, characteristic of an outer arm dynein . Electron microscopic observation of the axonemes after solubilization using ATP and Vi revealed axonemes partially lacking outer arm dyneins . These results suggest that the solubilized protein is an outer arm dynein and also that a dynein-ADP-phosphate complex decreases its affinity with the adjacent microtubules within axonemes . Limited digestion with chymotrypsin revealed that each solubilized dynein has a similar conformation, but it is markedly different from that of dynein in the absence of ATP or a phosphate analogue . The solubilized dynein obtained by the addition of Vi and ATP to axonemes was digested by UV irradiation to yield at least five new polypeptides (240, 230, 225, 180 and 160 kDa) but the dyneins solubilized by BeFx (or AlFx) in the presence of ATP did not produce any photocleavage products under the same conditions.

Orig Life Evol Biosph, 2003 Dec, 33(6), 621 - 31
Using thermal inactivation kinetics to calculate the probability of extreme spore longevity: implications for paleomicrobiology and lithopanspermia; Nicholson WL; Thermal inactivation kinetics with extrapolation were used to model the survival probabilities of spores of various Bacillus species over time periods of millions of years at the historical ambient temperatures (25-40 degrees C) encountered within the 250 million-year-old Salado formation, from which the putative ancient spore-forming bacterium Salibacillus marismortui strain 2-9-3 was recovered . The model indicated extremely low-to-moderate survival probabilities for spores of mesophiles . but surprisingly high survival probabilities for thermophilic spores . The significance of the results are discussed in terms of the survival probabilities of (i) terrestrial spores in ancient geologic samples and (ii) spores transported between planets within impact ejecta.

Int J Biol Macromol, 2003 Nov, 33(1-3), 141 - 8
Catalytic properties and mode of action of three endo-beta-glucanases from Talaromyces emersonii on soluble beta-1,4- and beta-1,3;1,4-linked glucans; McCarthy T et al.; In this paper, we present the first detailed analysis of the modes of action of three purified, thermostable endo-beta-D-glucanases (EG V-VII) against a range of soluble beta-linked glucans . Studies indicated that EG V-VII, purified to homogeneity from a new source, the thermophilic fungus Talaromyces emersonii, are strict beta-glucanases that exhibit maximum activity against mixed-link 1,3;1,4-beta-D-glucans . Time-course hydrolysis studies of 1,4-beta-D-glucan (carboxymethylcellulose; CMC), 1,3;1,4-beta-D-glucan from barley (BBG) and lichenan confirmed the endo-acting nature of EG V-VII and verified preference for 1,3;1,4-beta-D-glucan substrates . The results suggest that EG VI and EG VII belong to EC 3.2.1.6, as both enzymes also exhibit activity against 1,3-beta-glucan (laminaran), in contrast to EG V . Although cellobiose, cellotriose and glucose were the main glucooligosaccharide products released, the range and relative amount of each product was dependent on the particular enzyme, substrate and reaction time . Kinetic constants (Km, Vmax, kcat and kcat/Km) determined for EG V-VII with BBG as substrate yielded similar Km and Vmax values for EG V and EG VI . EG VII exhibited highest affinity for BBG (Km value of 9.1 mg ml(-1)) and the highest catalytic efficiency (kcat/Km of 12.63 s(-1) mg(-1) ml).

Int J Biol Macromol, 2003 Nov, 33(1-3), 129 - 34
Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan; Vardakou M et al.; Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX) . WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases . At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T . aurantiacus and 7.1 nM for the xylanase from S . thermophile . Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme . Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC) . The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution . The xylanase from T . aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S . thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3) . These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.

Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 629 - 34
Molecular mechanisms of the metabolite 4-hydroxytamoxifen of the anticancer drug tamoxifen: use of a model microorganism; Monteiro JP et al.; A strain of the thermophilic eubacterium Bacillus stearothermophilus was used as a model system to identify membrane mediated cytotoxic effects of 4-hydroxytamoxifen, following previous studies with tamoxifen . With this experimental approach we attempted to further clarify tamoxifen and 4-hydroxytamoxifen membrane interactions often evoked as responsible for their multiple cellular effects . Bacterial growth and the oxygen consumption rate provided quantitative data of the cytotoxic action of hydroxytamoxifen . The effects of hydroxytamoxifen on the physical properties of bacterial lipid membrane preparations were also evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene . Cultures of B . stearothermophilus grown in a complex medium containing hydroxytamoxifen in the concentration range of 1 to 7 microM exhibited progressively longer lag adapting periods, decreased specific growth rates and lower growth yields, as compared to control cultures . Hydroxytamoxifen also affected the electron redox flow of B . stearothermophilus protoplasts and induced significant perturbation of the structural order of bacterial lipid dispersions . We concluded that the bacterial model provides useful information about the nature and repercussion of membrane physical interactions of this lipophilic drug, on the basis of an easy and economic methodology.

Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 595 - 601
Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan; Martins JD et al.; Microorganisms are very powerful tools for the supply of information about the toxic effects of lipophilic compounds, since an impairment of cell growth usually occurs as a result of perturbations related, in most cases, with the partition of toxicants in membranes . The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify alpha- and beta-endosulfan interactions with the membrane possibly related with the insecticide toxicity . Two approaches have been pursued: (a) bacterial growth is followed and the effects of endosulfan isomers determined; (b) biophysical studies with the fluorescent fluidity probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to assess the effects of alpha- and beta-endosulfan on the organization of the membrane lipid bilayer . The effects on growth were quantitatively evaluated by determination of growth parameters, namely the lag phase, the specific growth rate and the cell density reached by cultures in the stationary phase . Growth inhibition by alpha and beta-endosulfan dependent on the concentration is diminished or removed by the addition of 2.5 mM Ca2+ to bacterial cultures . Fluorescence DPH polarization consistently showed opposite effects of Ca2+ and alpha- and beta-endosulfan on the physical state of bacterial polar lipid dispersions.

J Biomol Screen, 2002 Dec, 7(6), 547 - 53
Colorimetric assays for biodegradation of polycyclic aromatic hydrocarbons by fungal laccases; Alcalde M et al.; Polycyclic aromatic hydrocarbons (PAHs) are highly toxic organic pollutants widely distributed in terrestrial and aquatic environments . In the present work, 2 colorimetric assays for laccase-catalyzed degradation of PAHs were developed based on studies of the oxidation of 12 aromatic hydrocarbons by fungal laccases from Trametes versicolor and Myceliophthora thermophila . Using a sodium borohydride water-soluble solution, the authors could reduce the single product of laccase-catalyzed anthracene biooxidation into the orange-colored 9,10-anthrahydroquinone, which is quantifiable spectrophotometrically . An assay using polymeric dye (Poly R-478) as a surrogate substrate for lignin degradation by laccase in the presence of mediator is also presented . The decolorization of Poly R-478 was correlated to the oxidation of PAHs mediated by laccases . This demonstrates that a ligninolytic indicator such as Poly R-478 can be used to screen for PAH-degrading laccases; it will also be useful in screening mutant libraries in directed evolution experiments . Poly R-478 is stable and readily soluble . It has a high extinction coefficient and low toxicity toward white rot fungi, yeast, and bacteria, which allow its application in a solid-phase assay format.

Environ Technol, 2003 Sep, 24(9), 1183 - 90
Hydrolysis and acidogenesis of particulate organic material in mesophilic and thermophilic anaerobic digestion; Kim M et al.; The purpose of this study was to evaluate the effect of pH and inorganic nutrient supplementations for anaerobic hydrolysis and acidogenesis of particulate organic materials at both mesophilic (35 degrees C) and thermophilic (55 degrees C) temperatures . Hydrolysis and acidogenesis of a synthetic sludge was observed in batch operation for the evaluation of the pH effect . pH was uncontrolled in one reactor and controlled at 4.5, 5.5, and 6.5 in the other three reactors at both temperatures . The greatest degree of hydrolysis and acidogenesis occurred when the pH was controlled at 6.5 . The pH of the uncontrolled reactor dropped to 3.4 at both temperatures severely retarding hydrolysis and acidogenesis . Concentrations of acetic and n-butyric acids predominated with lower concentrations of propionic acid at both temperatures in all reactors . Lactic acid was produced as the earliest intermediate but as the reaction proceeded, short chain VFAs were produced as final end products with a decrease in lactic acid . The higher the pH, the earlier this trend was observed . For the controlled reactors at pH 6.5, the soluble COD production and the VSS reduction peaked in 4 days at 55 degrees C whereas it took about 11 days at 35 degrees C to obtain the same result . During the linear SCOD production period at a pH of 6.5 the hydrolysis rate of the thermophilic reactor was greater than that for mesophilic . Thermophilic conditions appeared to be more sensitive to pH than mesophilic ones for both hydrolysis and acidogenesis . Additional experiments were conducted to establish the effect of inorganic nutrient (Ca, Fe, Co, and Ni) supplementation on hydrolysis and acidogenesis at both temperatures . It has, prior to this, been assumed that only methanogenesis benefited from trace metal supplementation . However, the results demonstrated the importance of inorganic nutrient supplementation to optimize hydrolysis and acidogenesis at both temperatures.

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