Thermophile

J Biochem Mol Biol, 2002 Nov 30, 35(6), 568 - 75
New action pattern of a maltose-forming alpha-amylase from Streptomyces sp . and its possible application in bakery; Ammar YB et al.; An a-Amylase (EC 3.2.1.1) was purified that catalyses the production of a high level of maltose from starch without the attendant production of glucose . The enzyme was produced extracellularly by thermophilic Streptomyces sp . that was isolated from Thailand's soil . Purification was achieved by alcohol precipitation, DEAE-Cellulose, and Gel filtration chromatographies . The purified enzyme exhibited maximum activity at pH 6-7 and 60 degrees C . It had a relative molecular mass of 45 kDa, as determined by SDS-PAGE . The hydrolysis products from starch had alpha-anomeric forms, as determined by 1H-NMR . This maltose-forming alpha-Amylase completely hydrolyzed the soluble starch to produce a high level of maltose, representing up to 90% . It hydrolyzed maltotetrose and maltotriose to primarily produce maltose (82% and 62% respectively) without the attendant production of glucose . The high maltose level as a final end-product from starch and maltooligosaccharides, and the unique action pattern of this enzyme, indicate an unusual maltose-forming system . After the addition of the enzyme in the bread-baking process, the bread's volume increased and kept its softness longer than when the bread had no enzyme.

Curr Protein Pept Sci, 2002 Dec, 3(6), 601 - 14
Electrostatics in protein binding and function; Sinha N et al.; Protein electrostatic properties stem from the proportion and distribution of polar and charged residues . Polar and charged residues regulate the electrostatic properties by forming short-range interactions, like salt-bridges and hydrogen-bonds, and by defining the over-all electrostatic environment in the protein . Electrostatics play a major role in defining the mechanisms of protein-protein complex formation, molecular recognitions, thermal stabilities, conformational adaptabilities and protein movements . For example:- Functional hinges, or flexible regions of the protein, lack short-range electrostatic interactions; Thermophilic proteins have higher electrostatic interactions than their mesophilic counter parts; Increase in binding specificity and affinity involve optimization of electrostatics; High affinity antibodies have higher, and stronger, electrostatic interactions with their antigens; Rigid parts of proteins have higher and stronger electrostatic interactions . In this review we address the significance of electrostatics in protein folding, binding and function . We discuss that the electrostatic properties are evolutionally selected by a protein to perform an specific function . We also provide bona fide examples to illustrate this . Additionally, using continuum electrostatic and molecular dynamics approaches we show that the "hot-spot" inter-molecular interactions in a very specific antibody-antigen binding are mainly established through charged residues . These "hot-spot" molecular interactions stay intact even during high temperature molecular dynamics simulations, while the other inter-molecular interactions, of lesser functional significance, disappear . This further corroborates the significance of charge-charge interactions in defining binding mechanisms . High affinity binding frequently involves "electrostatic steering" . The forces emerge from over-all electrostatic complementarities and by the formation of charged and polar interactions . We demonstrate that although the high affinity binding of barnase-barstar and anti-hen egg white lysozyme (HEL) antibody-HEL complexes involve different molecular mechanisms, it is electrostatically regulated in both the cases . These observations, and several other studies, suggest that a fine tuning of local and global electrostatic properties are essential for protein binding and function.

Water Environ Res, 2002 Sep-Oct, 74(5), 494 - 507
Changing mesophilic wastewater sludge digestion into thermophilic operation at Terminal Island Treatment Plant; Iranpour R et al.; This paper describes the progress up to June 2000 for thermophilic digestion of wastewater sludge at the Los Angeles, California, Bureau of Sanitation's Terminal Island Treatment Plant . The development of the microorganism culture has followed a course similar to that seen at other successful plants for establishment of a stable, well-balanced thermophilic culture in a large digester, but at an accelerated pace . This study began with rapid heating, increasing the temperature of the 4500 m3 (1.2 mil . gal) digester to the target temperature of 55 degrees C at approximately 3 degrees C/d . A method of feeding to maximize the rate of culture development was used as feeding accelerated to approximately 400 m3/d (0.1 mgd) . An initial rise of acid concentration (primarily acetate) was seen . Within two weeks, acid concentration declined and stabilized, indicating that acidogenic and methanogenic microbial communities came into balance . Coliform data indicate that digester disinfection was stably effective from the middle of April . The salmonella tests done to date satisfy the U.S . Environmental Protection Agency (U.S . EPA) class A specification . Testing with helminth ova and enteric viruses before and after the digester shows satisfaction of class A standard for those organisms . The present combination of low volatile fatty acids and low hydrogen sulfide is good news for odor control . The data show increases in volatile solids destruction and estimated gas production, compared with the previous mesophilic operation; however, large uncertainties have been calculated from the data . As the digester is now operating successfully at the current feed rate, there seems to be no barriers to processing the entire sludge production of the plant . Other results indicate that the U.S . EPA requirements for exceptional quality class A biosolids are likely to be achieved.

J Med Microbiol, 2002 Dec, 51(12), 1117 - 27
Improved molecular identification of Thermoactinomyces spp . associated with mushroom worker's lung by 16S rDNA sequence typing; Xu J et al.; Mushroom worker's lung (MWL) is a hypersensitivity pneumonitis or allergic alveolitis caused by a type III IgG-mediated immunopathogenic inflammatory reaction in the host due to the inhalation of several thermophilic organisms, including Thermoactinomyces spp . It is difficult to distinguish phenotypically the eight species of this genus; therefore, this study sought to develop an improved molecular means of identifying Thermoactinomyces spp . associated with MWL by partial 16S rDNA PCR amplification and direct sequencing . Hypervariable regions within the 16S rRNA gene, which could be employed as signature sequences of the eight individual species, were identified and employed with highly conserved flanking primers to allow initial PCR amplification, before direct DNA sequencing of the 16S rDNA amplicons . A novel 24-mer 16S rDNA oligonucleotide upstream primer was designed from in silico alignments of all Thermoactinomyces spp . and was employed in combination with downstream (reverse) 16S rDNA primers . This permitted the successful identification of all four isolates associated with mushroom workers' lung . The method may be useful in the identification of Thermoactinomyces spp . associated with allergic alveolitis or pneumonitis associated with occupational exposure in agricultural and horticultural environments.

Environ Technol, 2002 Oct, 23(10), 1127 - 33
Comparison of activated sludge processes at different temperatures: 35 degrees C, 27-55 degrees C, and 55 degrees C; Suvilampi J et al.; The performance of mesophilic (35 degrees C; referred to as R1) and thermophilic (55 degrees C; R3) laboratory activated sludge processes (ASPs) as well as ASP with a fluctuating temperature (27-56 degrees C; R2) was compared . During the 124-day runs, in R1 and R3 hydraulic retention time was gradually reduced from 18 h to 3 h, corresponding to an increase in volumetric loading rate from 2 to 10 kg soluble COD m(-3) d(-1); in R2 hydraulic retention time was gradually reduced from 18 to 4.5 h, corresponding to an increase in volumetric loading rate from 2 to 7.5 kg soluble COD m(-3) d(-1) . R1 removed on average 85% of soluble COD (GF50-filtered) that was approximately 10% more than R3 . In R2 SCOD removal was dependent on the operating temperature, being comparable to R1 and R3 at respective temperature . However, the COD for 0.45 microm-filtered (bacteria-free) effluent samples was lower for R3 than for R1, indicating the role of free bacteria on effluent quality . Furthermore, 24 h post-aeration of R3 effluent at 35 degrees C decreased SCOD (GF50-filtrated) markedly (43% removal), whereas at 55 degrees C no SCOD removal occurred, which suggest mesophilic post-treatment ability to remove thermophilically recalcitrant matter or, more probably, the ability of free bacteria to aggregate more efficiently under lower temperatures . The results indicate that temperature may not be as crucial a factor in high temperature biological wastewater treatment as previously believed . On the other hand, in thermophilic ASP the importance of solids separation is emphasized.

J Biol Chem, 2003 Mar 7, 278(10), 8420 - 8 Epub 2002 Dec 02.
Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae; Edman M et al.; In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC ) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC ), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively . Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified . A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA . Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases . Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase . Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes . Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E . coli inner membrane and cytoplasm in vivo . In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

J Biol Chem, 2003 Feb 21, 278(8), 6101 - 10 Epub 2002 Dec 02.
Nickel in subunit beta of the acetyl-CoA decarbonylase/synthase multienzyme complex in methanogens . Catalytic properties and evidence for a binuclear Ni-Ni site; Gencic S et al.; The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C-C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates . The ACDS beta subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C-C bond activation . To investigate the role of nickel in this process two forms of the Methanosarcina thermophila beta subunit were overexpressed in anaerobically grown Escherichia coli . Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays . However, high activity developed during incubation with NiCl(2) . The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper . Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel . The kinetics of nickel incorporation matched the time course for enzyme activation . Other divalent metal ions could not substitute for nickel in yielding catalytic activity . Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C-C bond activation by the beta subunit in the absence of other ACDS subunits . Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO . In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species . Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation . The results are consistent with an Ni(2){Fe(4)S(4)} arrangement at the active site . A mechanism for C-C bond activation is proposed that includes a specific role for the Fe(4)S(4) center and accounts for the absolute requirement for nickel.

Arch Biochem Biophys, 2002 Dec 15, 408(2), 177 - 83
Structural energetics of MgADP binding to the isolated beta subunit of F1-ATPase from thermophilic Bacillus PS3; Perez-Hernandez G et al.; The energetics of binding of MgADP to the isolated beta subunit of F(1)-ATPase from thermophilic Bacillus (Tbeta) was characterized by high-precision isothermal titration calorimetry . The reaction was enthalpically driven, with a DeltaCp of -36cal(molK)(-1) . To gain insight into the molecular basis of this small DeltaCp, we analyzed the changes in accessible surface areas (DeltaASA) between the structures of empty and MgADP-filled beta subunits, extracted from the crystal structure of bovine heart F(1) . Consistent with the experimental DeltaCp, the DeltaASA was small (-775A(2)) . We used a reported surface area model developed for protein reactions to calculate DeltaCp and DeltaH from DeltaASA, obtaining good agreement with the experimental values . Conversely, using the same model, a DeltaASA of -770A(2) was estimated from experimental DeltaCp and DeltaH for the Tbeta-MgADP complex . Our structural-energetic study indicates that on MgADP binding the isolated Tbeta subunit exhibits intrinsic structural changes similar to those observed in F(1).

Arch Biochem Biophys, 2003 Jan 1, 409(1), 52 - 8
Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus; Puchkaev AV et al.; Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme . Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified . The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values . The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue . The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.

Biochemistry, 2002 Dec 10, 41(49), 14482 - 8
Valine 114 replacements in archaeal elongation factor 1 alpha enhanced its ability to interact with aminoacyl-tRNA and kirromycin; Masullo M et al.; Valine 114 in the D(109)AAILVVA sequence of elongation factor 1alpha from the archaeon Sulfolobus solfataricus (SsEF-1alpha) was substituted with an acidic (V114E), basic (V114K), or cavity-forming (V114A) residue, and the effects on the biochemical properties of the factor were investigated . This sequence is well-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structure of SsEF-1alpha, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequence G(13)XXXXGK{T,S} {Vitagliano, L., Masullo, M., Sica, F., Zagari, A., and Bocchini, V . (2001) EMBO J . 20, 5305-5311} . These mutants displayed functions absent in the wild-type factor . In fact, although they exhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1alpha, V114K and V114A exhibited an affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than that elicited by SsEF-1alpha but similar to that of eubacterial EF-Tu . V114E instead displayed not only a weaker binding capability for aa-tRNA but also a lower affinity for GDP . The intrinsic GTPase activity of V114E was drastically reduced compared to those of SsEF-1alpha, V114K, and V114A . Interestingly, the decreased intrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed for the G13A mutant of SsEF-1alpha {Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., and Bocchini, V . (2002) Biochemistry 41, 628-633} . Finally, the V114A substitution showed only a marginal effect on both the thermostability and thermophilicity of SsEF-1alpha, whereas V114K and V114E replacements strongly destabilized the molecule.

Plant Cell Physiol, 2002 Nov, 43(11), 1366 - 73
Low-molecular-mass polypeptide components of a photosystem II preparation from the thermophilic cyanobacterium Thermosynechococcus vulcanus; Kashino Y et al.; Using a recently introduced electrophoresis system {Kashino et al . (2001) Electrophoresis 22: 1004}, components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus) . PsbE, the large subunit polypeptide of cytochrome b(559), showed an apparent molecular mass much lower than the expected one . The unusually large mobility could be attributed to the large intrinsic net electronic charge . All other Coomassie-stained polypeptides were identified by N-terminal sequencing . In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes . Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN . The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae . These results strongly suggest that PsbN is not a member of the PSII complex . It is also shown that cyanobacteria have cytochrome b(559) in the high potential form as in higher plants.

Biochim Biophys Acta, 2002 Dec 2, 1556(2-3), 168 - 74
Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 A: insights into the role of solvent molecules in thermal stability and co-factor structure; Adir N et al.; The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 A resolution based on the previously determined lower resolution structure (PDB entry 1I7Y) . The improved data was collected using synchrotron radiation at 100 K . The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules . The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described . The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested . All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains . Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation . On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.

J Mol Biol, 2002 Dec 6, 324(4), 871 - 86
Structure of the mammalian ribosome-channel complex at 17A resolution; Morgan DG et al.; The co-translational translocation of proteins into the endoplasmic reticulum (ER) lumen and the biogenesis of membrane proteins require ribosome binding to a membrane channel formed by the Sec61p complex . We now report the 17A structure of a mammalian ribosome-channel complex derived from ER membranes . Atomic models of the ribosomal subunits were aligned to the programmed ribosome from Thermus thermophilus, to provide a common reference frame . The T.thermophilus ribosome, and by extension all known high resolution subunit models, were then docked within our map of the ribosome-channel complex . The structure shows that the ribosome contains a putative tRNA in the exit site, and a comparison with a non-programmed, yeast ribosome suggests that the L1 stalk may function as a gate in the tRNA exit path . We have localized six major expansion segments in the large subunit of the vertebrate ribosome including ES27, and suggest a function for ES30.The large ribosomal subunit is linked to the channel by four connections . We identified regions in the large subunit rRNA and four proteins that may help form the connections . These regions of the ribosome probably serve as a template to guide the assembly of the asymmetric translocation channel . Three of the connections form a picket fence that separates the putative translocation pore from the attachment site of an additional membrane component . The ribosome-channel connections also create an open junction that would allow egress of a nascent chain into the cytosol . At a threshold that is appropriate for the entire complex, the channel is rather solid and the lumenal half of the putative translocation pore is closed . These data suggest that the flow of small molecules across the membrane may be impeded by the channel itself, rather than the ribosome-channel junction.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 206 - 16 Epub 2002 Sep 28.
Redox properties of the photosystem II cytochromes b559 and c550 in the cyanobacterium Thermosynechococcus elongatus; Roncel M et al.; Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus with a histidine tag on the CP43 protein of PSII . Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential ( E'(m)) of +390 mV in about half of the centers and +275 mV in the other half . The high-potential form, whose E'(m)is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation . The E'(m) of the low-potential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH . The possibility that the heme of Cyt b559 in T . elongatus is in a more hydrophobic environment is discussed . Cyt c550 has a higher E'(m)when bound to the PSII core (-80 mV at pH 6.0) than after its extraction from the complex (-240 mV at pH 6.0) . The E'(m) of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58 mV/pH unit is observed when the pH decreases below pH 9.0 . Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme . From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.

FEBS Lett, 2002 Dec 4, 532(1-2), 231 - 6
Thermostability of membrane protein helix-helix interaction elucidated by statistical analysis; Schneider D et al.; A prerequisite for the survival of (micro)organisms at high temperatures is an adaptation of protein stability to extreme environmental conditions . In contrast to soluble proteins, where many factors have already been identified, the mechanisms by which the thermostability of membrane proteins is enhanced are almost unknown . The hydrophobic membrane environment constrains possible stabilizing factors for transmembrane domains, so that a difference might be expected between soluble and membrane proteins . Here we present sequence analysis of predicted transmembrane helices of the genomes from eight thermophilic and 12 mesophilic organisms . A comparison of the amino acid compositions indicates that more polar residues can be found in the transmembrane helices of thermophilic organisms . Particularly, the amino acids aspartic acid and glutamic acid replace the corresponding amides . Cysteine residues are found to be significantly decreased by about 70% in thermophilic membrane domains suggesting a non-specific function of most cysteine residues in transmembrane domains of mesophilic organisms . By a pair-motif analysis of the two sets of transmembrane helices, we found that the small residues glycine and serine contribute more to transmembrane helix-helix interactions in thermophilic organisms . This may result in a tighter packing of the helices allowing more hydrogen bond formation.

Food Addit Contam, 2002 Nov, 19(11), 1043 - 50
Distribution and stability of aflatoxin M1 during production and storage of yoghurt; Govaris A et al.; Yoghurt from cow's milk artificially contaminated with aflatoxin M1 (AFM1) at levels of 0.050 and 0.100 g l(-1) was fermented to reach pHs 4.0 and 4.6 . Yoghurt fermented to pH 4.6 was also used for preparing strained yoghurt . Yoghurts were stored at 4 degrees C for up to 4 weeks . Analysis of AFM1 in milk, yoghurt, strained yoghurt and yoghurt whey was carried out using immunoaffinity column extraction and liquid chromatography coupled with fluorometric detection . AFM1 levels in yoghurt samples showed a significant decrease (p < 0.01) compared with those initially added to milk . Growth of culture lactic acid bacteria was not affected in the AFM1 contaminated yoghurts, with the exception of Streptococcus thermophilus that showed a significantly (p < 0.01) lower increase in the yoghurt containing the toxin at high concentration . Following fermentation, AFM1 was significantly lower (p < 0.01) in yoghurts with pH 4.0 than in yoghurts with pH 4.6 at both contamination levels . During refrigerated storage, AFM1 was rather more stable in yoghurts with pH 4.6 than with pH 4.0 . The percentage loss of the initial amount of AFM1 in milk was estimated at about 13 and 22% by the end of the fermentation, and 16 and 34% by the end of storage for yoghurts with pHs 4.6 and 4.0, respectively . The percentage distribution ratio of AFM1 in strained yoghurt/yoghurt whey of the initial toxin present in the yoghurt was about 90/10 and 87/13 for the lower and the higher contamination levels, respectively.

Eukaryot Cell, 2002 Aug, 1(4), 583 - 93
New class of cargo protein in Tetrahymena thermophila dense core secretory granules; Haddad A et al.; Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion . The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores . Following exocytosis, the cores generally disassemble to diffuse in the cell environment . The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules . However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis . Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration) . By structural criteria, it is unrelated to the previously characterized lattice-forming proteins . It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.

Eukaryot Cell, 2002 Apr, 1(2), 293 - 303
Role of histone deacetylation in developmentally programmed DNA rearrangements in Tetrahymena thermophila; Duharcourt S et al.; In Tetrahymena, as in other ciliates, development of the somatic macronucleus during conjugation involves extensive and reproducible rearrangements of the germ line genome, including chromosome fragmentation and excision of internal eliminated sequences (IESs) . The molecular mechanisms controlling these events are poorly understood . To investigate the role that histone acetylation may play in the regulation of these processes, we treated Tetrahymena cells during conjugation with the histone deacetylase inhibitor trichostatin A (TSA) . We show that TSA treatment induces developmental arrests in the early stages of conjugation but does not significantly affect the progression of conjugation once the mitotic divisions of the zygotic nucleus have occurred . Progeny produced from TSA-treated cells were examined for effects on IES excision and chromosome breakage . We found that TSA treatment caused partial inhibition of excision of five out of the six IESs analyzed but did not affect chromosome breakage at four different sites . TSA treatment greatly delayed in some cells and inhibited in most the excision events in the developing macronucleus . It also led to loss of the specialized subnuclear localization of the chromodomain protein Pdd1p that is normally associated with DNA elimination . We propose a model in which underacetylated nucleosomes mark germ line-limited sequences for excision.

J Appl Microbiol, 1997 Mar, 82(3), 325 - 34
A gene encoding for an alpha-amylase from thermophilic Bacillus sp . strain TS-23 and its expression in Escherichia coli; Lin LL et al.; An alpha-amylase gene from Bacillus sp . strain TS-23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli . A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space . Analysis and activity staining of the concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a mol . wt of approximately 65,000 . The amylase gene (amyA) consisted of an open reading frame of 1,845 bp encoding a protein of 613 amino acids with a calculated mol . wt of 69,543 . The predicted amino acid sequence showed high homology with Bacillus species, E . coli and Salmonella typhimurium alpha-amylases . Deletion of 96 amino acids from the C-terminal portion of the amylase did not result in the loss of amylolytic activity . The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E . coli system and refolded to yield an activable enzyme.

J Appl Microbiol, 1997 Mar, 82(3), 273 - 80
Inhibition of Listeria monocytogenes by piscicolin 126 in milk and Camembert cheese manufactured with a thermophilic starter; Wan J et al.; The effect of bacteriocin, piscicolin 126, on the growth of Listeria monocytogenes and cheese starter bacteria was investigated in milk and in Camembert cheese manufactured from milk challenged with 10(2) cfu ml(-1) L . monocytogenes . In milk incubated at 30 degrees C, piscicolin 126 added in the range of 512-2,048 AU ml(-1) effectively inhibited growth of L . monocytogenes for more than 20 d when challenged with approximately 10(2) cfu ml(-1) L . monocytogenes . At higher challenge levels (10(4) and 10(6) cfu ml(-1)), piscicolin 126 reduced the viable count of L . monocytogenes by 4-5 log units immediately after addition of the bacteriocin; however, growth of Listeria occurred within 24 h . The minimum inhibitory concentration (MIC) of piscicolin 126 against lactic acid cheese starter bacteria was generally greater than 204,800 AU ml(-1) , and the viable count and acid production of these starter cultures in milk were not affected by the addition of 2,048 AU ml(-1) piscicolin 126 . Camembert cheeses made from milk challenged with L . monocytogenes and with added piscicolin 126 showed a viable count of L . monocytogenes 3-4 log units lower than those without piscicolin 126 . Inactivation of piscicolin 126 by proteolytic enzymes from cheese starter bacteria and mould together with the emergence of piscicolin 126-resistant isolates was responsible for the recovery of L . monocytogenes in the cheeses during ripening.

Prep Biochem Biotechnol, 2002 Nov, 32(4), 355 - 62
Role of cation-pi interactions to the stability of thermophilic proteins; Gromiha MM et al.; Elucidating the factors responsible for exhibiting extreme thermal stability of thermophilic proteins is very important for an understanding of the mechanism of protein stability, as well as to design stable proteins . In this work, we have analyzed the influence of cation-pi interactions to enhance the stability from mesophilic to thermophilic proteins . The favorable residue pairs forming such a system of interactions have been brought out . We found that the Tyr has a greater number of such interactions with Lys in thermophilic proteins . Specifically, the same Lys would experience a greater number of cation-pi interactions with several Tyr residues in thermophiles . On the other hand, the influence of Phe in making cation-pi interactions is higher in mesophiles than in thermophiles . Further, a network of cation-pi interactions are maintained by Lys in thermophiles, whereas Arg plays a major role in mesophilic proteins . Moreover, atoms that have a substantial positive charge in both Lys and Arg make a more significant contribution for cation-pi interactions than do cationic group atoms.

Can J Microbiol, 2002 Sep, 48(9), 848 - 52
Monitoring of Bacillus thermodenitrificans OHT-1 in compost by whole cell hybridization; Hatsu M et al.; Thermophilic aerobic composting is a widely practiced method for the disposal of exhaust materials . We isolated a thermophilic bacteria strain from a compost sample under aerobic conditions at 60 degrees C . On the basis of its 16S rRNA sequence and physiological characteristics, this strain was identified as Bacillus thermodenitrificans OHT-1 . An 18-subunit oligonucleotide probe for 16S rRNA, labeled with fluorescein isothiocyanate, was developed for the detection of B . thermodenitrificans . Spores and vegetative cells of B . thermodenitrificans OHT-1 were detected in liquid culture and laboratory compost by whole cell hybridization using this oligonucleotide probe . The results obtained by whole cell hybridization were evaluated in growth experiments of B . thermodenitrificans OHT-1 in laboratory compost and were used to enumerate spores and vegetative cells.

J Appl Microbiol, 2002, 93(6), 965 - 75
Purification and characterization of a thermophilic and acidophilic chitinase from Microbispora sp . V2; Nawani NN et al.; AIM: Purification and characterization of a chitinase from Microbispora sp . V2 . METHODS AND RESULTS: The chitinase from Microbispora sp . V2 was purified to homogeneity by gel filtration chromatography with 4.6% recovery . It had a molecular weight of 35 kDa and showed maximum activity towards p-nitrophenyl-beta-d-N,N'-diacetylchitobiose, indicating a chitobiosidase activity . The enzyme had a pH optimum of 3.0 and temperature optimum of 60 degrees C . It was stable in a wide pH range from 3.0 to 11.0, retaining 61% activity at pH 3.0 and 52% activity at pH 11.0 . It retained 71% activity at 30 degrees C and 45% activity at 50 degrees C, up to 24 h . The enzyme activity was not inhibited by any of the metal ions tested except Hg2+, in the presence of which only 10% activity was retained . CONCLUSIONS: The 35 kDa chitinase from Microbispora sp . V2 has an acidic pH optimum and a high temperature optimum . It is fairly stable and active, and degrades chitin efficiently, although the growth of the culture and enzyme production is slow . SIGNIFICANCE AND IMPACT OF THE STUDY: This report is the first detailed study of a chitinase from Microbispora sp . V2, isolated from hot springs . The chitinase from Microbispora sp . V2 may have potential applications in the recycling of chitinous wastes, particularly due to its thermophilic and acidophilic character . Studies at molecular level may provide further insight on the chitinolytic system of Microbispora spp . with respect to the number and types of chitinases and their regulation.

J Am Chem Soc, 2002 Dec 4, 124(48), 14442 - 9
Virtual screening for binding of phenylalanine analogues to phenylalanyl-tRNA synthetase; Wang P et al.; Although incorporation of nonnatural amino acids provides a powerful means of controlling protein structure and function, experimental investigations of amino acid analogues for utilization by the protein biosynthetic machinery can be costly and time-consuming . In this paper, we describe a computational protocol (HierDock) for predicting the relative energies of binding of phenylalanine analogues to phenylalanyl-tRNA synthetase (PheRS) . Starting with the crystal structure of Thermus thermophilus PheRS without bound ligand, HierDock predicts the binding site of phenylalanine (Phe) within 1.1 A of that revealed by the crystal structure of PheRS cocrystallized with Phe . The calculated binding energies of Phe analogues in PheRS, using HierDock, correlate well with the translational activities of the same analogues in Escherichia coli . HierDock identifies p-fluorophenylalanine and 3-thienylalanine as especially good substrates for PheRS, in agreement with experiment . These results suggest that the HierDock protocol may be useful for virtual screening of amino acid analogues prior to experiment.

J Appl Microbiol, 1997 Feb, 82(2), 259 - 66
Microtitre plate hybridization system for detection of thermophilic Campylobacter rRNA; Lamoureux M et al.; A microtitre plate nucleic acid probe hybridization system was developed for the detection of ribosomal RNA from thermophilic Campylobacter (Camp . jejuni, Camp . coli, Camp . lari and Camp . upsaliensis) . A specific DNA probe obtained by amplification of 23S rRNA sequences using the polymerase chain reaction technique was immobilized on a microtitre plate, and used for hybridization with target 23S rRNA from cell lysates . The RNA-DNA hybrids thus formed in the wells were detected by an immunoenzymatic assay using a monoclonal antiRNA-DNA hybrid antibody . The sensitivity of this system was 2.7 x 10(4) cells ml(-1) . This simple, sensitive and inexpensive hybridization and immunoenzymatic assay system should facilitate the detection of Campylobacter in food and clinical samples.

J Appl Microbiol, 1997 Feb, 82(2), 219 - 24
Seasonality of thermophilic Campylobacter populations in chickens; Wallace JS et al.; The small intestines and caeca of chickens were monitored for seasonal variation of thermophilic campylobacters over a 12-month period . There was a significant seasonal fluctuation in the carriage rate which correlated with the following environmental parameters: sunshine hours (P = 0.0003) and minimum (P = 0.007) and maximum temperatures (P = 0.003) . The number of campylobacters in the small intestine and caeca also showed significant seasonal variation (P = 0.0008); however, the periodicity in the caeca was significantly different from that in the small intestine (P = 0.007) . The numbers of Campylobacter in the caeca were significantly higher than those in the small intestine (P = 0.001) . No significant correlation was found between the numbers of campylobacters in the caeca and small intestines and the environmental parameters monitored.

Biotechnol Bioeng, 2003 Jan 20, 81(2), 241 - 52
Hydrolysis of lactose by free and immobilized beta-galactosidase from Thermus sp . strain T2; Ladero M et al.; The hydrolysis of lactose by a beta-galactosidase from the thermophilic microorganism Thermus sp . strain T2, both in solution and immobilized on a commercial silica-alumina, has been studied . The enzyme has been previously produced by Escherichia coli JM101 harboring the plasmid pBGT1, which contains the codifying gene under the promoters lpp(P) and lac(PQ) . The enzyme was immobilized on the support activated with tris-hydroxymethylphosphine (THP) . Activity and stability of the free and the immobilized enzyme towards pH and temperature were tested . To study the activity at different pH and temperature values, lactose was used as substrate . To check the stability, the enzyme was incubated either in buffer BP or in a solution of lactose in buffer BM at different pH and temperatures, being the remaining activity tested by withdrawing samples and determining their activity toward ONPG at 70 degrees C in buffer BP . Afterward, runs were performed to obtain kinetic models adequate for the description of the hydrolysis of lactose by the free and the immobilized enzyme . These data were fitted to the kinetic models proposed (all based on the Michaelis-Menten mechanism) by non-linear regression, being the models and their parameters compared to determine the effect of the immobilization on the kinetic behavior of the enzyme . Both the free and the immobilized enzyme are competitively inhibited by galactose, while glucose inhibited only the action of the free enzyme, in an uncompetitive way . The immobilization step seems to eliminate the inhibition by glucose . Moreover, the immobilization reduced to a half the inhibitory action of galactose . In general, the immobilization reduced the activity of the enzyme, but increased its thermal stability . Finally, a comparison between the kinetic behavior of this thermophilic enzyme and enzymes of mesophile microorganisms previously studied by us (E . coli and K . fragilis) and by other authors (Aspergillus niger) is performed .

Appl Environ Microbiol, 2002 Dec, 68(12), 6388 - 91
AbiA, a lactococcal abortive infection mechanism functioning in Streptococcus thermophilus; Tangney M et al.; The lactococcal abortive infection mechanisms AbiA and AbiG were introduced into Streptococcus thermophilus 4035, and a range of phages capable of infecting this host were examined for sensitivity to these mechanisms . AbiA proved effective against six phages when examined at a growth temperature of 30 degrees C but had no effect on any of the phages when tested at 37 or 42 degrees C . AbiG failed to affect any of the S . thermophilus phages at 30, 37, or 42 degrees C.

Appl Environ Microbiol, 2002 Dec, 68(12), 6300 - 9
Soil microbial community structure across a thermal gradient following a geothermal heating event; Norris TB et al.; In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity . Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees . Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain . The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils . Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions . Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil . A soil microcosm laboratory experiment simulated the geothermal heating event . As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks . The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.

Appl Environ Microbiol, 2002 Dec, 68(12), 6013 - 20
Isolation and characterization of metal-reducing thermoanaerobacter strains from deep subsurface environments of the Piceance Basin, Colorado; Roh Y et al.; Five bacterial strains were isolated from anaerobic enrichment cultures that had originated from inoculations with samples collected from the deep subsurface environments of the millions-of-years-old, geologically and hydrologically isolated Piceance Basin in Colorado . Small-subunit rRNA gene-based analyses indicated that all of these bacteria were closely related to Thermoanaerobacter ethanolicus, with similarities of 99.4 to 99.5% . Three isolates (X513, X514, and X561) from the five bacterial strains were used to examine physiological characteristics . These thermophilic bacteria were able to use acetate, glucose, hydrogen, lactate, pyruvate, succinate, and xylose as electron donors while reducing Fe(III), cobalt(III), chromium(VI), manganese(IV), and uranium(VI) at 60 degrees C . One of the isolates (X514) was also able to utilize hydrogen as an electron donor for Fe(III) reduction . These bacteria exhibited diverse mineral precipitation capabilities, including the formation of magnetite (Fe(3)O(4)), siderite (FeCO(3)), rhodochrosite (MnCO(3)), and uraninite (UO(2)) . The gas composition of the incubation headspace and the ionic composition of the incubation medium exerted profound influences on the types of minerals formed . The susceptibility of the thermophilic Fe(III)-reducing cultures to metabolic inhibitors specific for ferric reductase, hydrogenase, and electron transport indicated that iron reduction by these bacteria is an enzymatic process.

Biochemistry, 2002 Dec 3, 41(48), 14421 - 9
The beta G156C substitution in the F1-ATPase from the thermophilic Bacillus PS3 affects catalytic site cooperativity by destabilizing the closed conformation of the catalytic site; Bandyopadhyay S et al.; Fluorescence titrations of the alpha(3)(betaG(156)C/Y(345)W)(3)gamma, alpha(3)(betaE(199)V/Y(345)W)(3)gamma, and alpha(3)(betaY(345)W)(3)gamma subcomplexes of TF(1) with nucleotides show that the betaG(156)C substitution substantially lowers the affinity of catalytic sites for ATP and ADP with or without Mg(2+), whereas the betaE(199)V substitution increases the affinity of catalytic sites for nucleotides . Whereas the alpha(3)(betaG(156)C)(3)gamma and alpha(3)(betaE(199)V)(3)gamma subcomplexes hydrolyze 2 mM ATP at 2% and 0.7%, respectively, of the rate exhibited by the wild-type enzyme, the alpha(3)(betaG(156)C/E(199)V)(3)gamma hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme . The alpha(3)(betaG(156)C)(3)gamma, alpha(3)(betaG(156)C/E(199)V)(3)gamma, and alpha(3)(betaG(156)C/E(199)V/Y(345)W)(3)gamma subcomplexes resist entrapment of inhibitory MgADP in a catalytic site during turnover . Product {(3)H}ADP remains tightly bound to a single catalytic site when the wild-type, betaE(199)V, betaY(345)W, and betaE(199)V/Y(345)W subcomplexes hydrolyze substoichiometric {(3)H}ATP, whereas it is not retained by the betaG(156)C and betaG(156)C/Y(345)W subcomplexes . Less firmly bound, product {(3)H}ADP is retained when the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze substoichiometric {(3)H}ATP . The Lineweaver-Burk plot obtained with the betaG(156)C mutant is curved downward in a manner indicating that its catalytic sites act independently during ATP hydrolysis . In contrast, the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze ATP with linear Lineweaver-Burk plots, indicating cooperative trisite catalysis . It appears that the betaG(156)C substitution destabilizes the closed conformation of a catalytic site hydrolyzing MgATP in a manner that allows release of products in the absence of catalytic site cooperativity . Insertion of the betaE(199)V substitution into the betaG(156)C mutant restores cooperativity by restricting opening of the catalytic site hydrolyzing MgATP for product release until an open catalytic site binds MgATP.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 521 - 7
Community structure and function in prokaryotic marine plankton; Fuhrman JA; Molecular biodiversity studies of microbial communities have provided invaluable information on the existence of heretofore unknown organisms and on community composition . Cloning and 'fingerprinting' techniques have been used many times to study prokaryote community composition of marine plankton . There are still many opportunities for new discoveries in this area, but the results have also opened new questions about the activities of these organisms and their function, going beyond just listing taxa or counting organisms . Rarely can the broad function be inferred from phylogenetic position alone (e.g . cyanobacteria) . The recent discovery of abundant non-cyanobacterial marine phototrophs points to our inability to link phylogenetic position with function in a detailed way . One approach we have found fruitful is to combine fluorescence in situ hybridization with microautoradiography, a technique dubbed STARFISH . A recent application has shown that ubiquitous archaea from the deep sea, phylogenetically related to extreme thermophiles, are active in the uptake of amino acids from ambient (nanomolar) concentrations . This suggests the group is at least partly heterotrophic and able to compete successfully with bacteria for nutrients . Other as-yet uncultivated groups are also amenable to similar studies.

Ann N Y Acad Sci, 2002 Oct, 974, 598 - 609
Space-grown protein crystals are more useful for structure determination; Ng JD; The usefulness of X-ray data derived from space-grown protein crystals for calculating a more accurate structure is reviewed here for three model proteins . These include the plant sweetening protein, thaumatin, from Thaumatococcus daniellii; the aspartyl-tRNA synthetase from Thermus thermophilus; and pea lectin from Pisum sativum . In all three cases, X-ray diffraction data collected from protein crystals obtained under reduced gravity lead to better defined initial electron density maps, facilitating model building and improved crystallographic statistics . With thaumatin, the phasing power of the anomalous scattering atom, sulfur, is used to determine protein crystal quality in terms of its usefulness for ab initio structure determination . Thaumatin crystals grown under microgravity provided improved phasing statistics compared to those of Earth-grown crystals . Consequently, generating a de novo protein model of higher quality was facilitated using X-ray diffraction data from space-grown crystals . This lends evidence to the possibility that a microgravity environment can favor protein crystal growth and, subsequently, be more useful for structure determination.

J Biol Chem, 2003 Jan 31, 278(5), 3048 - 54 Epub 2002 Nov 19.
Characterization of an endonuclease IV 3'-5' exonuclease activity; Kerins SM et al.; Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups . By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV . Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity . The activity was detected in purified preparations of the endonuclease IV protein from E . coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions . Induction of either endonuclease IV in an E . coli overexpression system resulted in induction of the exonuclease activity, and the E . coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity . Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions . Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity . Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.

Comp Biochem Physiol A Mol Integr Physiol, 2002 Nov, 133(3), 519 - 27
Effects of temperature on the metabolic response to feeding in Python molurus; Wang T et al.; As ectothermic vertebrates, reptiles undergo diurnal and seasonal changes in body temperature, which affect many biological functions . In conjunction with a general review regarding the effects of temperature on digestion in reptiles, we describe the effects of various temperatures (20-35 degrees C) on the metabolic response to digestion in the Burmese python (Python molurus) . The snakes were fed mice amounting to 20% of their body weight and gas exchange (oxygen uptake and CO(2) production) were measured until digestion had ended and gas exchange returned to fasting levels . Elevated temperature was associated with a faster and larger metabolic increase after ingestion, and the time required to return to fasting levels was markedly longer at low temperature . The factorial increase between fasting oxygen consumption (VO(2)) and maximal VO(2) during digestion was, however, similar at all temperatures studied . Furthermore, the integrated SDA response was not affected by temperature suggesting the costs associated with digestion are temperature-independent . Other studies on reptiles show that digestive efficiency is only marginally affected by temperature and we conclude that selection of higher body temperatures during digestion (postprandial thermophilic response) primarily reduces the time required for digestion.

Biochem J, 2003 Mar 1, 370(Pt 2), 651 - 9
Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima; Kluskens LD et al.; The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium . The pel A gene of T . maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity . Gel filtration indicated that the native form of PelA is tetrameric . Highest activity (422 units/mg, with a K(m) of 0.06 mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate . In the tradition of pectate lyases, PelA demonstrated full dependency on Ca(2+) for stability and activity . The enzyme is highly thermoactive and thermostable, operating optimally at 90 degrees C and pH 9.0, with a half-life for thermal inactivation of almost 2 h at 95 degrees C, and an apparent melting temperature of 102.5 degrees C . Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known . The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3-8) and the examination of the bond cleavage frequencies.

Z Naturforsch {C}, 2002 Sep-Oct, 57(9-10), 805 - 10
Exopolysaccharides produced by lactic acid bacteria of kefir grains; Frengova GI et al.; A Lactobacillus delbrueckii subsp . bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains . By associating the Lactobacillus delbrueckii subsp . bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp . lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed . The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp . bulgaricus HP1 . The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp . bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h . The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.

Biochemistry, 2002 Nov 26, 41(47), 14054 - 65
Breaking and re-forming the disulfide bond at the high-potential, respiratory-type Rieske {2Fe-2S} center of thermus thermophilus: characterization of the sulfhydryl state by protein-film voltammetry; Zu Y et al.; A disulfide bond, adjacent to the {2Fe-2S} cluster, is conserved in all high-potential Rieske proteins from the respiratory and photosynthetic cytochrome bc(1) and b(6)f complexes but is absent from the low-potential, bacterial dioxygenase Rieske proteins . The role of the disulfide is unclear, since cysteine mutants have resulted in only apoprotein . The high stability of the soluble Thermus thermophilus Rieske protein permits chemical reduction of the disulfide bond and characterization of the sulfhydryl (dithiol) form by protein-film voltammetry . The effect of disulfide reduction on the cluster potential is small (DeltaE(0)' <or= -0.04 V) and attributed to relaxation of the disulfide tether between the protein loops ligating the cluster, including possible mechanical strain release and hydrogen-bonding modification . Above pH 6 an additional decrease in potential of the sulfhydryl form is assigned to the nearby negatively charged thiolates (DeltaE(0)' -0.16 to -0.12 V); the histidine-ligand nitrogen pKs are correspondingly increased . Entropies of reduction for the native and dithiolate forms are equal (-48 +/- 5 J K(-1) mol(-1), pH 7-8); thus changes in reduction potential are enthalpic in origin . Following sulfhydryl alkylation the cluster redox properties mirror those of the native protein (DeltaE(0)' approximately -0.1 V) over all pHs . While a sustained electrode potential of -0.85 V fails to reduce the disulfide, the free sulfhydryls recombine upon an oxidative excursion, at low pH, to restore the native redox properties . This unique behavior is attributed to preorganization of the two thiolate groups upon uptake of one or more protons by the sulfhydryl pair.

New Microbiol, 2002 Oct, 25(4), 469 - 76
Effects of temperatures, pH-values, ultra-violet light, ethanol and chloroform on the growth of isolated thermophilic Bacillus phages; Hazem A; Seven thermophilic Bacillus phages were characterized with reference to their host range, time of appearance, morphology of plaques, thermal inactivation, stability, lipid presence and inactivation by ultraviolet irradiation . Response surface methodology was adapted to describe the response of growth parameters to environmental changes . Most phages are susceptible to temperatures above 60 degrees C and inactivated immediately at 103 degrees C . Most phages are resistant to pH ranges 5 to 9 and almost all to pH 7 to 8 . Both phages 46 and 80 were highly resistance to UV exposure for 13 minutes and 20 minutes, respectively . The presence of chloroform or 75% ethanol showed no effect on almost all isolated phages that indicate of possibility of the absence of lipids . The isolated phages were slow in their growth, possibly due to the lower gross growth efficiency.

Mikrobiol Z, 2002 Jul-Aug, 64(4), 11 - 8
{Stability of genetic markers in methane-oxidizing bacteria}; Romanovskaia VA et al.; A number of Methylococcus thermophilus 111p clones have been obtained which have acquired resistance to tetracycline . The stability of maintenance of marker resistance in these clones and also in already designed Methylomonas rubra 15sh mutants has been investigated . Chromosomal markers resistance to antibiotics or formaldehyde were maintained in the marked strains Methylococcus thermophilus 111p and Methylomonas rubra 15sh after storage in nonselective conditions . The markers of resistance to antibiotics, which were coded by plasmids (pAS8-121 and pULB113), were not always preserved in Methylomonas rubra and Methylococcus thermophilus . The stability of maintenance of chromosomal markers in the investigated methane oxidizing bacteria testifies to the fact that they can be used in laboratory and industrial practice for testing the marked bacteria on selective media . The collection of the marked bacteria-mutants Methylomonas rubra 15sh and Methylococcus thermophilus 111p has been created . These strains stably support the marker resistance to various antibiotics or formaldehyde in unselective conditions.

Appl Microbiol Biotechnol, 2002 Nov, 60(3), 320 - 6 Epub 2002 Oct 12.
Esterases from Bacillus subtilis and B . stearothermophilus share high sequence homology but differ substantially in their properties; Henke E et al.; A novel esterase from Bacillus subtilis (BsubE) was cloned, functionally expressed in Escherichia coli and biochemically characterized . BsubE shows high homology (74% identity, >95% homology) to an esterase from the thermophilic B . stearothermophilus (BsteE) . Both enzymes were efficiently expressed in E . coli, using a L-rhamnose-expression system {11,500 units/l (BsteE), 3,400 units/l (BsubE)} and were purified by Ni-nitrilotriacetic acid chromatography, yielding specific activities of 70 units/mg (BsteE) and 40 units/mg (BsubE), as determined by the hydrolysis of p-nitrophenyl acetate . Despite the high homology, both esterases revealed remarkable differences in their properties . As expected, the esterase from the thermophilic organism showed significantly higher temperature stability . Whereas BsteE showed highest activity at 65-70 degrees C, BsubE was almost inactivated at 50 degrees C . Moreover, both enzymes showed quite different substrate patterns in the hydrolysis of various esters . Whilst the B . subtilis esterase accepted esters with a branched alcohol moiety well, the B . stearothermophilus esterase was more useful in the hydrolysis of substrates with a sterically demanding carboxylic acid group . BsteE showed excellent enantioselectivity ( E>100) in the kinetic resolution of menthyl acetate and even accepted the bulky menthyl benzoate as substrate ( E=19) . In contrast, BsubE converted 1-phenethylacetate with higher selectivity ( E>150 vs E=8).

Appl Microbiol Biotechnol, 2002 Nov, 60(3), 300 - 5 Epub 2002 Oct 12.
Development of a membrane dialysis bioreactor and its application to a large-scale culture of a symbiotic bacterium, Symbiobacterium thermophilum; Ueda K et al.; A simple membrane dialysis bioreactor was developed for a large-scale axenic culture of Symbiobacterium thermophilum, a symbiotic thermophile that requires co-cultivation with an associating thermophilic Bacillus strain S for normal growth . The bioreactor consisted of an outer- and an inner-coaxial cylindrical compartment bordered across a dialyzing membrane, which enabled a 1 l-scale dialysis culture with exchange of low molecular metabolites between the two compartments to be performed . Using the bioreactor, growth characteristics of S . thermophilum and Bacillus strain S were assessed under two medium conditions . The growth of S . thermophilum was measured by quantitative PCR because the bacterium formed no visible colonies and gave abnormally low turbidity . In medium containing 2% tryptone peptone, S . thermophilum proliferated up to 4x10(7) cells/ml, and strict dependence on the co-culture with Bacillus strain S was observed . On the other hand, medium containing 0.5% yeast extract not only facilitated the growth of S . thermophilum in the co-culture (6x10(7) cells/ml), but also allowed limited pure growth independent of Bacillus strain S (1x10(7) cells/ml), implying that some component of yeast extract can partially replace the growth requirement of S . thermophilum supplied by Bacillus strain S . Both the oxidative redox potential values and the cell morphology in the independently growing culture suggested the occurrence of marked unbalanced growth possibly caused by significant metabolic changes . The bioreactor is applicable to the analyses of culturing characteristics in symbiotic systems between free-living microorganisms.

J Biol Chem, 2003 Feb 7, 278(6), 4087 - 95 Epub 2002 Nov 14.
Proprotein processing within secretory dense core granules of Tetrahymena thermophila; Bradshaw NR et al.; In the ciliate Tetrahymena thermophila, the polypeptides stored in secretory dense core granules (DCGs) are generated by proteolytic processing of precursors, and the mature products assemble as a crystal . Previous observations suggested that this maturation involves precise cleavage at distinct motifs by a small number of enzymes . To test these inferences, we analyzed the determinants for site-specific processing of pro-Grl1p (Granule lattice protein 1) by complete gene replacement with modified alleles . Contrary to the predictions of previous models, none of the component amino acids in a putative processing motif was necessary for targeted cleavage . Indeed, cleavage at a range of alternative positions near the native site was consistent with normal DCG assembly . Furthermore, substitution of other classes of processing site motifs did not perturb DCG structure or function . These results suggest that processing can be catalyzed by multiple proteases, for which substrate accessibility may be the prime determinant of site specificity . Consistent with this, inhibition of either subtilisin or cathepsin family proteases resulted in delayed processing of pro-Grl1p.

FEMS Microbiol Lett, 2002 Nov 5, 216(2), 263 - 8
Meiothermus rosaceus sp nov isolated from Tengchong hot spring in Yunnan, China; Chen C et al.; A rosy-pigmented Gram-negative, thermophilic bacterium with an optimum growth temperature of about 55 degrees C was isolated from Tengchong hot springs in Yunnan province, China . Its growth scarcely occurred below 40 degrees C or above 70 degrees C . Phylogenetic and secondary structural analyses of 16S rRNA and DNA-DNA hybridization showed that the organism represented a new species of the genus Meiothermus . This new species could be distinguished easily from other species of the genus Meiothermus by the following phenotypic characteristics: rosy pigment, expanded body, sucrose and maltose were not utilized, gelatin and starch were not hydrolyzed . On the basis of the above data, the name Meiothermus rosaceus sp . nov . was proposed for the species represented by the strain RH9901(T)(CCTCC-AB200291).

FEMS Microbiol Lett, 2002 Nov 5, 216(2), 249 - 53
Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme; Hansen T et al.; The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli . The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits . Rate dependence (at 80 degrees C) on glucose-6-phosphate and NADP(+) followed Michaelis-Menten kinetics with apparent K(m) values of 0.15 mM and 0.03 mM, respectively; apparent V(max) values were about 20 U mg(-1) . The enzyme also reduced NAD(+) (apparent K(m) 12 mM, V(max) 12 U mg(-1)) . The 1000-fold higher catalytic activity (k(cat)/K(m)) with NADP(+) over NAD(+) defines the G6PD as NADP(+) specific in vivo . G6PD activity was competitively inhibited by NADPH with a K(i) value of 0.11 mM . With a temperature optimum of 92 degrees C the enzyme is the most thermoactive G6PD described.

Nat Struct Biol, 2002 Dec, 9(12), 928 - 33
Monovalent cations mediate formation of native tertiary structure of the Tetrahymena thermophila ribozyme; Takamoto K et al.; The formation of individual tertiary contacts of the Tetrahymena L-21 Sca I ribozyme has been monitored by hydroxyl radical footprinting and its global conformation by analytical ultracentrifugation as a function of monovalent ion concentration in the absence of divalent ions . Advanced methods of data analysis, which allow the hydroxyl radical reactivity of every nucleotide to be quantified, permit monitoring of each and every structural element of the RNA . Monovalent ion-mediated global compaction of the ribozyme is accompanied by the formation of native tertiary contacts; most native tertiary contacts are evident except several that are located near where divalent ions are observed in crystallographic structures . Non-native tertiary contacts are also observed at low but not high concentrations of monovalent ions . In light of recent studies that have shown that the presence of monovalent ions greatly accelerates the Mg2+-dependent folding of the Tetrahymena ribozyme, the present studies suggest that Na+ concentration changes not only the starting position of the RNA on its folding funnel but also pushes it deep into the well by forming native tertiary contacts and, thus, favoring fast and correct folding pathways.

J Mol Microbiol Biotechnol, 2002 Nov, 4(6), 533 - 8
Export of Thermus thermophilus cytoplasmic beta-glycosidase via the E . coli Tat pathway; Gerard F et al.; The Tat pathway is distinct from the Sec machinery given its unusual capacity to export folded proteins, which contain a twin-arginine (RR) signal peptide, across the plasma membrane . The functionality of the Tat pathway has been demonstrated for several Gram-negative and Gram-positive mesophilic bacteria . To assess the specificity of the Tat system, and to analyze the capacity of a mesophilic bacterial Tat system to translocate cytoplasmic proteins from hyperthermophilic bacteria, we fused the Thermus thermophilus beta-glycosidase (Glc) to the twin-arginine signal peptide of the E . coli TorA protein . When expressed in E . coli, the thermophilic RR-Glc chimera was successfully synthesized and efficiently translocated into the periplasm of the wild type strain . In contrast, the beta-glycosidase accumulated within the cytoplasm of all the tat mutants analyzed . The beta-glycosidase synthesized in these strains exhibited thermophilic properties . These results demonstrated, for the first time, the capacity of the E . coli Tat system to export cytoplasmic hyperthermophilic protein, implying an important potential of the Tat system for the production of thermostable enzymes used in bioprocessing applications.

Chemosphere, 2002 Nov, 49(6), 597 - 604
Investigations on the binding mechanism of the herbicide simazine to dissolved organic matter in leachates of compost; Ertunc T et al.; 14C-labelled simazine was composted together with biowaste on a pilot (m3) scale . The herbicide was quickly bound to the compost matrix . By aqueous extraction of 29 and 200 days old compost (equivalent to thermophilic and mesophilic phase of composting) only 4.2% and 3.1% respectively of the radioactivity in the compost samples could be extracted with water . Analysis of the extracts using high-performance size exclusion chromatography (HPSEC) revealed that the dissolved organic matter (DOM) had molecular weights ranging between 2 and 28 kDa . The amount of DOM-associated radioactivity increased from 53% (day 29) to 65% (day 200) of total extractable radioactivity . The type of binding of the 14C-labelled residues and the DOM was elucidated by silylation of humic matter and subsequent HPSEC . The data demonstrated that besides polar metabolites also intact simazine was bound to the DOM . A distinct shift from rather weak interactions to strong covalent linkages of simazine and its metabolites with increasing age of the compost was observed . The results showed that only low amounts of free simazine and its degradates can be extracted with water . We concluded that the shift towards stable covalent linkages is equivalent to a detoxification of the contaminant in aged compost . Consequently, the use of the analysed compost in its mature stage should not pose an environmental risk to the groundwater or the subsoil.

Virology, 2002 Oct 10, 302(1), 21 - 32
Transcription analysis of Streptococcus thermophilus phages in the lysogenic state; Ventura M et al.; The transcription of prophage genes was studied in two lysogenic Streptococcus thermophilus cells by Northern blot and primer-extension experiments . In the lysogen containing the cos-site phage Sfi21 only two gene regions of the prophage were transcribed . Within the lysogeny module an 1.6-kb-long mRNA started at the promoter of the phage repressor gene and covered also the next two genes, including a superinfection exclusion (sie) gene . A second, quantitatively more prominent 1-kb-long transcript was initiated at the promoter of the sie gene . Another prophage transcript of 1.6-kb length covered a group of genes without database matches that were located between the lysin gene and the right attachment site . The rest of the prophage genome was transcriptionally silent . A very similar transcription pattern was observed for a S . thermophilus lysogen containing the pac-site phage O1205 as a prophage . Prophages from pathogenic streptococci encode virulence genes downstream of the lysin gene . We speculate that temperate phages from lactic streptococci also encode nonessential phage genes ("lysogenic conversion genes") in this region that increase the ecological fitness of the lysogen to further their own evolutionary success . A comparative genome analysis revealed that many temperate phages from low GC content Gram-positive bacteria encode a variable number of genes in that region and none was linked to known phage-related function . Prophages from pathogenic streptococci encode toxin genes in this region . In accordance with theoretical predictions on prophage-host genome interactions a prophage remnant was detected in S . thermophilus that had lost most of the prophage DNA while transcribed prophage genes were spared from the deletion process.

Microbiology, 2002 Nov, 148(Pt 11), 3413 - 21
Cell-wall proteinases PrtS and PrtB have a different role in Streptococcus thermophilus/Lactobacillus bulgaricus mixed cultures in milk; Courtin P et al.; The manufacture of yoghurt relies on the simultaneous utilization of two starters: Streptococcus thermophilus and Lactobacillus delbrueckii subsp . bulgaricus (Lb . bulgaricus) . A protocooperation usually takes place between the two species, which often results in enhanced milk acidification and aroma formation compared to pure cultures . Cell-wall proteinases of Lactococcus lactis and lactobacilli have been shown to be essential to growth in milk in pure cultures . In this study, the role of proteinases PrtS from S . thermophilus and PrtB from Lb . bulgaricus in bacterial growth in milk was evaluated; a negative mutant for the prtS gene of S . thermophilus CNRZ 385 was constructed for this purpose . Pure cultures of S . thermophilus CNRZ 385 and its PrtS-negative mutant were made in milk as well as mixed cultures of S . thermophilus and Lb . bulgaricus: S . thermophilus CNRZ 385 or its PrtS-negative mutant was associated with several strains of Lb . bulgaricus, including a PrtB-negative strain . The pH and growth of bacterial populations of the resulting mixed cultures were followed, and the Lactobacillus strain was found to influence both the extent of the benefit of Lb . bulgaricus/S . thermophilus association on milk acidification and the magnitude of S . thermophilus population dominance at the end of fermentation . In all mixed cultures, the sequential growth of S . thermophilus then of Lb . bulgarius and finally of both bacteria was observed . Although proteinase PrtS was essential to S . thermophilus growth in milk in pure culture, it had no effect on bacterial growth and thus on the final pH of mixed cultures in the presence of PrtB . In contrast, proteinase PrtB was necessary for the growth of S . thermophilus, and its absence resulted in a higher final pH . From these results, a model of growth of both bacteria in mixed cultures in milk is proposed.

J Biol Chem, 2003 Jan 17, 278(3), 1864 - 71 Epub 2002 Nov 08.
Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium, Thermus thermophilus HB27, and evolutionary implication of beta-decarboxylating dehydrogenase; Miyazaki J et al.; Although the presence of an enzyme that catalyzes beta-decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through alpha-aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently . A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase was cloned from Thermus thermophilus HB27 . The gene product produced in recombinant Escherichia coli cells demonstrated homoisocitrate dehydrogenase (HICDH) activity . A knockout mutant of the gene showed an AAA-auxotrophic phenotype, indicating that the gene product is involved in lysine biosynthesis through AAA . We therefore named this gene hicdh . HICDH, the gene product, did not catalyze the conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine biosynthetic reaction, but it did recognize isocitrate, a related compound in the tricarboxylic acid cycle, as well as homoisocitrate as a substrate . It is of interest that HICDH catalyzes the reaction with isocitrate about 20 times more efficiently than the reaction with the putative native substrate, homoisocitrate . The broad specificity and possible dual function suggest that this enzyme represents a key link in the evolution of the pathways utilizing citrate derivatives . Site-directed mutagenesis study reveals that replacement of Arg(85) with Val in HICDH causes complete loss of activity with isocitrate but significant activity with 3-isopropylmalate and retains activity with homoisocitrate . These results indicate that Arg(85) is a key residue for both substrate specificity and evolution of beta-decarboxylating dehydrogenases.

J Bacteriol, 2002 Dec, 184(23), 6465 - 71
Aquifex aeolicus PilT, homologue of a surface motility protein, is a thermostable oligomeric NTPase; Herdendorf TJ et al.; Bacterial surface motility works by retraction of surface-attached type IV pili . This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems . In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified . A . aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein . This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered . The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro . Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a approximately equal 5- to 6-subunit oligomer . Potential implications of the NTPase activity of PilT in pilus retraction are discussed.

Int J Food Microbiol, 2003 Feb 15, 80(3), 217 - 22
Synthesis and utilisation of folate by yoghurt starter cultures and probiotic bacteria; Crittenden RG et al.; Thirty-two bacterial isolates from species commonly used in yoghurts and fermented milks were examined for their ability to synthesise or utilise folate during fermentation of skim milk . The organisms examined included the traditional yoghurt starter cultures, Lactobacillus delbrueckii subsp . bulgaricus and Streptococcus thermophilus, and probiotic lactobacilli, bifidobacteria, and Enterococcus faecium . Folate was synthesised by S . thermophilus, bifidobacteria, and E . faecium . S . thermophilus was the dominant producer, elevating folate levels in skim milk from 11.5 ng g(-1) to between 40 and 50 ng g(-1) . Generally, lactobacilli depleted the available folate in the skim milk . Fermentations with mixed cultures showed that folate production and utilisation by the cultures was additive . Fermentations using a combination of Bifidobacterium animalis and S . thermophilus resulted in a six-fold increase in folate concentration . Although increased folate levels in yoghurts and fermented milks are possible through judicious selection of inoculum species, the folate levels remain relatively low in terms of recommended daily allowance .

Biochimie, 2002 May-Jun, 84(5-6), 447 - 54
On the interaction of colicin E3 with the ribosome; Zarivach R et al.; Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery . Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E . coli numbering system) . The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death . The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay . A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction . Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone

FEMS Microbiol Lett, 2002 Oct 29, 216(1), 43 - 7
Cloning vectors for Streptococcus thermophilus derived from a native plasmid; Su P et al.; A 3.5-kb native plasmid (pND103) was identified in Streptococcus thermophilus ST2-1 . Preliminary sequence analysis indicated that pND103 belongs to group I S . thermophilus plasmids . A region of approximately 2 kb appears to contain three components: a plus origin of replication (ori) typical of plasmids that replicate via rolling circle replication; a gene encoding a replication protein (rep); and a gene encoding a small heat shock protein (hsp) . pND103 was then used to construct S . thermophilus/Escherichia coli hybrid cloning vectors by ligating different portions of pND103 to an origin-probe vector (pND330) composed of pUC19 and a Gram-positive erythromycin resistance gene . The shuttle vectors (pND913, pND914 and pND915) were successfully introduced back into plasmid-free S . thermophilus ST3-1 as well as to Lactococcus lactis LM0230 and E . coli JM109 . Segregational and structural stability study indicated that these vectors can be maintained in these hosts . The results indicated that pND913, pND914 and pND915 are potential shuttle cloning vectors for S . thermophilus.

Eur J Biochem, 2002 Nov, 269(22), 5590 - 8
Characterization of the exopolysaccharide produced by Streptococcus thermophilus 8S containing an open chain nononic acid; Faber EJ et al.; The exopolysaccharide produced by Streptococcus thermophilus 8S in reconstituted skimmed milk is a heteropolysaccharide containing d-galactose, d-glucose, d-ribose, and N-acetyl-d-galactosamine in a molar ratio of 2 : 1 : 1 : 1 . Furthermore, the polysaccharide contains one equivalent of a novel open chain nononic acid constituent, 3,9-dideoxy-d-threo-d-altro-nononic acid, ether-linked via C-2 to C-6 of an additional d-glucose per repeating unit . Methylation analysis and 1D/2D NMR studies (1H and 13C) performed on the native polysaccharide, and mass spectrometric and NMR analyses of the oligosaccharide obtained from the polysaccharide by de-N-acetylation followed by deamination and reduction demonstrated the 'hepta'saccharide repeating unit to be: -->4)-alpha-D-Galp-(1-->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1--7')-Sub-(1-->4)-beta-D-GalpNAc-(1--> in which Sug is 6-O-(3',9'-dideoxy-d-threo-d-altro-nononic acid-2'-yl)-alpha-d-glucopyranose.

Eur J Biochem, 2002 Nov, 269(22), 5536 - 46
Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species; Arnorsdottir J et al.; The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp . PA44 has been successfully cloned, sequenced and expressed in Escherichia coli . The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa . The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence . Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity . The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases . With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same . Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues . The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index . These factors may contribute to the adaptation of these proteinases to different temperature conditions.

Syst Appl Microbiol, 2002 Oct, 25(3), 450 - 5
Three novel halotolerant and thermophilic Geobacillus strains from shallow marine vents; Maugeri TL et al.; During a polyphasic taxonomic analysis performed on isolates from shallow marine hydrothermal vents of Eolian Islands (Italy), three thermophilic, halotolerant bacilli, designated as strain 1bw, strain 5-2 and strain 10-1, could not be affiliated to any described species . Physiological and biochemical characteristics, membrane lipids composition, mol % G+C content, and phylogenetic relationships determined on the basis of the 16S rRNA gene sequence analysis, placed these strains within the genus Geobacillus . The three strains were only moderately related to species of Geobacillus and their relatives, members of Saccharococcus . Determination of the relatedness among each other at a higher taxonomic level by DNA-DNA reassociation experiments demonstrated the three isolates to represent three different novel Geobacillus genomospecies . The taxonomic novelty of these three marine strains was substantiated by their physiological properties and by fatty acid patterns that did not match closely those of any Geobacillus type strain . These three novel strains could be of interest to biotechnology because of their ability to produce exopolysaccharides and to adhere on polystirene, characteristics undescribed so far for other Geobacillus species . They are also able to utilise hydrocarbons such as gas oil, kerosene and mineral lubricating oil . Strain 5-2 is tolerant to zinc.

Syst Appl Microbiol, 2002 Oct, 25(3), 319 - 25
Production and characterization of exopolysaccharides excreted by thermophilic bacteria from shallow, marine hydrothermal vents of Flegrean Ares (Italy); Nicolaus B et al.; Thermophilic microorganisms (4001-4014), described as aerobic or facultatively anaerobic, endospore forming with growth optima temperatures in the range of 60 to 80 degrees C, have been isolated from hot marine springs around Ischia and from hydrothermal vents in the gulf of Naples . Mucous colonies are been selected for the recovery of new strains producing exopolysaccharides (EPS) . To induce the biosynthesis of new exopolysaccharides, different sugars were tested as carbon sources in the media . The production of EPS in the strain 4009 reached 60 mg/l using trehalose as carbon source, increasing the yield of about 1000 fold . The 4001-EPS was a mannan with a molecular weight of 380.000 D and with a complex primary structure . In fact, the analysis of the permethylated polysaccharide in GC-MS, showed the presence of mannose, glucose, galactose, mannosamine in the relative ratio of 1:0.1:tr :tr, respectively . Nuclear magnetic resonance spectrum of the exopolysaccharide confirmed the presence of a repetitive unity formed by seven monosaccharides, six with alpha gluco/galacto configuration and one residue with beta conformation.

Water Res, 2002 Oct, 36(17), 4369 - 85
Comparative process stability and efficiency of anaerobic digestion; mesophilic vs . thermophilic; Kim M et al.; The comparative process stability and efficiency of mesophilic (35 degrees C) and thermophilic anaerobic digestion (55 degrees C) has been evaluated for four different reactor configurations, which are: daily batch-fed single-stage continuously stirred tank reactor (CSTR), continuously fed single-stage CSTR, daily batch-fed two-phase CSTR, and daily batch-fed non-mixed single-stage reactor . The results are discussed for three periods: (1) start-up, (2) steady state, and (3) organic loading rate (OLR) increase until reactor failure (pH below 5.5) . During the start-up, the single-stage CSTRs at both temperatures showed the least stability, while the non-mixed single-stage reactors reached steady state in the shortest time with relatively stable pH and low volatile fatty acid (VFA) . In the case of the thermophilic non-mixed reactor, efficient removal of propionate occurred but supplementation of nutrients (Ca, Fe, Ni, and Co) was required when VFA increased . The results imply the importance of inorganic nutrients bioavailability . The comparative results of the reactor performance at steady state clearly showed the superior performance of the thermophilic non-mixed reactor with respect to lower VFA, higher gas production and volatile solids removal implying that microbial consortia proximity can alleviate the problem of poor effluent quality in thermophilic system . During the OLR increase until reactor failure, all thermophilic reactors except the thermophilic non-mixed reactor showed increases in propionate concentrations as the OLR increased, while all mesophilic reactors except the mesophilic two-phase system showed little increase in VFA concentrations . When all reactors had the same conditions with OLR increase, the continuously fed reactors showed the lowest gas production, while the non-mixed reactors showed the highest gas production at both temperatures . It is hypothesized that the non-mixing reactor configuration has closer microbial consortia proximity than others . Therefore, the results in this study indicated the importance of microbial consortia proximity . A proposed model for the effect of the distance between two syntrophic bacteria reasonably matched the data in this study.

Arch Microbiol, 2002 Dec, 178(6), 538 - 47 Epub 2002 Oct 16.
Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe; Nam GW et al.; A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia . Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 degrees C and pH 7 with a maximum specific growth rate (0.14 h(-1)) in Thermotoga- Fervidobacterium(TF) medium . After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine . Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites . A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F . islandicum AW-1 . The enzyme exhibited activity toward casein and soluble keratin optimally at 100 degrees C and pH 9, and had a half-life of 90 min at 100 degrees C . The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol . Therefore, the enzyme from F . islandicum AW-1 is a novel, thermostable keratinolytic serine protease.

Biochem Biophys Res Commun, 2002 Nov 15, 298(5), 632 - 7
Thermostable aldehyde dehydrogenase from psychrophile, Cytophaga sp . KUC-1: enzymological characteristics and functional properties; Yamanaka Y et al.; We found the occurrence of NAD(P)(+)-dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp . KUC-1, and purified to homogeneity . About 50% of the enzyme activity remained even after heating at 50 degrees C for 65min and the highest activity was observed in the range of 55-60 degrees C . The enzyme was thermostable and thermophilic, although it was derived from a psychrophile . The circular dichroism at 222nm of the enzyme showed a peak at 32 degrees C . This temperature was closely similar to the transition temperature in the Arrhenius plots . The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R . The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues . The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different.

J Mol Biol, 2002 Nov 8, 323(5), 859 - 69
Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1; Tyndall JD et al.; We describe the first lipase structure from a thermophilic organism . It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold . The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability . Zinc binding is mediated by two histidine and two aspartic acid residues . These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana . The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.

J Biochem (Tokyo), 2002 Nov, 132(5), 759 - 65
Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8: open-closed conformational change and ammonia tunneling; Omi R et al.; Imidazole glycerol phosphate synthase (IGPs) catalyzes the fifth step in the histidine biosynthetic pathway located at the branch point to de novo purine biosynthesis . IGPs is a multienzyme comprising glutaminase and synthase subunits . The glutaminase activity, which hydrolyzes glutamine to give ammonia, is coupled with substrate binding to the synthase subunit . The three-dimensional structure of the IGPs from Thermus thermophilus HB8 has been determined at 2.3 A resolution, and compared with the previously determined structures for the yeast and Thermotoga maritima enzymes . The structure of each subunit is similar to that of the corresponding domain in the yeast enzyme or subunit in the T . maritima enzyme . However, the overall structure is significantly different from the yeast and T . maritima enzymes, indicating that IGPs may change the relative orientation between the two subunits and close the glutaminase site upon glutamine binding . The putative ammonia tunnel, which carries nascent ammonia from glutaminase to the synthase site, has a closed gate comprising a cyclic salt bridge formed by four charged residues of the synthase subunit . The side chain of Lys100 in the cyclic salt bridge might change its side chain direction to form new interactions with the main chain carbonyl group of glutamine from the synthase subunit and the hydoxyl group of tyrosine from the glutaminase subunit, resulting in the opening of the gate for ammonia transfer.

J Dairy Sci, 2002 Oct, 85(10), 2489 - 96
Production of pyroglutamic acid by thermophilic lactic acid bacteria in hard-cooked mini-cheeses; Mucchetti G et al.; Pyroglutamic acid is present in high amounts (0.5g/ 100g) in many cheese varieties-and particularly in extensively ripened Italian cheeses such as Grana Padano and Parmigiano Reggiano . An in vivo model system for cooked mini-cheese production and ripening acceleration was set up to demonstrate the ability of thermophilic lactic acid bacteria, used as a starter, to produce pyroglutamic acid (pGlu) . In mini-cheeses stored at 38 and 30 degrees C for up to 45 d, all starters tested produced different amounts of pGlu . In descending order of pGlu production, the bacteria analyzed were: Lactobacillus helveticus, Lactobacillus delbrueckii subsp . bulgaricus, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp . lactis . Evidence for the presence of glutamine to pGlu cyclase activity in lactic acid bacteria was provided . Cell lysates obtained from cultures of L . helveticus, L . delbrueckii subsp . bulgaricus, L . delbrueckii subsp . lactis, and S . thermophilus showed the ability to cyclize glutamine to pGlu, resulting in processing yields from 1.4 to 30.3%, depending on the subspecies . Formation of pGlu from free glutamine appeared to be similar to that observed using a glutamine-glutamine dipeptide substrate . Under the experimental conditions applied, pGlu aminopeptidase activity was only detected in L . helveticus . Thus, pGlu formation in long-ripened cooked cheese may depend on the activity of thermophilic lactic acid bacteria.

J Dairy Sci, 2002 Oct, 85(10), 2479 - 88
Effect of milk base and starter culture on acidification, texture, and probiotic cell counts in fermented milk processing; Sodini I et al.; In the present work, the compared effect of milk base and starter culture on acidification, texture, growth, and stability of probiotic bacteria in fermented milk processing, was studied . Two strains of probiotic bacteria were used, Lactobacillus acidophilus LA5 and L . rhamnosus LR35, with two starter cultures . One starter culture consisted only of Streptococcus thermophilus ST7 (single starter culture); the other was a yogurt mixed culture with S . thermophilus ST7 and L . bulgaricus LB12 (mixed starter culture) . For the milk base preparation, four commercial dairy ingredients were tested (two milk protein concentrates and two casein hydrolysates) . The resulting fermented milks were compared to those obtained with control milk (without enrichment) and milk added with skim milk powder . The performance of the two probiotic strains were opposite . L . acidophilus LA5 grew well on milk but showed a poor stability during storage . L . rhamnosus LR35 grew weakly on milk but was remarkably stable during storage . With the strains tested in this study, the use of the single starter culture and the addition of casein hydrolysate gave the best probiotic cell counts . The fermentation time was of about 11 h, and the probiotic level after five weeks of storage was greater than 106 cfu/ml for L . acidophilus LA5 and 10(7) cfu/ml for L . rhamnosus LR35 . However, an optimization of the level of casein hydrolysate added to milk base has to be done, in order to improve texture and flavor when using this dairy ingredient.

Eur J Cell Biol, 2002 Sep, 81(9), 517 - 24
External GTP binding and induction of cell division in starved Tetrahymena thermophila; Iwamoto M et al.; The extracellular nucleotide, guanosine 5'-triphosphate (GTP) is known to be a chemorepellent for ciliated protozoa such as Paramecium and Tetrahymena . Here, we studied the surface localization of GTP binding sites and also its effects on the cell division of Tetrahymena thermophila . When a ribose-modified and fluorescent analog of GTP, 2'-(or -3')-O-trinitrophenyl (TNP)-GTP was added to the cells starved in non-nutrient buffer, a remarkable fluorescence was observed at the compound cilia of the oral area, while it was weak at other cilia and the somatic membrane . Following transfer of the cells to the starvation medium, the intensity of TNP-GTP fluorescence at the oral area gradually increased and was saturated at 3-4 hours . Addition of GTP to the starved cell induced not only an avoiding reaction in swimming, but also induced a synchronous cell division that occurred 2 hours later . An attempt to search for other stimuli, which induced cell division, revealed that mechanical stimulation by a short period of centrifugation was almost as effective as the addition of GTP . The supernatant after centrifugation had the ability to induce cell division, and such activity was abolished after the supernatant was treated with the phosphatase, apyrase, suggesting the release of GTP by the mechanical stimulation . These results indicate that the released GTP binds mainly to the oral area and this then induces the cell division of starved T . thermophila.

Biochem Biophys Res Commun, 2002 Nov 1, 298(3), 383 - 91
Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase; Gruber G et al.; A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry . Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content . Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F(1) ATPase of the thermophilic bacterium PS3 (TF(1)) . The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1) . By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) ATPase activity . This is the first demonstration of a hydrolytically active hybrid-complex consisting of F(1) and V(1) subunits . The arrangement of subunit E in V(1) has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V(1) and an A(3)B(3)HEG-subcomplex of the V(1) ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V(1).

Mol Biol Evol, 2002 Nov, 19(11), 1881 - 90
Serpins in prokaryotes; Irving JA et al.; Members of the serpin (serine proteinase inhibitor) superfamily have been identified in higher multicellular eukaryotes (plants and animals) and viruses but not in bacteria, archaea, or fungi . Thus, the ancestral serpin and the origin of the serpin inhibitory mechanism remain obscure . In this study we characterize 12 serpin-like sequences in the genomes of prokaryotic organisms, extending this protein family to all major branches of life . Notably, these organisms live in dramatically different environments and some are evolutionarily distantly related . A sequence-based analysis suggests that all 12 serpins are inhibitory . Despite considerable sequence divergence between the proteins, in four of the 12 sequences the region of the serpin that determines proteinase specificity is highly conserved, indicating that these inhibitors are likely to share a common target . Inhibitory serpins are typically prone to polymerization upon heating; thus, the existence of serpins in the moderate thermophilic bacterium Thermobifida fusca, the thermophilic bacterium Thermoanaerobacter tengcongensis, and the hyperthermophilic archaeon Pyrobaculum aerophilum is of particular interest . Using molecular modeling, we predict the means by which heat stability in the latter protein may be achieved without compromising inhibitory activity.

J Biol Chem, 2003 Jan 31, 278(5), 3427 - 36 Epub 2002 Oct 30.
Structure and binding mode of a ribosome recycling factor (RRF) from mesophilic bacterium; Nakano H et al.; X-ray and NMR analyses on ribosome recycling factors (RRFs) from thermophilic bacteria showed that they display a tRNA-like L-shaped conformation consisting of two domains . Since then, it has been accepted that domain I, consisting of a three-helix bundle, corresponds to the anticodon arm of tRNA and domain II and a beta/alpha/beta sandwich structure, corresponds to the acceptor arm . In this study, we obtained a RRF from a mesophilic bacterium, Vibrio parahaemolyticus, by gene cloning and carried out an x-ray analysis on it at 2.2 A resolution . This RRF was shown to be active in an in vitro assay system using Escherichia coli polysomes and elongation factor G (EF-G) . In contrast, the above-mentioned RRFs from thermophilic bacteria were inactive in such a system . Analysis of the relative orientations between the two domains in the structures of various RRFs, including this RRF from mesophilic bacterium, revealed that domain II rotates about the long axis of the helix bundle of domain I . To elucidate the ribosome binding site of RRF, the peptide fragment (RRF-DI) corresponding to domain I of RRF was expressed and characterized . RRF-DI is bound to 70 S ribosome and the 50 S subunit with an affinity similar to that of wild-type RRF . But it does not bind to the 30 S subunit . These findings caused us to reinvestigate the concept of the mimicry of RRF to tRNA and to propose a new model where domain I corresponds to the acceptor arm of tRNA and domain II corresponds to the anticodon arm . This is just the reverse of a model that is now widely accepted . However, the new model is in better agreement with published biological findings.

Plant Cell Physiol, 2002 Oct, 43(10), 1182 - 8
Chill-induced inhibition of photosynthesis: genotypic variation within Cucumis sativus; Yu JQ et al.; Cucumber is generally a thermophilic species; however, cultivars have been selected for higher yield during winter cultivation in unheated glasshouses in temperate regions . We tested whether photosynthesis in these varieties had greater chilling tolerance . There was no difference in the instantaneous reduction of photosynthesis at low temperature between four winter glasshouse and four summer field cultivars . After 5 d of 10 degrees C and 100 micro mol m(-2) s(-1) photon flux density, the four field cultivars had a sustained depression of photosynthesis after returning to clement conditions . This inhibition was associated with reduced rates of CO(2) fixation and photosystem II (PSII) electron transport in the light, but not with sustained PSII photoinhibition . However, photosynthesis in the glasshouse genotypes was nearly identical to the pre-chill rates . Chill impacts on light-adapted chlorophyll fluorescence parameters, such as the quantum yield of PSII electron transport (phi(PSII)), correlated well with overall photosynthesis . This demonstrates the potential for using these fast and non-invasive techniques to screen for chill-tolerant genotypes, with the potential to further improve winter cucumber yield in unheated glasshouses.

Appl Environ Microbiol, 2002 Nov, 68(11), 5656 - 62
Metabolic engineering of acetaldehyde production by Streptococcus thermophilus; Chaves AC et al.; The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper . Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene . In S . thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde . In this work, several wild-type S . thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine . Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production . Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT . To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption . Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation . Subsequently, an S . thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P(LacA)) . When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed . These results show that, in S . thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions . These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S . thermophilus as starter culture.

Appl Environ Microbiol, 2002 Nov, 68(11), 5508 - 16
Molecular characterization of cadmium resistance in Streptococcus thermophilus strain 4134: an example of lateral gene transfer; Schirawski J et al.; Two genes (cadC(St) and cadA(St) {subscript St represents Streptococcus thermophilus}), located on the chromosome of S . thermophilus 4134, were shown to constitute a cadmium/zinc resistance cassette . The genes seem to be organized in an operon, and their transcription is cadmium dependent in vivo . The proposed product of the cadA open reading frame (CadA(St)) is highly similar to P-type cadmium efflux ATPases, whereas the predicted protein encoded by cadC(St) (CadC(St)) shows high similarity to ArsR-type regulatory proteins . The observed homologies and G+C content of this cassette and surrounding regions suggest that this DNA was derived from Lactococcus lactis and may have been introduced relatively recently into the S . thermophilus 4134 genome by a lateral gene transfer event . The complete cassette confers cadmium and zinc resistance to both S . thermophilus and L . lactis, but expression of cadA(St) alone is sufficient to give resistance . By using electrophoretic mobility shift assays it was shown that the CadC(St) protein is a DNA binding protein that binds specifically to its own promoter region, possibly to two copies of an inverted repeat, and that this CadC(St)-DNA interaction is lost in the presence of cadmium . Using lacZ fusion constructs it was shown that the cadmium-dependent expression of CadA(St) is mediated by the negative regulator CadC(St) . A model for the regulation of the expression of cadmium resistance in S . thermophilus is discussed.

Appl Environ Microbiol, 2002 Nov, 68(11), 5464 - 71
Characterization of the melA locus for alpha-galactosidase in Lactobacillus plantarum; Silvestroni A et al.; Alpha-galactosides are abundant sugars in legumes such as soy . Because of the lack of alpha-galactosidase (alpha-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence . To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous alpha-Gal is promising . In this framework, we characterized the melA gene for alpha-Gal in Lactobacillus plantarum . The melA gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers . The melA gene was cloned and expressed in Escherichia coli, yielding an active alpha-Gal . We show that melA is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose . Posttranscriptional regulation by the carbon source could also occur . Upstream of melA, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both alpha- and beta-galactosides in Streptococcus thermophilus . rafP is also expressed as a monocistronic mRNA . Downstream of melA, the lacL and lacM genes were identified that encode a heterodimeric beta-galactosidase . A putative galM gene identified in the same cluster suggests the presence of a galactose operon . These results indicate that the genes involved in galactoside catabolism are clustered in L . plantarum ATCC 8014 . This first genetic characterization of melA and of its putative associated transporter, rafP, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues.

J Agric Food Chem, 2002 Nov 6, 50(23), 6752 - 7
Volatile compounds in Hispánico cheese manufactured using a mesophilic starter, a thermophilic starter, and bacteriocin-producing Lactococcus lactis subsp . lactis INIA 415; Garde S et al.; The effect of the addition of Lactococcus lactis subsp . lactis INIA 415, a strain harboring the structural genes of nisin Z and lacticin 481, on the formation of volatile compounds in Hispanico cheese manufactured with a mesophilic starter or with the mesophilic starter and a thermophilic starter was investigated . Addition of bacteriocin-producing L . lactis subsp . lactis INIA 415 to milk enhanced the formation of 2-methyl-propanal, 2-methylbutanal, 3-methylbutanal, 2-methyl-1-propanol, 3-methyl-1-butanol, 1-octanol, 2-butanone, and 2,3-butanedione . On the other hand, addition of thermophilic starter enhanced the formation of acetaldehyde, ethanol, 3-methyl-2-buten-1-ol, ethyl butanoate, ethyl hexanoate, 2-butanone, and 2,3-butanedione in Hispanico cheese . Stepwise discriminant analysis using the relative abundances of volatile compounds classified cheeses by type of starter, with function 1 related to thermophilic starter and function 2 to bacteriocin producer.

Curr Microbiol, 2002 Dec, 45(6), 390 - 3
Cloning of a gene encoding acetate kinase from Methanosarcina mazei 2-P isolated from a Japanese paddy field soil; Tonouchi A et al.; The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli . The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene . The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase . The activity of the His(6)-tagged acetate kinase purified from E . coli JM109 was optimal at 35 degrees C.

Curr Microbiol, 2002 Dec, 45(6), 385 - 9
Modulation of the cell-surface proteinase activity of thermophilic lactobacilli by the Peptide supply; Hebert EM et al.; The proteolytic system of thermophilic lactobacilli is considered important for bacterial nutrition as well as for the formation of flavor and texture in fermented products . We investigated the influence of peptide content on the cell surface proteinase and intracellular aminopeptidase activities from seven thermophilic lactobacilli strains . The proteinase activities were remarkably reduced in cells grown in the peptide-rich medium MRS or in a chemically defined medium supplemented with Casitone compared with those found in a synthetic medium . The degree of inhibition observed was strain dependent . When proteinase activities were analyzed by their hydrolytic patterns of alpha- and beta-casein degradation, four types of P(III)-caseinolytic cleavage specificity were distinguished . Lactobacillus helveticus strains possessed aminopeptidase activities with broader specificity than those found in L . delbrueckii subsp . lactis strains . However, the aminopeptidase activities were not influenced by the peptide content of the medium.

J Biol Chem, 2003 Jan 3, 278(1), 608 - 16 Epub 2002 Oct 24.
Preliminary characterization and crystal structure of a thermostable cytochrome P450 from Thermus thermophilus; Yano JK et al.; The second structure of a thermophile cytochrome P450, CYP175A1 from the thermophilic bacterium Thermus thermophilus HB27, has been solved to 1.8-A resolution . The overall P450 structure remains conserved despite the low sequence identity between the various P450s . The CYP175A1 structure lacks the large aromatic network found in the only other thermostable P450, CYP119, thought to contribute to thermal stability . The primary difference between CYP175A1 and its mesophile counterparts is the investment of charged residues into salt-link networks at the expense of single charge-charge interactions . Additional factors involved in the thermal stability increase are a decrease in the overall size, especially shortening of loops and connecting regions, and a decrease in the number of labile residues such as Asn, Gln, and Cys.

J Biol Chem, 2002 Dec 20, 277(51), 49651 - 4 Epub 2002 Oct 24.
A lysine substitute for K+ . A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans; Belogurov GA et al.; The H(+) proton-translocating inorganic pyrophosphatase (H(+)-PPase) family is composed of two phylogenetically distinct types of enzymes: K(+)-dependent and K(+)-independent . However, to date, the sequence criteria governing this dichotomy have remained unknown . In this study, we describe the heterologous expression and functional characterization of H(+)-PPase from the thermophilic bacterium Carboxydothermus hydrogenoformans . Both PP(i)-hydrolyzing and PP(i)-energized H(+) translocation activities of the recombinant enzyme in Escherichia coli inner membrane vesicles are strictly K(+)-dependent . Here we deduce the K(+) requirement of all available H(+)-PPase sequences based on the K(+) dependence of C . hydrogenoformans H(+)-PPase in conjunction with phylogenetic analyses . Our data reveal that K(+)-independent H(+)-PPases possess conserved Lys and Thr that are absent in K(+)-dependent H(+)-PPases . We further demonstrate that a A460K substitution in C . hydrogenoformans H(+)-PPase is sufficient to confer K(+) independence to both PP(i) hydrolysis and PP(i)-energized H(+) translocation . In contrast, a A463T mutation does not affect the K(+) dependence of H(+)-PPase.

J Biol Chem, 2002 Dec 27, 277(52), 51003 - 7 Epub 2002 Oct 23.
Complete inhibition of the tentoxin-resistant F1-ATPase from Escherichia coli by the phytopathogenic inhibitor tentoxin after substitution of critical residues in the alpha - and beta -subunit; Schnick C et al.; Substitution of critical residues in the alpha- and beta-subunit can turn the typically resistant ATP synthase from the bacterium Escherichia coli into an enzyme showing high sensitivity to the phytopathogenic inhibitor tentoxin, which usually affects only certain sensitive plant species . In contrast to recent results obtained with the thermophilic F(1) (Groth, G., Hisabori, T., Lill, H., and Bald, D . (2002) J . Biol . Chem . 277, 20117-20119), substitution of a critical serine in the beta-subunit (betaSer(59)), which is supposed to provide an important intermolecular hydrogen bond in the binding site, was not sufficient on its own for conferring tentoxin sensitivity to the E . coli F(1) complex . Superimposition of the chloroplast F(1)-tentoxin inhibitor complex on a homology model of the E . coli F(1) complex provided detailed information on the critical residues in the alpha-subunit of the binding cleft and allowed us to model the binding site according to the steric requirements of the inhibitor . Substitution of the highly conserved residue alphaLeu(64) seems to be most important for allowing access of the inhibitor to the binding site . Combining this substitution with either additional replacements in the alpha-subunit (Q49A, L95A, E96Q, I273M) or the replacement of Ser(59) in the beta-subunit enhanced the sensitivity to the inhibitor and resulted in a complete inhibition of the E . coli F(1)-ATPase by the plant-specific inhibitor tentoxin.

Appl Biochem Biotechnol, 2002 Jul-Dec, 102-103(1-6), 359 - 66
Screening of thermoacidophilic autotrophic bacteria for covellite solubilization; Umrania VV et al.; In an attempt to obtain suitable bacterial isolates for bioleaching of copper from chalcopyrite and covellite, soil samples taken from areas of the metal industry were screened using an enrichment procedure specially run at acidic pH and thermophilic temperature range, to overcome the limitations of mesophiles employed for the purpose besides having economic and environmental advantages . Of a total of 47 isolates, the most promising 3 having resistance to copper toxicity were evolved by subjecting them to gradually increasing concentrations of CuSO4 by acclimatization runs conducted on an environmental shaker for 125 d at 65 degrees C . The isolates, JVCu-8, JVCu-10, and JVCu-12, exhibited significantly enhanced bioleaching and copper tolerance ability at pH 3.5 and 60-70 degrees C . The total solubilization of copper recorded was 87, 89.4, and 91.2% by JVCu-8, JVCu-10, and JVCu-12, respectively, and these isolates exhibited tolerance to CuSO4 concentrations of 6.9, 7.2, and 7.2%, respectively . The isolates morphologically resembled Thiobacillus and Sulfolobus.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 127 - 32
Genomic and proteomic analyses reveal multiple homologs of genes encoding enzymes of the methanol:coenzyme M methyltransferase system that are differentially expressed in methanol- and acetate-grown Methanosarcina thermophila; Ding YH et al.; Each of the genomic sequences of Methanosarcina acetivorans, Methanosarcina mazei, and Methanosarcina thermophila revealed two homologs of mtaA, three homologs of mtaB, and three homologs of mtaC encoding enzymes specific for methanogenesis from methanol . Two-dimensional gel electrophoretic analyses of polypeptides from M . thermophila established that methanol induces the expression of mtaA-1, mtaB-1, mtaB-2, mtaB-3, mtaC-1, mtaC-2, and mtaC-3 whereas mtaB-3 and mtaC-3 are constitutively expressed in acetate-grown cells . The gene product of one of three mttC homologs, encoding trimethylamine-specific methyltransferase I, was detected in methanol- but not acetate-grown M . thermophila . A postulated role for the multiple homologs is discussed.

Arch Biochem Biophys, 2002 Nov 1, 407(1), 125 - 34
Enhancement of transglycosylation activity by construction of chimeras between mesophilic and thermophilic beta-glucosidase; Goyal K et al.; The family 3 beta-glucosidase from Thermotoga maritima is a highly thermostable enzyme (85 degrees C) that displays transglycosylation activity . In contrast, the beta-glucosidase from Cellvibrio gilvus is mesophilic (35 degrees C) and displays no such transglycosylation activity . Both enzymes consist of two domains, an N-terminal and a C-terminal domain, and the amino acid identities between the two enzymes in these domains are 32.4 and 36.4%, respectively . In an attempt to identify the molecular basis underpinning the display of transglycosylation activity and the requirements for thermal stability, eight chimeric genes were constructed by shuffling the two parental beta-glucosidase genes at four selected borders, two in the N-terminal domain and two in the C-terminal domain . Of the eight chimeric genes constructed, only two chimeric enzymes (Tm578/606Cg and Tm638/666Cg) gave catalytically active forms and these were the ones shuffled in the C-terminal domain . For these active chimeric enzymes, 80% (Tm578/606Cg) and 88% (Tm638/666Cg) of their amino acid sequences originated from T . maritima . With regard to their thermal profiles, the two active chimeric enzymes, Tm578/606Cg and Tm638/666Cg, displayed profiles intermediate to those of the two parental enzymes as they were optimally active at 65 and 70 degrees C, respectively . These two chimeric enzymes were optimally active at pH 4.1 and 3.9, which is closer to that observed for the T . maritima enzyme (pH 3.2-3.5) than that for the C . gilvus enzyme (pH 6.2-6.5) . Kinetic parameters for the chimeric enzymes were investigated with five different substrates including pNP-beta-D-glucopyranoside . The kinetic parameters obtained for the chimeric enzymes were closer to those of the T . maritima enzyme than to those of the C . gilvus enzyme . Transglycosylation activity was observed for both chimeric enzymes and the activity of the Tm578/606Cg chimera was at a level twice that observed with the T . maritima enzyme . This study is an effective demonstration of the usefulness of chimeric enzymes in altering the characteristics of an enzyme.

Arch Biochem Biophys, 2002 Nov 1, 407(1), 117 - 24
Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside; Hazard A et al.; Here we report a fast, simple purification for thermophilic F1F0 ATP synthase (TF1F0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl beta-D-maltoside to yield enzyme with an ATPase activity of 41 micromol/min/mg, 2.5-fold higher than that previously reported . ATPase activity was 80% inhibited by the F0-reactive reagent dicyclohexylcarbodiimide, indicating that F1-F0 interactions were largely intact . To measure ATP-driven proton pumping activity, purified TF1F0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine . In the presence of valinomycin, a maximum ATP-driven deltapH of 0.8 units was obtained . To measure ATP synthesis activity, TF1F0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin . Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis . A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial approximately 30-min lag phase.

Lett Appl Microbiol, 2002, 35(5), 423 - 7
Genetic diversity in Swiss cheese starter cultures assessed by pulsed field gel electrophoresis and arbitrarily primed PCR; Jenkins JK et al.; AIMS: To assess intraspecific genetic heterogeneity among commercial Swiss cheese starter culture strains of Lactobacillus helveticus, Streptococcus thermophilus and Propionibacterium freudenreichii and to compare the efficacy of two genetic typing methods . METHODS AND RESULTS: Two genetic typing methods, pulsed field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR), were used . Nine Strep . thermophilus strains revealed eight PFGE and five AP-PCR genotypes . Seventeen Lactobacillus strains yielded 16 and five genotypes by PFGE and AP-PCR, respectively . Eleven Propionibacterium strains yielded 10 PFGE genotypes . Cluster analysis of PFGE profiles generated similarity coefficients for Strep . thermophilus, Lact . helveticus and Prop . freudenreichii strains of 29.5%, 60.3%, and 30.5%, respectively . Milk acidification rates for Strep . thermophilus and Lact . helveticus were determined . CONCLUSIONS: Pulsed field gel electrophoresis is more discriminatory than AP-PCR . The Lact . helveticus group is more homogeneous than the other species examined . Strains with > 87% similarity by PFGE consistently had the same acidification rate and AP-PCR profile . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strains sold for Swiss cheese manufacture in the United States are genetically diverse . Clustering of genetically related bacteria may be useful in identifying new strains with industrially relevant traits.

Sci Total Environ, 2002 Oct 7, 297(1-3), 105 - 8
Fate of Ah-receptor agonists in organic household waste during anaerobic degradation--estimation of levels using EROD induction in organ cultures of chick embryo livers; Engwall M et al.; The fate of 7-ethoxyresorufin O-deethylase (EROD)-inducing compounds in source-separated organic household waste subject to anaerobic degradation (i.e . mesophilic/thermophilic anaerobic treatment) was investigated using organ cultures of embryonic chicken livers from fertilised hen eggs . This bioassay reflects the combined effect of all EROD-inducing, possibly dioxin-like compounds in a sample, including chemicals that are seldom or never analysed . All samples tested induced EROD in the bioassay, indicating the presence of dioxin-like compounds . In the anaerobic processes, the amounts of acid-resistant EROD-inducing compounds coming out of the reactors were considerably higher than the incoming amounts, especially for the low-temperature (mesophilic) process . This apparent production of EROD-inducing compounds may be due to de novo synthesis or to an increase in the EROD-inducing potency of the compounds in the material.

Gene, 2002 Sep 4, 297(1-2), 51 - 60
Amino acid composition of genomes, lifestyles of organisms, and evolutionary trends: a global picture with correspondence analysis; Tekaia F et al.; Can we infer the lifestyle of an organism from the characteristic properties of its genome? More precisely, what are the relations between easily quantifiable properties from genomic sequences, such as amino-acid compositions, and more subtle characteristics concerning for example lifestyles or evolutionary trends? Here, we seek a global picture for such properties, based on a large number (56) of complete genomes, including significant numbers of representatives from the three domains of life . We consider the amino acid compositions of the predicted proteomes, and we use correspondence analysis, as a multivariate method to extract the relevant information from the large-scale data . From these analyses we derive a series of conclusions, concerning lifestyles, as well as physico-chemical and evolutionary trends: (1) correspondence analysis of the amino acid compositions permits discrimination between the three known lifestyles (mesophily/thermophily/hyperthermophily) . (2) For various organisms, amino-acid composition properties are essentially driven by GC content, and to a significantly lesser extent by growth temperatures associated with lifestyles . Roughly speaking, the respective contributions of these two components are 57 and 20% . It is notable that these proportions are essentially unchanged with respect to a previous analysis (Nature 393 (1998) 537), which involved only 15 genomes, available at the time . (3) In terms of amino acid compositional biases, two specific 'signatures' for thermophily (in a broad sense, including hyperthermophily) can be detected . First, thermophilic species display a relative abundance in glutamic acid (Glu), concomitantly with the depletion in glutamine . Second, in thermophilic species, the relative abundance in Glu (negative charge) is significantly correlated (Pearson correlation coefficient r=0.83 with P<0.0001), with the increase in the lumped 'pool' lysine+arginine (positive charges) . This correlation (absent in mesophiles) could be interpreted on a physico-chemical basis, relevant to the thermostability of proteins . (4) Statistically significant differences are observed between the average lengths of the genes in the surveyed species, which follow their distribution between the three domains of life . Also a significant difference is observed between the average lengths of thermophilic (283.0+/-5.8) versus mesophilic (340+/-9.4) genes . It is thus possible that the 'general' shortening of the primary sequences in thermophilic proteins plays a role in thermostability . (5) Considering various combinations of conservation properties (genes conserved exclusively in eukaryotes, in archaea, in bacteria, in combinations of two domains, etc.) correspondence analysis reveals a trend towards thermophilic-hyperthermophilic profiles for the most conserved subset of genes (ancient genes) . (6) When limited to the subset of species-specific genes, correspondence analysis leads to a different picture for the clustering of genomes following amino-acid compositions: for example, the 'core' specific part of a genome can bear lifestyle signatures different from those of the complete genome.Various results are discussed both on methodological and biological grounds . The evolutionary perspectives opened by our analyses are noted.

Int J Food Microbiol, 2002 Nov 15, 79(1-2), 105 - 12
Survival of Escherichia coli O157:H7 in traditional African yoghurt fermentation; Ogwaro BA et al.; Growth and survival of a nontoxigenic strain of Escherichia coli O157:H7 (ATCC 43888) was determined in traditionally fermented pasteurized milk . Preheated milk was inoculated with 1% (v/v) of a mixed culture of Lactobacillus delbrueckii ssp . bulgaricus (NCIMB 11778) and Streptococcus salivarius ssp . thermophilus (NCIMB 110368) and incubated at 25, 30, 37 or 43 degrees C for 24 h . E . coli O157:H7 (10(5) CFU/ml) were introduced into the milk pre- and post-fermentation . Fermented milk samples were subsequently stored at either 4 degrees C (refrigerator temperature) or 25 degrees C (to mimic African ambient temperature) for 5 days . After 24 h of fermentation, the pH of the samples fermented at the higher temperatures of 37-43 degrees C decreased from 6.8 to 4.4-4.0 ( +/- 0.2) whereas at the lower temperature of 25 degrees C, the pH decreased to pH 5.0 +/- 0.1 . During this period, viable counts for E . coli O157:H7 increased from 10(5) to 10(8) - 10(9) CFU/ml except in milk fermented at 43 degrees C wherein viability declined to 10(4) CFU/ml . In fermented (25-30 degrees C) milk stored at 4 degrees C for 5 days, E . coli O157:H7 viability decreased from 10(8-9) to 10(6-7) CFU/ml whereas milk fermented at 43 degrees C resulted in loss of detectable cells . In contrast, storage of fermented milk samples at 25 degrees C for 5 days eventually resulted in complete loss of viability irrespective of fermentation temperature . Stationary phase E . coli O157:H7 inoculated post-fermentation (25 and 43 degrees C) survived during 4 degrees C storage, but not 25 degrees C storage . Fermentation temperature and subsequent storage temperature are critical to the growth and survival of E . coli O157:H7 in traditional fermented products involving yoghurt starter cultures.

Biopolymers, 2002 Nov 15, 65(4), 263 - 73
Comparative analysis of thermoadaptation within the archaeal glyceraldehyde-3-phosphate dehydrogenases from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus; Charron C et al.; To gain insight into the molecular determinants of thermoadaptation within the family of archaeal glyceraldehyde-3-phosphate dehydrogenases (GAPDH), a homology-based 3-D model of the mesophilic GAPDH from Methanobacterium bryantii was built and compared with the crystal structure of the thermophilic GAPDH from Methanothermus fervidus . The homotetrameric model of the holoenzyme was initially assembled from identical subunits completed with NADP molecules . The structure was then refined by energy minimization and simulated-annealing procedures . PROCHECK and the 3-D profile method were used to appraise the model reliability . Striking molecular features underlying the difference in stability between the enzymes were deduced from their structural comparison . First, both the increase in hydrophobic contacts and the decrease in accessibility to the protein core were shown to discriminate in favor of the thermophilic enzyme . Besides, but to a lesser degree, the number of ion pairs involved in cooperative clusters appeared to correlate with thermostability . Finally, the decreased stability of the mesophilic enzyme was also predicted to proceed from both the lack of charge-dipole interactions within alpha-helices and the enhanced entropy of unfolding due to an increase in chain flexibility . Thus, archaeal GAPDHs appear to be governed by thermoadaptation rules that differ in some aspects from those previously observed within their eubacterial counterparts .

J Mol Recognit, 2002 Jul-Aug, 15(4), 188 - 96
tRNA discrimination by T . thermophilus phenylalanyl-tRNA synthetase at the binding step; Vasil'eva IA et al.; The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs . The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis . The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis . Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding . Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme . The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step . A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex . The highest affinity of T . thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs .

Extremophiles, 2002 Oct, 6(5), 351 - 8 Epub 2002 May 01.
Genes and derived amino acid sequences of S-layer proteins from mesophilic, thermophilic, and extremely thermophilic methanococci; Akca E et al.; Cells of methanococci are covered by a single layer of protein subunits (S-layer) in hexagonal arrangement, which are directly exposed to the environment and which cannot be stabilized by cellular components . We have isolated S-layer proteins from cells of Methanococcus vannielii ( T(opt.)=37 degrees C), Methanococcus thermolithotrophicus ( T(opt.)=65 degrees C), and Methanococcus jannaschii ( T(opt.)=85 degrees C) . The primary structure of the S-layer proteins was determined by sequencing the corresponding genes . According to the predicted amino acid sequence, the molecular masses of the S-layer proteins of the different methanococci are in a small range between 59,064 and 60,547 Da . Compared with its mesophilic counterparts, it is worth noting that in the S-layer protein of the extreme thermophile Mc . jannaschii the acidic amino acid Asp is predominant, the basic amino acid Lys occurs in higher amounts, and Cys and His are only present in this organism . Despite the differences in the growth optima and the predominance of some amino acids, the comparative total primary structure revealed a relatively high degree of identity (38%-45%) between the methanococci investigated . This observation indicates that the amino acid sequence of the S-layer proteins is significantly conserved from the mesophilic to the extremely thermophilic methanococci.

Appl Microbiol Biotechnol, 2002 Oct, 60(1-2), 128 - 33 Epub 2002 Aug 24.
Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp . strain A11-2; Konishi J et al.; A benzothiophene (BT) and dibenzothiophene (DBT) monooxygenase (TdsC), which catalyzes the oxidation of the sulfur atoms in BT and DBT molecules, was purified from Paenibacillus sp . strain A11-2 . The molecular mass of the purified enzyme and its subunit were determined to be 200 kDa and 43 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating a tetrameric structure . The N-terminal amino acid sequence of the purified TdsC completely matched the amino acid sequence deduced from the nucleotide sequence of the tdsC gene reported previously {Ishii et al . (2000) Biophys Biochem Res Commun 270:81-88} . The optimal temperature and pH for the TdsC reaction were 65 degrees C and pH 9, respectively . TdsC required NADH, FMN and TdsD, a NADH-dependent FMN oxidoreductase, for its activity, as was observed for TdsA . FAD, lumiflavin and/or NADPH had some effect on the maintenance of TdsC activity . A comparison of the substrate specificity of TdsC and DszC, the homologous monooxygenase purified from Rhodococcus erythropolis strain KA2-5-1, demonstrated a contrasting pattern towards alkylated DBTs and BTs.

Protein Sci, 2002 Nov, 11(11), 2727 - 34
Alpha-crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions; Nath D et al.; Alpha-crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins . For the functional in vitro analysis of alpha-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system . The mechanism of chaperone action of alpha-crystallin is less investigated . Here we studied the refolding of Gdn HCl-denatured Xyl II in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding . Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II . The alpha-crystallin.Xyl II complex exhibited no functional activity . Addition of ATP to the complex initiated the renaturation of Xyl II with 30%-35% recovery of activity . The nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP . Although the presence of Ca(2+) was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence . Experimental evidence indicated that the binding of ATP to the alpha-crystallin.Xyl II complex brought about conformational changes in alpha-crystallin facilitating the dissociation of xylanase molecules . This is the first report of the enhancement of alpha-crystallin chaperone functions by metal ions.

Protein Sci, 2002 Nov, 11(11), 2561 - 74
Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging; Bogin O et al.; Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH . In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH . Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH . Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH . To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure . The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.

Int J Food Microbiol, 2003 Jan 25, 80(2), 177 - 83
Preston and Park-Sanders protocols adapted for semi-quantitative isolation of thermotolerant Campylobacter from chicken rinse; Josefsen MH et al.; Human campylobacteriosis has become the major cause of foodborne gastrointestinal diseases in several European countries . In order to implement effective control measures in the primary production, and as a tool in risk assessment studies, it is necessary to have sensitive and quantitative detection methods.Thus, semi-quantitative detection of thermophilic Campylobacter spp . in 20 naturally contaminated chicken rinse samples was carried out using the two most common standard protocols: Preston and Park-Sanders, as proposed by Nordic Committee on Food Analysis (NMKL) and International Standard Organization (ISO), respectively . For both protocols, the chicken rinse samples were prepared in 500 ml buffered peptone water, as recommended in the ISO protocol no . 6887-2 . The results indicated that the Preston protocol was superior to the Park-Sanders protocol in supporting growth of Campylobacter spp.In conclusion, the established semi-quantitative assessment using Preston broth could be useful in monitoring the outcome of control programs or quantitative risk assessments.

J Mol Biol, 2002 Oct 25, 323(3), 463 - 73
TROSY-NMR studies of the 91kDa TRAP protein reveal allosteric control of a gene regulatory protein by ligand-altered flexibility; McElroy C et al.; The tryptophan biosynthesis genes of several Bacilli are controlled through terminator/anti-terminator transcriptional attenuation . This process is regulated by tryptophan-dependent binding of the trp RNA-binding attenuation protein (TRAP) to the leader region of the trp operon mRNA, precluding formation of the antiterminator RNA hairpin, and allowing formation of the less stable terminator hairpin . Crystal structures are available of TRAP in complex with tryptophan and in ternary complex with tryptophan and RNA . However, no structure of TRAP in the absence of tryptophan is available; thus, the mechanism of allostery remains unclear . We have used transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments to study the mechanism of ligand-mediated allosteric regulation in the 90.6kDa 11-mer TRAP . By recording a series of two-dimensional 15N-edited TROSY NMR spectra of TRAP from the thermophile Bacillus stearothermophilus over an extended range of temperatures, we have found tryptophan binding to be temperature-dependent, in agreement with the previously observed temperature-dependent RNA binding . Triple-resonance TROSY-based NMR spectra recorded at 55 degrees C have allowed us to obtain backbone resonance assignments for uniformly 2H,13C,15N-labeled TRAP in the inactive form and in the active form (free and bound to tryptophan) . On the basis of ligand-dependent differential line-broadening and chemical shift perturbations, coupled with the results of proteolytic sensitivity measurements, we infer that tryptophan-modulated protein flexibility (dynamics) plays a central role in TRAP function by altering its RNA-binding affinity . Furthermore, because the crystal structures show that the ligand is buried completely in the bound state, we speculate that such dynamic behavior may be important to enable rapid response to changes in intracellular tryptophan levels . Thus, we propose that allosteric control of TRAP is accomplished by ligand-altered protein dynamics.

J Food Prot, 2002 Oct, 65(10), 1597 - 604
Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries; Suarez VB et al.; Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp . bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000 . Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S . thermophilus strains . The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml . The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1) . They showed high burst size values and remarkably short latent periods . The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes . The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains . These results suggest that the naturally occurring cultures are inherently more phage resistant.

Cell Motil Cytoskeleton, 2002 Dec, 53(4), 281 - 8
Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila; Hennessey TM et al.; Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards . Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels . It is this increase in intraciliary Ca++ that triggers ciliary reversal . However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known . We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1) . In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli . In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells . However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency . Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact . Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants . We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming .

Structure (Camb), 2002 Oct, 10(10), 1415 - 23
Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8; Niwa H et al.; FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding . The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain . We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment . Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.

Inorg Chem, 2002 Oct 21, 41(21), 5544 - 54
Preparation of highly efficient manganese catalase mimics; Triller MU et al.; The series of compounds {Mn(bpia)(mu-OAc)}(2)(ClO(4))(2) (1), {Mn(2)(bpia)(2)(muO)(mu-OAc)}(ClO(4))(3).CH(3)CN (2), {Mn(bpia)(mu-O)}(2)(ClO(4))(2)(PF(6)).2CH(3)CN (3), {Mn(bpia)(Cl)(2)}(ClO)(4) (4), and {(Mn(bpia)(Cl))(2)(mu-O)}(ClO(4))(2).2CH(3)CN (5) (bpia = bis(picolyl)(N-methylimidazol-2-yl)amine) represents a structural, spectroscopic, and functional model system for manganese catalases . Compounds 3 and 5 have been synthesized from 2 via bulk electrolysis and ligand exchange, respectively . All complexes have been structurally characterized by X-ray crystallography and by UV-vis and EPR spectroscopies . The different bridging ligands including the rare mono-mu-oxo and mono-mu-oxo-mono-mu-carboxylato motifs lead to a variation of the Mn-Mn separation across the four binuclear compounds of 1.50 A (Mn(2)(II,II) = 4.128 A, Mn(2)(III,III) = 3.5326 and 3.2533 A, Mn(2)(III,IV) = 2.624 A) . Complexes 1, 2, and 3 are mimics for the Mn(2)(II,II), the Mn(2)(III,III), and the Mn(2)(III,IV) oxidation states of the native enzyme . UV-vis spectra of these compounds show similarities to those of the corresponding oxidation states of manganese catalase from Thermus thermophilus and Lactobacillus plantarum . Compound 2 exhibits a rare example of a Jahn-Teller compression . While complexes 1 and 3 are efficient catalysts for the disproportionation of hydrogen peroxide and contain an N(4)O(2) donor set, 4 and 5 show no catalase activity . These complexes have an N(4)Cl(2) and N(4)OCl donor set, respectively, and serve as mimics for halide inhibited manganese catalases . Cyclovoltammetric data show that the substitution of oxygen donor atoms with chloride causes a shift of redox potentials to more positive values . To our knowledge, complex 1 is the most efficient binuclear functional manganese catalase mimic exhibiting saturation kinetics to date.

J Bacteriol, 2002 Nov, 184(21), 6026 - 36
The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria; Proux C et al.; The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported . Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria . Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared . Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness . This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403 . A systematic dot plot analysis with 11 complete L . lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages . The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster . With respect to structural genes, four DNA homology groups could be defined within temperate L . lactis phages . Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L . lactis . It seems reasonable to base phage taxonomy on data from comparative genomics . However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics . For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera . We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes) . This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system . In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.

J Bacteriol, 2002 Nov, 184(21), 5955 - 65
The first archaeal ATP-dependent glucokinase, from the hyperthermophilic crenarchaeon Aeropyrum pernix, represents a monomeric, extremely thermophilic ROK glucokinase with broad hexose specificity; Hansen T et al.; An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity . The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa . The apparent K(m) values for ATP and glucose (at 90 degrees C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V(max) was about 35 U/mg . The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k(cat)/K(m)), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose . Divalent cations were required for maximal activity: Mg(2+), which was most effective, could partially be replaced with Co(2+), Mn(2+), and Ni(2+) . The enzyme had a temperature optimum of at least 100 degrees C and showed significant thermostability up to 100 degrees C . The coding function of open reading frame (ORF) APE2091 (Y . Kawarabayasi, Y . Hino, H . Horikawa, S . Yamazaki, Y . Haikawa, K . Jin-no, M . Takahashi, M . Sekine, S . Baba, A . Ankai, H . Kosugi, A . Hosoyama, S . Fukui, Y . Nagai, K . Nishijima, H . Nakazawa, M . Takamiya, S . Masuda, T . Funahashi, T . Tanaka, Y . Kudoh, J . Yamazaki, N . Kushida, A . Oguchi, and H . Kikuchi, DNA Res . 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A . pernix, was proved by functional expression in Escherichia coli . The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A . pernix . N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091 . The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors . This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.

Int J Food Microbiol, 2002 Dec 15, 79(3), 161 - 74
Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations; Degeest B et al.; To increase the exopolysaccharide (EPS) yields from Streptococcus thermophilus LY03 and to unravel the nature of the EPS degradation process, fermentation experiments were carried out with this strain in a customized MRS medium, using different additional carbohydrates or amino acids possibly related to growth and EPS production . No significant increase of the EPS yields or activities of the enzymes alpha-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase that are correlated with EPS production, or of the activity of dTDP-glucose pyrophosphorylase involved in the rhamnose synthetic branch of EPS biosynthesis, was observed . The EPS monomer composition remained unchanged for all experiments . Fermentations with a sudden temperature increase or lowered pH were carried out as well to try to avoid EPS degradation upon prolonged fermentation . It was demonstrated that EPS degradation took place enzymatically . Incubations of purified high-molecular-mass EPS with cell-free culture supernatant or cell extracts showed its degradation by enzymes with an endo-activity . This glycohydrolytic activity probably encompasses several enzymes having a molecular mass lower than 50,000 and 10,000 Da, and seems to be rather stable at high temperature and low pH . These results contribute to a better understanding of the physiological and chemical factors influencing EPS production and degradation.

Curr Protein Pept Sci, 2002 Feb, 3(1), 55 - 65
Initiation and inhibition of protein biosynthesis studies at high resolution; Zarivach R et al.; Analysis of the high resolution structure of the small subunit from Thermus thermophilus shed light on its inherent conformational variability and indicated an interconnected network of features allowing concerted movements during translocation . It also showed that conformational rearrangements may be involved in subunit association and that a latch-like movement guarantees processivity and ensures maximized fidelity . Conformational mobility is associated with the binding and the anti association function of initiation factor 3, and antibiotics interfering with prevent the initiation of the biosynthetic process . Proteins stabilize the structure mainly by their long basic extensions that penetrate into the ribosomal RNA . When pointing into the solution, these extensions may have functional roles in binding of non-ribosomal factors participating in the process of protein biosynthesis . In addition, although the decoding center is formed of RNA, proteins seem to serve ancillary functions such as stabilizing ist required conformation and assisting the directionality of the translocation.

Water Res, 2002 Sep, 36(15), 3860 - 6
Aerobic waste activated sludge (WAS) for start-up seed of mesophilic and thermophilic anaerobic digestion; Kim M et al.; Since there are very limited numbers of thermophilic anaerobic digesters being operated, it is often difficult to start up a new one using sludge from an existing reactor as a seed . However, for obvious reasons it seems few attempts have been made to compare the start-up performance of thermophilic anaerobic digestion using different sources of seed sludges . The purpose of this study was to evaluate the start-up performance of anaerobic digestion using aerobic waste activated sludge (WAS) from a plant which has no anaerobic digesters and mesophilic anaerobic digested sludge (ADS) as the seed source at both mesophilic (35 degrees C) and thermophilic (55 degrees C) temperatures . In this study, two experiments were conducted . First, thermophilic anaerobic reactors were seeded with WAS (VSS = 4400 mg/L) and ADS (VSS = 14,500 mg/L) to investigate start-up performance with a feed of acetate as well as propionate . The results show that WAS started to produce CH4 soon after acetate feeding without a lag time, while ADS had a lag time of 10 days . When the feed was changed to propionate, WAS removed propionate down to below the detection limit of 10 mg/L, while ADS removed little propionate and produced little CH4 . Second, in order to further compare the methanogenic activity of WAS and ADS, both mesophilic and thermophilic reactors were operated . WAS acclimated to anaerobic conditions shortly (< 5 days at both mesophilic and thermophilic) and after acclimating it produced more CH4 per unit amount of seeded VSS than ADS . WAS at mesophilic temperature biodegraded acetate at the same rate as for thermophilic . However WAS at mesophilic temperature biodegraded propionate at a much faster rate than at thermophilic . WAS as the seed source of anaerobic digestion resulted in much better performance than ADS at both mesophilic and thermophilic temperatures for both acetate and propionate metabolism.

Nat Struct Biol, 2002 Nov, 9(11), 855 - 61
Evolution of Tetrahymena ribozyme mutants with increased structural stability; Guo F et al.; Determining how large RNA molecules stabilize their tertiary structures is critical for understanding how they perform their biological functions . Here we use in vitro selection to identify active variants of the Tetrahymena ribozyme with increased stability . The mutant pool converged to a single family that shared nine mutations; an RNA representing the consensus sequence was structurally more stable by 10.5 degrees C and catalytically active at elevated temperatures . Remarkably, of the nine altered sites, most are already known to be involved in tertiary interactions, and the stabilizing mutations primarily improve the packing interactions in the molecular interior . The wild type ribozyme and the selected mutants provide pairs of mesophilic and thermophilic homologs for studying the origin of their thermal stability.

Microbiology, 2002 Oct, 148(Pt 10), 3307 - 15
Identification and characterization of single-stranded-DNA-binding proteins from Thermus thermophilus and Thermus aquaticus - new arrangement of binding domains; Dabrowski S et al.; Single-stranded-DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in bacteria, archaea and eukarya . This paper reports the identification and characterization of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and Thermus aquaticus . These proteins (TthSSB and TaqSSB), in contrast to their known counterparts from mesophilic bacteria, archaea and eukarya, are homodimers, and each monomer contains two ssDNA-binding domains with a conserved OB (oligonucleotide/oligosaccharide-binding) fold, as deduced from the sequence analysis . The N-terminal domain is located in the region from amino acid 1 to 123 and the C-terminal domain is located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively . Purified TthSSB or TaqSSB binds only to ssDNA and with high affinity . The binding site size for TaqSSB and TthSSB protein corresponds to 30-35 nucleotides . It is concluded that the SSBs of thermophilic and mesophilic bacteria, archaea and eukarya share a common core ssDNA-binding domain . This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

Environ Microbiol, 2002 Oct, 4(10), 603 - 11
First insight into the genome of an uncultivated crenarchaeote from soil; Quaiser A et al.; Molecular phylogenetic surveys based on the characterization of 16S rRNA genes have revealed that soil is an environment particularly rich in microbial diversity . A clade of crenarchaeota (archaea) has frequently been detected among many other novel lineages of uncultivated bacteria . In this study we have initiated a genomic approach for the characterization of uncultivated microorganisms from soil . We have developed a procedure based on a two-phase electrophoresis technique that allows the fast and reliable purification of concentrated and clonable, high molecular weight DNA . From this DNA we have constructed complex large-insert genomic libraries . Using archaea-specific 16S rRNA probes we have isolated a 34 kbp fragment from a 900 Mbp fosmid library of soil DNA . The clone contained a complete 16S/23S rRNA operon and 17 genes encoding putative proteins . Phylogenetic analyses of the rRNA genes and of several protein encoding genes (e.g . DNA polymerase, FixAB, glycosyl transferase) confirmed the specific affiliation of the genomic fragment with the non-thermophilic clade of the crenarchaeota . Content and structure of the genomic fragment indicated that the archaea from soil differ significantly from their previously studied uncultivated marine relatives . The protein encoding genes gave the first insights into the physiological potential of these organisms and can serve as a basis for future genomic and functional genomic studies.

Environ Microbiol, 2002 Oct, 4(10), 595 - 602
The frequency and characteristics of highly thermophilic bacteria in cool soil environments; Marchant R et al.; Following enrichment at 70 degrees C and 80 degrees C, five highly thermophilic aerobic eubacteria have been isolated from cool soil environments . These organisms show a temperature range for growth of 40-80 degrees C and have optimal and very high growth rates around 70 degrees C with generation times less than 30 min . All isolates are narrow rods, which stain Gram-negative, but have a Gram-positive cell wall structure and only one of five isolates is a spore former . All cultures contain a small proportion of previously unreported extremely long flexuous rods, which can be seen to divide eventually . Biochemical testing of five strains reveals a significant ability to utilize alkanes and some aromatic hydrocarbons . Using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of 16S rDNA the five strains were differentiated into three categories, which paralleled the biochemical results . 16S rDNA sequences showed high similarity with thermophilic Bacillus species now reclassified as Geobacillus . These bacteria are present in high numbers in apparently all soils and the question is raised of how these organisms, which are apparently unable to grow at the temperatures experienced in these cool soils, are so prominent.

Bioresour Technol, 2002 Dec, 85(3), 291 - 9
Super blue box recycling (SUBBOR) enhanced two-stage anaerobic digestion process for recycling municipal solid waste: laboratory pilot studies; Vogt GM et al.; The super blue box recycling (SUBBOR) process is an enhanced, multi-stage anaerobic digestion process for mixed municipal solid waste (MSW) and other biomass feedstock materials . The technology centers on enhanced high solids, thermophilic digestion after steam-pressure disruption of the ligno-cellulosic fiber components that are recalcitrant to conventional anaerobic digestion . Mixed MSW, rich in organic components but also containing inorganic materials, such as glass, aluminum and steel, as well as non-digestible plastic materials, has been laboratory pilot tested with a fully integrated process train designed to treat and recycle all of the MSW components . Methane yields from the MSW were 0.36 m3 CH4/kg volatile solids (VS) representing a 40% increase over the yield obtained from conventional single stage digestion . The secondary digestion step after steam pressure disruption also provided a 40% improvement in total solids and VS reduction . The residual organic fraction following two-stage digestion was fine in texture and was recovered as a clean peat fraction with reduced contents of heavy metal and other fugitive non-digested contaminants . Mass and energy balance determinations indicated a high degree of MSW diversion from landfill disposal (>80%) was achievable by the SUBBOR process as well as substantial net electrical and thermal energy production . Continuous long-term trials of the SUBBOR process at 25,000 tonnes/year are underway.

Nucleic Acids Res, 2002 Oct 1, 30(19), 4272 - 7
Synonymous codon usage is subject to selection in thermophilic bacteria; Lynn DJ et al.; The patterns of synonymous codon usage, both within and among genomes, have been extensively studied over the past two decades . Despite the accumulating evidence that natural selection can shape codon usage, it has not been possible to link a particular pattern of codon usage to a specific external selective force . Here, we have analyzed the patterns of synonymous codon usage in 40 completely sequenced prokaryotic genomes . By combining the genes from several genomes (more than 80 000 genes in all) into a single dataset for this analysis, we were able to investigate variations in codon usage, both within and between genomes . The results show that synonymous codon usage is affected by two major factors: (i) the overall G+C content of the genome and (ii) growth at high temperature . This study focused on the relationship between synonymous codon usage and the ability to grow at high temperature . We have been able to eliminate both phylogenetic history and lateral gene transfer as possible explanations for the characteristic pattern of codon usage among the thermophiles . Thus, these results demonstrate a clear link between a particular pattern of codon usage and an external selective force.

Protein Eng, 2002 Aug, 15(8), 683 - 8
Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H; Chon H et al.; A series of DNA-linked RNases H, in which the 15-mer DNA is cross-linked to the Thermus thermophilus RNase HI (TRNH) variants at positions 135, 136, 137 and 138, were constructed and analyzed for their abilities to cleave the complementary 15-mer RNA . Of these, that with the DNA adduct at position 135 most efficiently cleaved the RNA substrate, indicating that position 135 is the most appropriate cross-linking site among those examined . To examine whether DNA-linked RNase H also site-specifically cleaves a highly structured natural RNA, DNA-linked TRNHs with a series of DNA adducts varying in size at position 135 were constructed and analyzed for their abilities to cleave MS2 RNA . These DNA adducts were designed such that DNA-linked enzymes cleave MS2 RNA at a loop around residue 2790 . Of the four DNA-linked TRNHs with the 8-, 12-, 16- and 20-mer DNA adducts, only that with the 16-mer DNA adduct efficiently and site-specifically cleaved MS2 RNA . Primer extension revealed that this DNA-linked TRNH cleaved MS2 RNA within the target sequence.

J Lipid Res, 2002 Oct, 43(10), 1641 - 51
Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether membrane lipid of cosmopolitan pelagic crenarchaeota; Damste JS et al.; The basic structure and stereochemistry of the characteristic glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipid of cosmopolitan pelagic crenarchaeota has been identified by high field two-dimensional (2D)-NMR techniques . It contains one cyclohexane and four cyclopentane rings formed by internal cyclisation of the biphytanyl chains . Its structure is similar to that of GDGTs biosynthesized by (hyper)thermophilic crenarchaeota apart from the cyclohexane ring . These findings are consistent with the close phylogenetic relationship of (hyper)thermophilic and pelagic crenarchaeota based 16S rRNA . The latter group inherited the biosynthetic capabilities for a membrane composed of cyclopentane ring-containing GDGTs from the (hyper)thermophilic crenarchaeota . However, to cope with the much lower temperature of the ocean, a small but key step in their evolution was the adjustment of the membrane fluidity by making a kink in one of the bicyclic biphytanyl chains by the formation of a cyclohexane ring . This prevents the dense packing characteristic for the cyclopentane ring-containing GDGTs membrane lipids used by hyperthermophilic crenarchaeota to adjust their membrane fluidity to high temperatures.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 927 - 34
Production of a polyester degrading extracellular hydrolase from Thermomonospora fusca; Gouda MK et al.; The production of a polyester-degrading hydrolase from the thermophilic actinomycete Thermomonospora fusca was investigated with regard to its potential technical application . Only in the presence of a polyester (random aliphatic-aromatic copolyester from 1,4-butanediol, terephthalic acid, and adipic acid with around 40-50 mol % terephthalic acid in the acid component), the excretion of the extracellular enzyme could be achieved with an optimized synthetic medium using pectin and NH(4)Cl as nitrogen source . Compared to complex media, a significantly higher specific activity at comparable volumetric yields could be obtained, thus reducing the expenditure for purification . The activity profile in the medium is controlled by a complex process involving (1) induction of enzyme excretion, (2) enzyme adsorption on the hydrophobic polyester surface, (3) inhibition of enzyme generation by monomers produced by polyester cleavage, and (4) enzyme denaturation . Diafiltration with cellulose acetate membranes as the sole downstream processing step led to a product of high purity and with sufficient yield (60% of total activity) . Scaling-up from shaking flasks to a fermentor scale of 100 L revealed no specific problems . However, the excretion of the hydrolase by the actinomycete turned out to be inhibited by the degradation products (monomers) of the aliphatic-aromatic copolyester used as inductor for the enzyme production . The crude enzyme exhibited generally similar properties (temperature and pH optimum) as the highly purified hydrolase described previously; however, the storage capability and thermal stability is improved when the crude enzyme solution is diafiltrated.

Waste Manag Res, 2002 Aug, 20(4), 350 - 6
Anaerobic thermophilic treatment of cattle manure in UASB reactors; Castrillon L et al.; Cattle manure was characterised after filtration through a 1-mm sieve and subsequently treated in a 9-l volume Upflow Anaerobic Sludge Blanket (UASB) reactor made of transparent PVC at a thermophilic temperature (55 degrees C) . Different Hydraulic Retention Times (HRT) (22.5, 16, 10.6, 8.9 and 7.3 days) were employed and organic matter, total solids and metals were determined, as was the production of biogas . After screening, the COD of the manure subjected to anaerobic thermophilic treatment varied between values of 33,382 and 45,513 mgO2 l(-1) . The highest percentage of COD removal obtained was 79.7% for an HRT of 22.5 days and there was a fraction refractory to biodegradation of 11%, calculated using Chen & Hashimoto's model . Finally, the results obtained at a thermophilic temperature were compared with those obtained at a mesophilic temperature (obtained in a previous work) . The reduction in COD was slightly greater under mesophilic conditions, though the main advantage of thermophilic anaerobic treatment is the faster inactivation of viruses and bacteria.

Proteomics, 2002 Sep, 2(9), 1316 - 24
Proteomic study of cold shock protein in Bacillus stearothermophilus P1: Comparison of temperature downshifts; Sinchaikul S et al.; The thermophilic bacterium Bacillus stearothermophilus P1 is unique in its ability to thrive in extreme environments such as high temperatures or high pH conditions . The study of cold shock response is very interesting and interpreted as a shock response to express the genes involved in synthesis of specific proteins . This study investigated the study of cold shock protein of B . stearothermophilus P1 when the cell culture temperature shifted from 65 degrees C to 37 degrees C and 25 degrees C . Cell growth at 37 degrees C weakly increased in the previous 3 h and then slowly decreased . In contrast, cell growth at 25 degrees C was slowly decreased . The protein contents after temperature downshifts were analyzed by proteomic techniques using protein chip and two-dimensional (2-D) electrophoresis that are highly effective and useful for protein separation and identification . The different proteins after a temperature decrease from 65 degrees C to 37 degrees C and 25 degrees C were expressed on 2-D gel patterns and the cold shock protein was detected in the acidic area with the isoelectric point and molecular mass approximately 4.5 and 7.3 kDa, respectively . The NH(2)-terminal sequence of a major cold shock protein from B . stearothermophilus P1 was MQRGKVKWFNNEKGFGFIEVEGGSD, similar to other cold shock proteins from Bacillus sp . up to 96% identity, but different from the other bacteria with homology less than 80% identity.

Proteomics, 2002 Sep, 2(9), 1311 - 5
Proteomic analysis of a thermostable superoxide dismutase from Bacillus stearothermophilus TLS33; Sookkheo B et al.; Thermophilic bacterium Bacillus stearothermophilus TLS33 isolated from a hot spring in Chiang Mai, Thailand produces an extracellular superoxide dismutase (SOD) . SOD is a free radical metabolizing enzyme that protects the cell membrane from damage by the highly reactive superoxide free radicals . To identify the secreted SOD, we used the systematically proteomic approaches of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis and database searching . The bacterium was grown in a medium containing 0.1% w/v yeast extract and 0.1% w/v tryptone in 100% v/v base mixture at 65 degrees C for 72 h, by assessing their growth by protein and SOD activity . The bacterium produced the highest SOD activity at 65 degrees C for 48 h and the extracellular SOD was run on 2-D PAGE using broad range pH 3-10 immobilized pH gradients (IPGs) and narrow range pH 4-7 IPGs . The isoelectric point and molecular mass of the extracellular SOD were approximately 5.8 and 28 kDa, respectively . In addition, the NH(2)-terminal amino acid sequence was found to be P-F-E-L-P-A-L-P-Y-P-Y-D-A-L-E-P-P-I-I-D, which had a homology of approximately 85% to the Mn-SOD family and 65% to the Fe-SOD family.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1859 - 65
Thermovibrio ruber gen . nov., sp . nov., an extremely thermophilic, chemolithoautotrophic, nitrate-reducing bacterium that forms a deep branch within the phylum Aquificae; Huber H et al.; A novel, extremely thermophilic, chemolithoautotrophic bacterium was isolated from the submarine hydrothermal system off the beach of Lihir Island, Papua New Guinea . Cells of the organism were curved rods of about 1.5-3 microm in length and 0.5-0.8 microm in width . The bacterium grew within the temperature range 50-80 degrees C (optimum around 75 degrees C) and was an obligate anaerobe . Molecular hydrogen was used as the sole electron donor by the bacterium, and nitrate or elemental sulfur served as electron acceptors, producing ammonium or H2S, respectively . Complex organic substrates stimulated growth of the bacterium, but they could not be used as the sole energy source . Based on 16S-rDNA-based phylogenetic analyses and on physiological, biochemical and structural characteristics, the novel organism was found to represent a novel genus for which the name Thermovibrio is proposed . This novel genus, together with Desulfurobacterium thermolithotrophum, may represent a new order within the phylum Aquificae . Since cell pellets of the novel bacterium had an intense red colour, the name Thermovibrio ruber is proposed for the novel organism . The type strain of Thermovibrio ruber gen . nov., sp . nov . is ED11/3LLKT (= DSM 14644T = JCM 11468T).

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1837 - 43
Development of fluorescent adjacent hybridization probes and their application in real-time PCR for the simultaneous detection and identification of Fervidobacterium and Caloramator; Connolly GR et al.; A stretch of nucleotides consisting of a conserved region and an adjacent upstream variable region in the 16S rDNA of members of domain Bacteria was identified as a suitable target site for developing a real-time PCR adjacent hybridization assay . A single universal fluorogenic cyanin 5-labelled probe, CY5 1046+, targeting the conserved region, and two FITC-labelled probes, Calo and Fervi, targeting the variable region were designed and synthesized for the identification and differentiation of the thermophilic anaerobes Caloramator and Fervidobacterium . The simultaneous hybridization of probes CY5 1046+ and Fervi to the 16S rDNA target sites of Fervidobacterium species during PCR amplification resulted in an increase in fluorescence emission that was monitored continuously in a LightCycler . The differences in the temperature of disassociation of the hybridization probes (Tm) due to compositional variation in the nucleotide bases of the probe and target sequences enabled Fervidobacterium islandicum DSM 5733T to be differentiated from Fervidobacterium gondwanense ACM 5017T and Fervidobacterium nodosum ATCC 35602T . The simultaneous hybridization of probes CY5 1046+ and Calo to the 16S rDNA target sequence of Caloramator indicus ACM 3982T during PCR amplification also resulted in an increase in fluorescence emission . However, as the target sequence of C . indicus ACM 3982T is identical to those of the other three Caloramator species, Caloramator fervidus ATCC 43204T (formerly Clostridium fervidus), Caloramator proteoclasticus DSM 10124T and Caloramator coolhaasii DSM 12679T, further species discrimination on the basis of Tm will not be possible, making probe Calo a useful genus-specific probe . The devised method was subsequently used to detect and identify Fervidobacterium and Caloramator isolates from our previously uncharacterized culture collection and from enrichment cultures . The assay is cheap and flexible, as only a battery of inexpensive FITC probes for differentiating other members of domain Bacteria needs to be synthesized and DNA templates prepared by a simple lyse-boil method, in addition to purified DNA, can also be used.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1737 - 43
Thermovenabulum ferriorganovorum gen . nov., sp . nov., a novel thermophilic, anaerobic, endospore-forming bacterium; Zavarzina DG et al.; A thermophilic, anaerobic, spore-forming bacterium (strain Z-9801T) was isolated from a terrestrial hydrothermal source in the Uzon caldera on the Kamchatka peninsula . Cells of strain Z-9801T were straight, sometimes branched rods, 0.5-0.6 microm in diameter and 1.5-7.0 microm in length, with peritrichous flagella . The temperature range for growth was 45-76 degrees C, with an optimum at 63-65 degrees C . The pH range for growth was 4.8-8.2, with an optimum at 6.7-6.9 . The substrates utilized by strain Z-9801T included peptone, yeast extract, beef extract, Casamino acids, starch, pyruvate, melibiose, sucrose, fructose, maltose, xylose and ribose . The fermentation products from melibiose were ethanol, acetate, H2 and CO2 . Strain Z-9801T used H2 in the presence of Fe(III) and an organic electron donor . Strain Z-9801T reduced Fe(III), Mn(IV), nitrate, fumarate, sulfite, thiosulfate, elemental sulfur and 9,10-anthraquinone 2,6-disulfonate . The G+C content of strain Z-9801T DNA was 36 mol% . 16S rDNA sequence analysis revealed that the isolated organism forms a separate branch within the Bacillus/Clostridium group . On the basis of physiological properties and phylogenetic analysis, it is proposed that strain Z-9801T (= DSM 14006T = UNIQEM 210T) should be assigned to a novel species of a new genus, Thermovenabulum ferriorganovorum gen . nov., sp . nov.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1729 - 35
Pelotomaculum thermopropionicum gen . nov., sp . nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium; Imachi H et al.; An anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium, strain SI(T), isolated previously from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor, was characterized . The strain could grow fermentatively on pyruvate and fumarate in pure culture . The strain grew on propionate, ethanol, lactate, 1-butanol, 1-pentanol, 1,3-propanediol, 1-propanol and ethylene glycol in co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain deltaH(T) . The optimum temperature for growth was 55 degrees C and the pH optimum was 7.0 . The G+C content of the DNA was 52.8 mol % . Strain SI(T) contained MK-7 and MK-7(H4) as the major quinones and contained iso-C15:0 as the major fatty acid . Based on 16S rDNA sequence analysis, strain SI(T) formed a novel lineage within the gram-positive, spore-forming, sulphate-reducing bacterial group Desulfotomaculum . However, the strain lacked the ability to conduct dissimilatory sulphate reduction . Instead, it could reduce fumarate to succinate with concomitant growth on several organic substances as electron donor . These phenotypic and genetic properties support the formation of a novel species of a new genus, for which the name Pelotomaculum thermopropionicum gen . nov., sp . nov . is proposed . The type strain is strain SI(T) (= DSM 13744T = JCM 10971T).

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1715 - 22
Petrotoga olearia sp . nov . and Petrotoga sibirica sp . nov., two thermophilic bacteria isolated from a continental petroleum reservoir in Western Siberia; L'Haridon S et al.; Strictly anaerobic, thermophilic bacteria (strains SL24T, SL25T, SL27, SL29 and SL32) were isolated from a deep, continental oil reservoir in Western Siberia (Russia) . These motile, rod-shaped organisms were surrounded by a sheath-like structure, a feature characteristic of the Thermotogales . On the basis of partial 16S rDNA sequences (500 nucleotides), strains SL25T, SL27, SL29 and SL32 were identical . Therefore, only strains SL24T and SL25T were studied in detail . The optimum temperature for growth of both strains was 55 degrees C . Their optimum pH for growth was 7.5 and their optimum NaCl concentration was between 20 and 30 g l(-1) . The novel isolates reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide . The G+C contents of the genomic DNA of strains SL24T and SL25T were respectively 35 and 33 mol% . Phylogenetically, both strains are most closely related to Petrotoga miotherma, there being 98.9-99.4% similarity between their 16S rDNA sequences . Phenotypic properties and DNA-DNA hybridization experiments indicate that the strains belong to two novel species, for which the names Petrotoga olearia (type strain SL24T = DSM 13574T = JCM 11234T) and Petrotoga sibirica (type strain SL25T= DSM 13575T = JCM 11235T) are proposed.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1675 - 80
Thermanaeromonas toyohensis gen . nov., sp . nov., a novel thermophilic anaerobe isolated from a subterranean vein in the Toyoha Mines; Mori K et al.; A novel thermophilic, strictly anaerobic, thiosulfate-reducing bacterium, designated strain ToBE(T), was isolated from a geothermal aquifer at a depth of 550 m in the Toyoha Mines (Hokkaido, Japan) . The cells of this bacterium were rod-shaped (0.6 x 2.6 microm), non-motile and sporulating . Strain ToBE(T) was able to grow on formate, lactate, pyruvate or various sugars in the presence of thiosulfate as an electron acceptor . The strain could grow at 55-73 degrees C and pH 5.5-8.5 . The optimum temperature and pH for the growth were 70 degrees C and pH 6.5 . The G+C content of the DNA was 49.6 mol % . The major quinone and cellular fatty acids were respectively menaquinone-7 and iso-C15:0 and iso-C17:0 . Analysis of the 16S rDNA revealed that the isolate was a member of the gram-positive bacteria and was related to the genus Thermoanaerobacter . However, the phylogenetic tree showed that the strain was distant from any other known bacteria, with sequence similarities of less than 90% . On the basis of phenotypic features and phylogenetic analysis, the name Thermanaeromonas toyohensis gen . nov., sp . nov., is proposed for the isolate, with strain ToBE(T) (= DSM 14490T = JCM 11376T) as the type strain.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1647 - 54
Meiothermus taiwanensis sp . nov., a novel filamentous, thermophilic species isolated in Taiwan; Chen MY et al.; Two novel filamentous bacterial isolates, strains WR-30T and WR-220, with an optimum growth temperature of approximately 55-60 degrees C were isolated from Wu-rai hot springs in the northern part of Taiwan . These isolates were aerobic, thermophilic, non-sporulating, red-pigmented and heterotrophic and formed extremely long, filamentous trichomes from cells of different lengths . Phylogenetic analysis of 16S rDNA, DNA-DNA hybridization, morphological and biochemical features and fatty acid composition revealed that the isolates represent a novel species of the genus Meiothermus . The name Meiothermus taiwanensis sp . nov . is proposed for this novel species . The type strain of M . taiwanensis is strain WR-30T (= ATCC BAA-399T = CCRC 17170T = DSM 14542T); strain WR-220 (= ATCC BAA400 = CCRC 17171 = DSM 14543) is a reference strain.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1621 - 8
Caminicella sporogenes gen . nov., sp . nov., a novel thermophilic spore-forming bacterium isolated from an East-Pacific Rise hydrothermal vent; Alain K et al.; A novel thermophilic, anaerobic, strictly chemoorganoheterotrophic bacterium, designated as AM1114T, was isolated from a deep-sea hydrothermal vent sample from the East-Pacific Rise (EPR 13 degrees N) . The cells were long (3-10 microm) rods, motile with peritrichous flagella, and exhibited a gram-negative cell wall ultrastructure . In the late stationary phase of growth, cells formed an ovoid, refractile, terminal endospore . They grew at 45-65 degrees C inclusive (optimum 55-60 degrees C; doubling time approx . 45 min), at pH 4.5-8.0 inclusive (optimum pH 7.5-8.0) and at sea salt concentrations of 20-60 g l(-1) inclusive (optimum 25-30 g l(-1)) . Strain AM1114T was an obligately heterotrophic bacterium able to ferment a mixture of 20 amino acids, complex proteinaceous substrates (such as yeast extract, brain-heart infusion or peptone), and carbohydrates such as glucose, galactose or maltose . The main fermentation products on glucose/yeast extract/peptone/sulfur medium were hydrogen, carbon dioxide, butyrate, ethanol, acetate, formate and L-alanine . The G+C content of the genomic DNA (determined by thermal denaturation) was 24.2+/-1 mol% . Phylogenetic analyses of the 16S rRNA gene located the strain within cluster XI of the lineage encompassing the genus Clostridium and related genera (sensu Collins et al., 1994), in the bacterial domain . On the basis of 16S rDNA sequence comparisons and physiological and biochemical characteristics, it is proposed that the isolate should be described as a novel genus, namely Caminicella gen . nov., of which Caminicella sporogenes sp . nov . is the type species . The type strain is AM1114T (= DSM 14501T = CIP 107141T).

Water Sci Technol, 2002, 46(4-5), 83 - 9
Use of thermophilic biological aerobic technology for industrial waste treatment; Rozich AF et al.; Thermophilic aerobic treatment systems offer unique advantages for treatment of high strength organic waste streams and slurries/sludges . These systems combine the best features of conventional aerobic and anaerobic processes that include rapid biodegradation kinetics and low biological solids production, respectively . Application of these processes can result in substantial economic benefit by reducing residuals processing and disposal costs . These systems have not been widely applied for industrial waste treatment, therefore the goal of this paper to show the advantages of applying thermophilic aerobic treatment to these streams . Also included in the paper is a discussion of the process benefits along with design/application considerations and industrial case histories.

Water Sci Technol, 2002, 46(4-5), 447 - 53
The contribution of thermophilic anaerobic digestion to the stable operation of wastewater sludge treatment; Zabranska J et al.; Thermophilic anaerobic digestion of sewage sludge has been successfully operated in full-scale tanks almost three years . The higher loading capacity and specific biogas production rate in comparison with mesophilic digestion was proved . Thermophilic anaerobic sludge is also more resistant against foaming problems . Biogas from thermophilic tanks contains less hydrogen sulphide and other malodorous substances . Pathogens removal rate is apparently more efficient in the thermophilic process.

Water Sci Technol, 2002, 46(4-5), 209 - 14
Effects of volatile fatty acids on a thermophilic anaerobic hydrogen fermentation process degrading peptone; Cheng SS et al.; Hydrogen fermentation using glucose as a single substrate caused abrupt pH drops and the gradual losses of hydrogen producers, which in turn led to system failure . In this study the use of a proteinaceous substrate, peptone, avoided the abrupt pH drops in the reactive system and allowed for further exploration of volatile fatty acids (VFAs) and pH effects on the hydrogen fermentation process . Our results showed that: (1) during the hydrogen fermentation tests, the abrupt pH drops were avoided thus system stability increased due to the production of ammonia from the peptone fermented, (2) pH control was not necessary and the addition of acetate to the process had little effect on the hydrogen fermentation process, (3) at the extreme pHs the addition of acetate either lengthened the lag phase (pH < or = 6) or slowed the hydrogen production rate (pH > or = 8), and both situations were not desired, and (4) high VFA content in the system sped up the consumption of hydrogen gas . Results of this study suggested that the hydrogen fermentation using the protein-containing substances as substrate was beneficial in maintaining the system pH . As long as the pH was maintained around 6-8, system inhibition due to VFAs accumulation was minimized . Thus, the optimal operation of a hydrogen fermentation process would be achievable via the control of substrate composition at a certain carbohydrate-to-protein ratio.

Biochem Biophys Res Commun, 2002 Oct 4, 297(4), 950 - 5
The beta subunit of Aquifex aeolicus leucyl-tRNA synthetase is responsible for cognate tRNA recognition; Gouda M et al.; The active form of the leucyl-tRNA synthetase from an extreme thermophile Aquifex aeolicus has a heterodimeric (alpha/beta type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases . In an attempt to clarify the individual roles of each subunit in the function of leucyl-tRNA synthetase, several elementary activities were separately measured using each of the subunits alone or the reconstructed alpha/beta complex . It was found that the beta subunit alone is capable of recognizing its cognate tRNA, while the leucyl-adenylate formation and the overall leucyl-tRNA formation are detected only when both of the subunit proteins coexisted.

J Biochem Mol Biol, 2002 Sep 30, 35(5), 498 - 507
Two flexible loops in subtilisin-like thermophilic protease, thermicin, from Thermoanaerobacter yonseiensis; Jang HJ et al.; A gene that encodes a thermostable protease, coined thermicin, has been isolated from Thermoanaerobacter yonseiensis that is expressed and characterized in E . coli . In order to elucidate the molecular characteristics on thermostability of the enzyme, molecular modeling and mutagenesis technology were applied . In the modeling structure, the structural core, including the active site, was well conserved; whereas, the two loop regions were unique when compared to thermitase . The mutant enzyme with the small loop deleted (D190-I196), based on modeling structural information, showed identical enzyme activity . However, when the large loop was deleted (P233-P244), a little lower K(m) and even a lower kcat was found . This indicates that the large loop could influence catalytic activity . However, the unfolding temperature (T(m)), which was determined by a differential-scanning calorimetry for the mutant enzyme deleted the small loop, was 96 degrees C . This is 14 degrees C lower than that for the parent thermicin . These results suggest that the small loop may play a role in maintaining the proper folding of the enzyme at high temperatures, whereas the large loop might be related to catalysis.

J Agric Food Chem, 2002 Oct 9, 50(21), 6017 - 22
Physicochemical properties and bacterial resistance of biodegradable milk protein films containing agar and pectin; Letendre M et al.; Free-standing sterilized edible films based on milk proteins, namely calcium caseinate and whey protein isolate, and polysaccharides, namely pectin and agar, were developed . Cross-linking of the proteins was achieved by the combination of thermal and radiative treatments . Autoclaving pectin and agar prior to their addition to the protein solutions generated films with an improved (P < or = 0.05) puncture strength . The presence of proteins and pectin-agar in the film formulation enhanced (P < or = 0.05) the moisture barrier of the films by 18% . A strain of Streptococcus thermophilus was used to assess the biodegradability behavior of the cross-linked films . Microbiological counts and soluble nitrogen analysis confirmed the biodegradability property of the milk protein films containing autoclaved pectin and agar.

J Cell Biol, 2002 Sep 30, 158(7), 1195 - 206
Tetrahymena thermophila contains a conventional gamma-tubulin that is differentially required for the maintenance of different microtubule-organizing centers; Shang Y et al.; The gene (GTU1) encoding Tetrahymena thermophila gamma-tubulin was cloned and analyzed . GTU1 is a single-copy, essential gene encoding a conventional gamma-tubulin . HA-tagged GTU1p localizes to four microtubule-organizing centers (MTOCs) in vegetative cells: basal bodies (BBs), macronuclear envelopes, micronuclear envelopes, and contractile vacuole pores . gamma-Tubulin function was studied by placing the GTU1 gene under control of an inducible-repressible promoter . Overexpression of GTU1 had no detectable effect on cell growth or morphology . Depletion of gamma-tubulin resulted in marked changes in cell morphology and in MT bundling . MTOCs showed different sensitivities to gamma-tubulin depletion, with BBs being the most sensitive . gamma-Tubulin was required not only for the formation of new BBs but also for maintenance of mature BBs . BBs disappeared in stages, first losing gamma-tubulin and then centrin and glutamylated tubulin . When GTU1 expression was reinduced in depleted cells, BBs reformed rapidly, and the normal, highly organized structure of the Tetrahymena cell cortex was reestablished, indicating that the precise patterning of the cortex can be formed de novo.

Protein Expr Purif, 2002 Oct, 26(1), 131 - 8
Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T . aquaticus-expression and purification; Dabrowski S et al.; Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya . We report here the identification, expression, and purification of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and T . aquaticus . The nucleotide (nt) sequence revealed that T . thermophilus SSB (TthSSB) and T . aquaticus (TaqSSB) consist of 264 and 266 amino acids, respectively, and have a molecular weight of 29.87 and 30.03kDa, respectively . The homology between these protein, is very high-82% identity and 90% similarity . They are the largest known prokaryotic SSB proteins . TthSSB and TaqSSB monomers have two putative ssDNA-binding sequences: N-terminal (located in the region from amino acids 1 to 123) and C-terminal (located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively) . PCR-derived DNA fragment containing the complete structural gene for TthSSB or TaqSSB protein was cloned into an expression vector . The clones expressing SSB-like proteins were selected and cloned DNA fragments were verified to be authentic by sequencing several clones . The purification was carried out using reduction of contamination by the host protein with heat treatment, followed by QAE-cellulose and ssDNA-cellulose column chromatography . We found our expression and purification system to be quite convenient and efficient, and will use it for production of thermostable SSB-proteins for crystallography study . We have applied the use of TthSSB and TaqSSB protein to increase the amplification efficiency with a number of diverse templates . The use of SSB protein may prove to be generally applicable in improving the PCR efficiency.

Biochemistry, 2002 Oct 8, 41(40), 12115 - 23
Different unfolding pathways for mesophilic and thermophilic homologues of serine hydroxymethyltransferase; Bhatt AN et al.; To determine how much information can be transferred from folding and unfolding studies of one protein to another member of the same family or between the mesophilic and thermophilic homologues of a protein, we have characterized the equilibrium unfolding process of the dimeric enzyme serine hydroxymethyltransferase (SHMT) from two sources, Bacillus subtilis (bsSHMT) and Bacillus stearothermophilus (bstSHMT) . Although the sequences of the two enzymes are highly identical ( approximately 77%) and homologous (89%), bstSHMT shows a significantly higher stability against both thermal and urea denaturation than bsSHMT . The GdmCl-induced unfolding of bsSHMT was found to be a two-step process with dissociation of the native dimer, resulting in stabilization of a monomeric species, followed by the unfolding of the monomeric species . A similar unfolding pathway has been reported for Escherichia coli aspartate aminotransferase, a member of the type I fold family of PLP binding enzymes such as SHMT, the sequence of which is only slightly identical ( approximately 14%) with that of SHMT . In contrast, for bstSHMT, a highly cooperative unfolding without stabilization of any monomeric intermediate was observed . These studies suggest that mesophilic proteins of the same structural family even sharing a low level of sequence identity may follow a common unfolding mechanism, whereas the mesophilic and thermophilic homologues of the same protein despite having a high degree of sequence identity may follow significantly different unfolding mechanisms.

Chemistry, 2002 Oct 4, 8(19), 4498 - 505
A novel open-chain nononic acid linked by an ether bond to glucose as a polysaccharide constituent; Faber EJ et al.; A novel sugar constituent was isolated from the heteropolysaccharide excreted by Streptococcus thermophilus 8 S when grown in skimmed milk . The structure and absolute configuration were determined by means of chemical analysis, mass spectrometry, NMR spectroscopy, along with molecular dynamics simulations, and was shown to be 6-O-(3',9'-dideoxy-D-threo-D-altro-nononic acid-2'-yl)-D-glucopyranose.

J Mol Evol, 2002 Oct, 55(4), 445 - 59
Molecular evolution of the lysine biosynthetic pathways; Velasco AM et al.; Among the different biosynthetic pathways found in extant organisms, lysine biosynthesis is peculiar because it has two different anabolic routes . One is the diaminopimelic acid pathway (DAP), and the other over the a-aminoadipic acid route (AAA) . A variant of the AAA route that includes some enzymes involved in arginine and leucine biosyntheses has been recently reported in Thermus thermophilus (Nishida et al . 1999) . Here we describe the results of a detailed genomic analysis of each of the sequences involved in the two lysine anabolic routes, as well as of genes from other routes related to them . No evidence was found of an evolutionary relationship between the DAP and AAA enzymes . Our results suggest that the DAP pathway is related to arginine metabolism, since the lysC, asd, dapC, dapE, and lysA genes from lysine biosynthesis are related to the argB, argC, argD, argE, and speAC genes, respectively, whose products catalyze different steps in arginine metabolism . This work supports previous reports on the relationship between AAA gene products and some enzymes involved in leucine biosynthesis and the tricarboxylic acid cycle (Irvin and Bhattacharjee 1998; Miyazaki et al . 2001) . Here we discuss the significance of the recent finding that several genes involved in the arginine (Arg) and leucine (Leu) biosynthesis participate in a new alternative route of the AAA pathway (Miyazaki et al . 2001) . Our results demonstrate a clear relationship between the DAP and Arg routes, and between the AAA and Leu pathways.

Curr Opin Microbiol, 2002 Oct, 5(5), 525 - 32
The origin of DNA genomes and DNA replication proteins; Forterre P; In recent years, it has became clear that most proteins involved in cellular DNA precursor synthesis or DNA replication have been 'invented' more than once, indicating that the transition from RNA to DNA genomes was more complex than previously thought . Several authors have suggested that DNA viruses, which often encode their own version of these proteins, played an important role in this process . The nature of the genome of the last universal cellular ancestor (LUCA) -- that is, RNA or DNA, prokaryotic-like or eukaryotic-like -- remains in dispute . A hyperthermophilic LUCA would have suggested a circular, double-stranded DNA genome; however, recent data favor a mesophilic or moderately thermophilic LUCA.

Eur J Biochem, 2002 Oct, 269(19), 4830 - 8
Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3 oxidase from T . thermophilus; Hellwig P et al.; The caa3-oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS and Fourier-transform infrared (FT-IR) spectroscopic approach . In this oxidase the electron donor, cytochrome c, is covalently bound to subunit II of the cytochrome c oxidase . Oxidative electrochemical redox titrations in the visible spectral range yielded a midpoint potential of -0.01 +/- 0.01 V (vs . Ag/AgCl/3m KCl, 0.218 V vs . SHE') for the heme c . This potential differs for about 50 mV from the midpoint potential of isolated cytochrome c, indicating the possible shifts of the cytochrome c potential when bound to cytochrome c oxidase . For the signals where the hemes a and a3 contribute, three potentials, = -0.075 V +/- 0.01 V, Em2 = 0.04 V +/- 0.01 V and Em3 = 0.17 V +/- 0.02 V (0.133, 0.248 and 0.378 V vs . SHE', respectively) could be obtained . Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of -0.22 V +/- 0.01 V for heme a3-CN- and of Em2 = 0.00 V +/- 0.02 V and Em3 = 0.17 V +/- 0.02 V for heme a (-0.012 V, 0.208 V and 0.378 V vs . SHE', respectively) . The three phases of the potential-dependent development of the difference signals can be attributed to the cooperativity between the hemes a, a3 and the CuB center, showing typical behavior for cytochrome c oxidases . A stronger cooperativity of CuB is discussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping . We thus studied the FT-IR spectroscopic properties of this enzyme to identify alternative protonatable sites . The vibrational modes of a protonated aspartic or glutamic acid at 1714 cm-1 concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases . Furthermore modes at positions characteristic for tyrosine vibrations have been identified . Electrochemically induced FT-IR difference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the nu(C=O) modes, which reflect the high degree of similarity of heme a3 to other typical heme copper oxidases . A comparison with previously studied cytochrome c oxidases is presented and on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3 upon electron transfer to/from the redox active centers discussed.

Syst Appl Microbiol, 2002 Aug, 25(2), 198 - 206
Rubritepida flocculans gen . nov., sp . nov., a new slightly thermophilic member of the alpha-1 subclass of the Proteobacteria; Alarico S et al.; A bacterial isolate, with an optimum growth temperature of about 50 degrees C, was recovered from the hot spring at Egerszalok in Hungary . Phylogenetic analyses using the 16S rRNA gene sequence of strain H-8T indicated that the new organism represented a new genus and species of alpha-1 subclass of the Proteobacteria . The major fatty acids of strain H-8T are 16:0, 18:1 omega7c; the rare fatty acid 19:0 20H cyclo 11,12 is also present . Ubiquinone 9 is the major respiratory quinone, the polar lipids are phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol in addition to two unidentified aminolipids . The new isolate forms red-colored colonies, flocculates in liquid media, is heterotrophic and strictly aerobic . Thiosulfate is oxidized to sulfate, but an increase in biomass could not be measured because of the flocculating behavior . Bacteriochloropyll a was detected by direct spectrophotometric analysis when the organism was grown at 30 degrees C, but could not be detected after growth at 50 degrees C . pufL and pufM genes were present . Heterotrophic growth of strain H-8T occurs on a few carbohydrates, amino acids and organic acids . On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strain H-8T represents a new genus and a new species most closely related to Roseococcus thiosulfatophilus for which we propose the name Rubritepida flocculans.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 1), 1729 - 33 Epub 2002 Sep 26.
Crystal contacts engineering of aspartyl-tRNA synthetase from Thermus thermophilus: effects on crystallizability; Charron C et al.; To understand how surface residues in a protein structure influence crystal growth, packing arrangement and crystal quality, crystal surfaces were modified and crystallizability of seven different mutants investigated . The model was aspartyl-tRNA synthetase-1 from Thermus thermophilus, a homodimer (M(r) 122000) with a subunit of 580 amino acids . Engineering concerned modification of amino acids involved in packing contacts in the orthorhombic lattice (P2(1)2121) of the synthetase . Comparison of the crystallization behaviour of the mutants indicates a correlation between disruption/addition of packing interactions and crystallizability of the mutants: disruption or modification of lattice contacts prevents crystallization or leads to crystals of poor quality . In contrast, addition of potential contacts leads to well-shaped crystals of same space group and cell parameters than wild-type crystals.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 1), 1674 - 80 Epub 2002 Sep 26.
From conventional crystallization to better crystals from space: a review on pilot crystallogenesis studies with aspartyl-tRNA synthetases; Lorber B et al.; Aspartyl-tRNA synthetases were the model proteins in pilot crystallogenesis experiments . They are homodimeric enzymes of Mr approximately 125 kDa that possess as substrates a transfer RNA, ATP and aspartate . They have been isolated from different sources and were crystallized either as free proteins or in association with their ligands . This review discusses their crystallisability with emphasis to crystal quality and structure determination . Crystallization in low diffusivity gelled media or in microgravity environments is highlighted . It has contributed to prepare high-resolution diffracting crystals with better internal order as reflected by their mosaicity . With AspRS from Thermus thermophilus, the better crystalline quality of the space-grown crystals within APCF is correlated with higher quality of the derived electron density maps . Usefulness for structural biology of targeted methods aimed to improve the intrinsic physical quality of protein crystals is highlighted.

J Biol Chem, 2002 Dec 6, 277(49), 47160 - 6 Epub 2002 Sep 25.
The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity; Beinker P et al.; ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system . The mechanism of protein reactivation and interaction with the DnaK system remains unclear . ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior . The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown . We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins . In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein . In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected . These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts . It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus . This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.

FEMS Microbiol Lett, 2002 Sep 10, 214(2), 271 - 5
A PCR-based method for identification of lactobacilli at the genus level; Dubernet S et al.; We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level . One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains . Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species . Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

Prikl Biokhim Mikrobiol, 2002 Jul-Aug, 38(4), 413 - 8
{Immunoenzyme analysis of decomposition of herbicides by soil and wood-rot fungi}; Koroleva OV et al.; The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicillium sp . 13 and noncellulolytic ones Humicola lanuginosa spp . 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26 . Detection of atrazine in liquid fungal cultures was performed by using enzyme immunoassay technique . Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp . 13) of fungal growth with atrazine were observed on solid agar media . Hyphomycete Mycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine . Neither of thermophilic strains was capable of atrazine consumption in three-week cultivation . In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80-90% in 40 days . Mycelia sterilia INBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days . The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.

Chembiochem, 2002 Jul 2, 3(7), 604 - 17
Close-range electrostatic interactions in proteins; Kumar S et al.; Two types of noncovalent bonding interactions are present in protein structures, specific and nonspecific . Nonspecific interactions are mostly hydrophobic and van der Waals . Specific interactions are largely electrostatic . While the hydrophobic effect is the major driving force in protein folding, electrostatic interactions are important in protein folding, stability, flexibility, and function . Here we review the role of close-range electrostatic interactions (salt bridges) and their networks in proteins . Salt bridges are formed by spatially proximal pairs of oppositely charged residues in native protein structures . Often salt-bridging residues are also close in the protein sequence and fall in the same secondary structural element, building block, autonomous folding unit, domain, or subunit, consistent with the hierarchical model for protein folding . Recent evidence also suggests that charged and polar residues in largely hydrophobic interfaces may act as hot spots for binding . Salt bridges are rarely found across protein parts which are joined by flexible hinges, a fact suggesting that salt bridges constrain flexibility and motion . While conventional chemical intuition expects that salt bridges contribute favorably to protein stability, recent computational and experimental evidence shows that salt bridges can be stabilizing or destabilizing . Due to systemic protein flexibility, reflected in small-scale side-chain and backbone atom motions, salt bridges and their stabilities fluctuate in proteins . At the same time, genome-wide, amino acid sequence composition, structural, and thermodynamic comparisons of thermophilic and mesophilic proteins indicate that specific interactions, such as salt bridges, may contribute significantly towards the thermophilic-mesophilic protein stability differential.

Appl Environ Microbiol, 2002 Oct, 68(10), 4812 - 9
The exopolyphosphatase gene from sulfolobus solfataricus: characterization of the first gene found to be involved in polyphosphate metabolism in archaea; Cardona ST et al.; Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms . Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain . We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus . The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa) . The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60 degrees C . The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs . In contrast, informatic analysis revealed that the deduced amino acid sequence of S . solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs . To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the ARCHAEA:

Cell, 2002 Sep 20, 110(6), 689 - 99
Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena; Mochizuki K et al.; During development of the somatic macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres . We identified a gene, TWI1, in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination . We also found that small RNAs were specifically expressed prior to chromosome rearrangement during conjugation . These RNAs were not observed in TWI1 knockout cells and required PDD1, another gene required for rearrangement, for expression . We propose that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing.

J Biochem Mol Biol, 2002 May 31, 35(3), 320 - 9
Cloning, expression, and characterization of thermostable DNA polymerase from Thermoanaerobacter yonseiensis; Kim DJ et al.; A gene, coined tay, for a thermostable DNA polymerase from the novel, extremely thermophilic bacterium Thermoanaerobacter yonseiensis was cloned and expressed in E . coli . Using a DNA polymerase homologous PCR product as a hybridization probe, tay was isolated and sequenced to consist of 2,616 nucleotides that encode 872 amino acids . A database analysis showed that DNA polymerase, coined Tay, from T . yonseiensis shared a 39 percent to 47 percent identity in the amino acid sequence with those from other DNA polymerases . Tay was overexpressed in E . coli as a fusion protein with a poly-histidine tag at the Cterminus . It was purified by heat treatment, followed by a Ni(2+)-chelate column . The molecular weight of purified Tay was approximately 97 kDa, as shown by SDS PAGE, and it showed high DNA polymerase activity and thermostability . However, it had no 3'-->5' exonuclease activity

J Am Chem Soc, 2002 Oct 2, 124(39), 11574 - 5
Influence of amino acid side chain packing on Fe-methionine coordination in thermostable cytochrome C; Yamamoto Y et al.; Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins . The strength of heme axial coordinations was found to correlate with the overall protein thermostability.

J Biotechnol, 2002 Oct 23, 99(2), 111 - 9
Characterization of a thermostable levansucrase from Bacillus sp . TH4-2 capable of producing high molecular weight levan at high temperature; Ben Ammar Y et al.; A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp . from Thailand soil . The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies . The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa . This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability . The optimum temperature and pH were 60 degrees C and 6.0, respectively . The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+) . The optimum temperature for levan production was 50 degrees C . Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C .

Biochem Biophys Res Commun, 2002 Sep 27, 297(3), 616 - 24
Specific binding of a protein to a novel DNA element in the cyanobacterial small heat-shock protein gene; Kojima K et al.; Previously, it was shown that transcription of the small heat-shock protein gene, hspA, from the thermophilic cyanobacterium Synechococcus vulcanus is transiently heat-inducible at a vegetative promoter that lacks any known regulatory DNA elements . A novel regulatory mechanism that suppresses the expression of hspA under non-heat-shock condition has been postulated . In this study, it is demonstrated that a protein(s) in the extract of unstressed cells of the thermophilic cyanobacterium Thermosynechococcus elongatus, a cyanobacterium closely related to S . vulcanus, specifically binds to a 5'-untranslated region of the hspA gene . An AT-rich imperfect inverted-repeat sequence (ACAAgcAAA-TTTagTTGT) as a target for a putative DNA-binding protein has been identified . The DNA-binding activity in the cell as well as in the cell extract was lost much more quickly at a heat-shock temperature than a normal growth temperature . In a cell, the activity was restored within 45 min after a heat-shock by the heat-induced synthesis and stabilization of a DNA-binding protein . It is proposed that the inverted repeat is a specific target for a DNA-binding protein and that it plays a role in the regulation of the cyanobacterial hspA gene expression.

Nat Struct Biol, 2002 Oct, 9(10), 750 - 5
All-atom homology model of the Escherichia coli 30S ribosomal subunit; Tung CS et al.; Understanding the structural basis of ribosomal function requires close comparison between biochemical and structural data . Although a large amount of biochemical data are available for the Escherichia coli ribosome, the structure has not been solved to atomic resolution . Using a new RNA homology procedure, we have modeled the all-atom structure of the E . coli 30S ribosomal subunit . We find that the tertiary structure of the ribosome core, including the A-, P- and E-sites, is highly conserved . The hypervariable regions in our structure, which differ from the structure of the 30S ribosomal subunit from Thermus thermophilus, are consistent with the cryo-EM map of the E . coli ribosome.

Avian Dis, 2002 Jul-Sep, 46(3), 542 - 6
Enhancement of chicken resistance against Escherichia coli infection by oral administration of Bifidobacterium thermophilum preparations; Kobayashi C et al.; Three types of Bifidobacterium thermophilum extract were prepared and fed to 2-wk-old chickens to evaluate their usefulness in enhancing the defense activity of the chickens against pathogenic Escherichia coli . All three preparations resulted in significant reduction (P < 0.05) of E . coli numbers in the lungs of the treated chicken groups compared with the control nontreated group . Besides, improvement in the survival rate was observed in the treated chicken groups, especially the one administered the enzyme-digested B . thermophilum extract sample . Concanavalin A-stimulated lymphocytes from the latter group demonstrated significantly higher proliferation activity compared with those from the control group . These results suggest that oral administration of B . thermophilum preparations may be used to enhance the resistance of chickens against E . coli infection.

Gene, 2002 Jul 24, 295(1), 1 - 6
Alteration of cell morphology and viability in a recA mutant of Streptococcus thermophilus upon induction of heat shock and nutrient starvation; Giliberti G et al.; We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation . Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses . Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S . thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.

DNA Res, 2002 Aug 31, 9(4), 123 - 30
Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1; Nakamura Y et al.; The entire genome of a thermophilic unicellular cyanobacterium, Thermosynechococcus elongatus BP-1, was sequenced . The genome consisted of a circular chromosome 2,593,857 bp long, and no plasmid was detected . A total of 2475 potential protein-encoding genes, one set of rRNA genes, 42 tRNA genes representing 42 tRNA species and 4 genes for small structural RNAs were assigned to the chromosome by similarity search and computer prediction . The translated products of 56% of the potential protein-encoding genes showed sequence similarity to experimentally identified and predicted proteins of known function, and the products of 34% of these genes showed sequence similarity to the translated products of hypothetical genes . The remaining 10% lacked significant similarity to genes for predicted proteins in the public DNA databases . Sixty-three percent of the T . elongatus genes showed significant sequence similarity to those of both Synechocystis sp . PCC 6803 and Anabaena sp . PCC 7120, while 22% of the genes were unique to this species, indicating a high degree of divergence of the gene information among cyanobacterial strains . The lack of genes for typical fatty acid desaturases and the presence of more genes for heat-shock proteins in comparison with other mesophilic cyanobacteria may be genomic features of thermophilic strains . A remarkable feature of the genome is the presence of 28 copies of group II introns, 8 of which contained a presumptive gene for maturase/reverse transcriptase . A trace of genome rearrangement mediated by the group II introns was also observed.

J Inorg Biochem, 2002 Sep 20, 91(4), 491 - 501
Thermophilic cytochrome P450 (CYP119) from Sulfolobus solfataricus: high resolution structure and functional properties; Park SY et al.; Crystal structures of a thermostable cytochrome P450 (CYP119) and a site-directed mutant, (Phe24Leu), from the acidothermophilic archaea Sulfolobus solfataricus were determined at 1.5-2.0 A resolution . We identify important crystallographic waters in the ferric heme pocket, observe protein conformational changes upon inhibitor binding, and detect a unique distribution of surface charge not found in other P450s . An analysis of factors contributing to thermostability of CYP119 of these high resolution structures shows an apparent increase in clustering of aromatic residues and optimum stacking . The contribution of aromatic stacking was investigated further with the mutant crystal structure and differential scanning calorimetry.

Tsitologiia, 2002, 44(6), 561 - 9
{Internal symmetry of the mirror type in the primary structure of ribozymes}; Shpakov AO; Internal symmetry of the mirror type has been first found in molecules of RNA referred to as ribozymes . For identification and investigation of the internal symmetry in RNA primary structure, two methods were developed, dot matrix and scanning, respectively . The methods are based on a comparison of normal and reversible nucleotide sequences . The objects of our study was ribozymes from introns of Tetrahymena thermophila and hepatitis delta virus, and also a group of related ribozymes, possessing both cleavage and ligation activities . The centers of internal symmetry are mainly localized in the catalytic sites and other important regions of ribozyme molecules . A positive correlation was found between the conservativity and symmetry of the primary structure of ribozymes . However, this correlation was not so clear in comparison with the correlation in the case of proteins . As reported earlier (Shpakov, 1995, 2001), the internal symmetry was discovered in protein and DNA molecules . The obtained data enable us to consider the internal symmetry as a common characteristics of nucleotide and amino acid sequences of the biopolymers.

EMBO Rep, 2002 Oct, 3(10), 982 - 7 Epub 2002 Sep 13.
Composition of the central stalk of the Na+-pumping V-ATPase from Caloramator fervidus; Chaban Y et al.; The Na+-pumping V-ATPase complex of the thermophilic bacterium Caloramator fervidus was purified and dissociated under controlled conditions . The structure of purified V1-ATPase subcomplexes differing in subunit composition was analyzed by electron microscopy and single particle analysis of 50 000 projections . Difference mapping of subcomplex projections revealed the presence and position of two subunits in the central stalk . A density with an elongated shape similar to the gamma subunit of F-ATPases is partly located within V1 and corresponds, most likely, to subunit E . Subunit E is connected to the membrane-bound part V0 via subunit C, a spherical density that is connected to the center of V0 . The presence of subunit C makes the central stalk substantially longer in comparison to the F-ATPases, in which the gamma subunit connects directly to F0.

Appl Microbiol Biotechnol, 2002 Sep, 59(6), 737 - 45 Epub 2002 Jun 22.
Isolation and characterization of a moderate thermophile, Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization; Kayser KJ et al.; An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C . Similar to other biodesulfurization-competent organisms, M . phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC . The specific desulfurization activity of the 50 degrees C M . phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1) . M . phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth . The dszABC operon of M . phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8 . The presence of the R . erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M . phlei GTIS10 . Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R . erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M . phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C . In M . phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase . The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile . These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.

Appl Microbiol Biotechnol, 2002 Sep, 59(6), 672 - 8 Epub 2002 Jul 26.
Redox-mediated decolorization of synthetic dyes by fungal laccases; Claus H et al.; Laccases from the lignin-degrading basidiomycetes Trametes versicolor, Polyporus pinisitus and the ascomycete Myceliophthora thermophila were found to decolorize synthetic dyes to different extents . Differences were attributed to the specific catalytic properties of the individual enzymes and to the structure of the dyes . Due to their higher oxidative capacities, the laccases from the two basidiomycetes decolorized dyes more efficiently than that of the ascomycete . The azo dye Direct Red 28, the indigoid Acid Blue 74 and anthraquinonic dyes were directly enzymatically decolorized within 16 h . The addition of 2 mM of the redox-mediator 1-hydroxybenzotriazole further improved and facilitated the decolorization of all nine dyes investigated . Laccases decolorized dyes both individually and in complex mixtures in the presence of bentonite or immobilized in alginate beads . Our data suggest that laccase/mediator systems are effective biocatalysts for the treatment of effluents from textile, dye or printing industries.

J Mol Biol, 2002 Sep 20, 322(3), 635 - 44
L22 ribosomal protein and effect of its mutation on ribosome resistance to erythromycin; Davydova N et al.; The ribosomal protein L22 is a core protein of the large ribosomal subunit interacting with all domains of the 23S rRNA . The triplet Met82-Lys83-Arg84 deletion in L22 from Escherichia coli renders cells resistant to erythromycin which is known as an inhibitor of the nascent peptide chain elongation . The crystal structure of the Thermus thermophilus L22 mutant with equivalent triplet Leu82-Lys83-Arg84 deletion has been determined at 1.8A resolution . The superpositions of the mutant and the wild-type L22 structures within the 50S subunits from Haloarcula marismortui and Deinococcus radiodurans show that the mutant beta-hairpin is bent inward the ribosome tunnel modifying the shape of its narrowest part and affecting the interaction between L22 and 23S rRNA . 23S rRNA nucleotides of domain V participating in erythromycin binding are located on the opposite sides of the tunnel and are brought to those positions by the interaction of the 23S rRNA with the L22 beta-hairpin . The mutation in the L22 beta-hairpin affects the orientation and distances between those nucleotides . This destabilizes the erythromycin-binding "pocket" formed by 23S rRNA nucleotides exposed at the tunnel surface . It seems that erythromycin, while still being able to interact with one side of the tunnel but not reaching the other, is therefore unable to block the polypeptide growth in the drug-resistant ribosome.

Can J Microbiol, 2002 Jul, 48(7), 626 - 34
The evaluation of mixtures of yeast and potato extracts in growth media for biomass production of lactic cultures; Gaudreau H et al.; The effectiveness of yeast extracts (YE) and potato extracts (PE) to promote growth of seven lactic cultures was evaluated by automated spectrophotometry (AS) . Two aspects of the growth curve were analysed: (1) maximum biomass obtained (using ODmax) and (2) highest specific growth rate mu(max)) Eleven lots from the same PE-manufacturing process were examined for lot-to-lot variability . The ODmax values of three of the seven strains were significantly affected by lot source, but mu(max) was not significantly affected . The growth of bacteria was systematically lower in base medium containing 100% PE than in base medium containing 100% YE for both ODmax or mu(max) data, which could be related to the lower content in nitrogen-based compounds in PE . In AS assays, highest OD values for Lactobacillus casei EQ28, Lactobacillus rhamnosus R-011, Lactobacillus plantarum EQ12, and Streptococcus thermophilus R-083 were obtained with a mixture of PE and YE . Fermentations (2 L) were also carried out to determine the accuracy of AS to predict biomass levels obtained under fermentation trials . In these fermentations, replacement of 50% YE with PE was shown to enable good growth of S . thermophilus . With L . rhamnosus R-011, a high correlation (R2 = 0.95) was found between ODmax data obtained in the AS assays and that of the 2-L bioreactor when the same growth medium was used for both series of fermentations . However, AS was not as efficient when industrial media were used for the bioreactor assays . The relationship was still good for ODmax between AS data and that of the bioreactor data with L . rhamnosus R-011 in industrial LBS medium (R2 = 0.87), but was very poor with the S . thermophilus R-083 on Rosell #43 industrial medium (R2 = 0.33) . Since PE cost 40% less than YE, there are strong economic advantages in considering such a partial replacement of YE by PE.

Plant Physiol, 1997 May, 114(1), 231 - 236
Characterization and Purification of an Aldose Reductase from the Acidophilic and Thermophilic Red Alga Galdieria sulphuraria; Gross W et al.; The acidophilic and thermophilic red alga Galdieria sulphuraria is able to grow heterotrophically on at least six different pentoses . These pentoses are reduced in the cell to pentiols by an NADP-dependent aldose reductase . The pentiols are then introduced into the oxidative pentose phosphate pathway via NAD-dependent polyol dehydrogenases and pentulokinases . The aldose reductase was purified 130-fold to apparent homogeneity by column chromatography . The enzyme is a homodimer of about 80 kD, as estimated by size-exclusion chromatography and from the sedimentation behavior . The Michaelis constant values for D-xylose (27 mM), D-ribose (29 mM), D-lyxose (30 mM), and D-arabinose (38 mM) were about three to five times lower than for the L-forms of the sugars . The activity of the enzyme with hexoses, deoxysugars, and sugar phosphates was only about 5 to 10% of the rate with pentoses . In the reverse reaction the activity was low and only detectable with pentiols . No activity was measured with NAD(H) as the cosubstrate in either direction.

Environ Microbiol, 2002 Sep, 4(9), 501 - 9
Compatible solutes of organisms that live in hot saline environments; Santos H et al.; The accumulation of organic solutes is a prerequisite for osmotic adjustment of all microorganisms . Thermophilic and hyperthermophilic organisms generally accumulate very unusual compatible solutes namely, di-myo-inositol-phosphate, di-mannosyl-di-myo-inositol-phosphate, di-glycerol-phosphate, mannosylglycerate and mannosylglyceramide, which have not been identified in bacteria or archaea that grow at low and moderate temperatures . There is also a growing awareness that some of these compatible solutes may have a role in the protection of cell components against thermal denaturation . Mannosylglycerate and di-glycerol-phosphate have been shown to protect enzymes and proteins from thermal denaturation in vitro as well, or better, than compatible solutes from mesophiles . The pathways leading to the synthesis of some of these compatible solutes from thermophiles and hyperthermophiles have been elucidated . However, large numbers of questions remain unanswered . Fundamental and applied interest in compatible -solutes and osmotic adjustment in these organisms, drives research that, will, in the near future, allow us to understand the role of compatible solutes in osmotic protection and thermoprotection of some of the most fascinating organisms known on Earth.

Biochim Biophys Acta, 2002 Sep 10, 1555(1-3), 111 - 5
Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry; van Montfort BA et al.; A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated . For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa . As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) . The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete . The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.

J Protein Chem, 2002 Jul, 21(5), 333 - 7
Substrate size selectivity of 20S proteasomes: analysis with variable-sized synthetic substrates; Hortin GL et al.; Proteasomes are tubular complexes with proteolytic activities on their lumenal surfaces so that large substrates should be sterically hindered from reaching the catalytic sites . Here we examine effects of substrate size on rates of cleavage by 20S proteasomes of Methanosarcina thermophila . Synthetic chromogenic substrates of variable size were prepared by linking a constant substrate group (Ala-Ala-Phe-p-nitroanilide) to a linear polymer (methoxypolyethylene glycol) with variable chain length . The smallest macromolecular substrates were cleaved more efficiently than free tripeptide substrate, and cleavage of macromolecular substrates was saturable, whereas cleavage of free tripeptide substrate was not, indicating mechanistic differences between the cleavage of large and small substrates . Rates of macromolecular substrate cleavage decreased progressively up to 10-fold as the size of the polymeric component of substrates increased . Macromolecular synthetic substrates appear to be better models of proteasome action on natural protein substrates and demonstrate substrate size selectivity of proteasomes.

Extremophiles, 2002 Aug, 6(4), 325 - 31 Epub 2002 Mar 26.
Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from Alicyclobacillus acidocaldarius and Archaeoglobus fulgidus by random mutagenesis and screening; Manco G et al.; The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively . The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop . In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction . The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations . Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.

Extremophiles, 2002 Aug, 6(4), 319 - 23 Epub 2002 Mar 26.
Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile, Bacillus pallidus; Bustard MT et al.; The aerobic biodegradation of high-concentration, to 24 g l(-1), 2-propanol (IPA) by a thermophilic isolate ST3, identified as Bacillus pallidus, was successfully carried out for the first time . This solvent-tolerant B . pallidus utilized IPA as the sole carbon source within a minimal salts medium . Cultivation was carried out in 100-ml shake flasks at 60 degrees C and compared with cultivation within a 1-l stirred tank reactor (STR) . Specific growth rate (micro) was about 0.2 h(-1) for both systems, with a maximum cell density of 2.4 x 10(8) cells ml(-1) obtained with STR cultivation . During exponential growth and stationary phase, IPA biodegradation rates were found to be 0.14 and 0.02 g l(-1) h(-1), respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l(-1) h(-1) were achievable in the STR . Generation of acetone, the major intermediate in aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization . Acetone levels reached a maximum of 2.2-2.3 g l(-1) after 72 and 58 h for 100-ml and 1-l systems, respectively . Both IPA and acetone were completely removed from the medium following 160 and 175 h, respectively, during STR growth, although this was not demonstrated within shake-flask reactions . Growth of B . pallidus on acetone or IPA alone demonstrated that the maximum growth rate ( micro ) obtainable was 0.247 h(-1) at 4 g l(-1) acetone and 0.202 h(-1) at 8 g l(-1) IPA within shake-flask cultivation . These results indicate the potential of the solvent-tolerant thermophile B . pallidus ST3 in the bioremediation of hot solvent-containing industrial waste streams.

Extremophiles, 2002 Aug, 6(4), 301 - 8 Epub 2002 Mar 19.
Isolation of a novel Thermus thermophilus metal efflux protein that improves Escherichia coli growth under stress conditions; Spada S et al.; The mechanisms of metal ion transport in thermophilic organisms are poorly understood . Phage display-based screening of a Thermus thermophilus genomic library in Escherichia coli led to the identification of a novel metal cation efflux protein . The Thermus protein showed extensive sequence and putative structural conservation to Czr and Czc proteins in mesophilic bacterial and mammalian species . Expression of the gene in E . coli led to increased resistance to zinc and cadmium ions, but not to cobalt, in an effect that was apparently caused by increased efflux of metals from the cell . This increased resistance was inducible by zinc and cadmium and, to a lesser extent, by cobalt . Furthermore, E . coli cells containing the Thermus gene exhibited improved cell physiology and delayed cell lysis during recombinant protein production, leading to accumulation of higher levels of recombinant protein . The molecular basis and potential application of the findings are discussed.

Extremophiles, 2002 Aug, 6(4), 291 - 9 Epub 2002 Mar 15.
Cloning and transcriptional analysis of the Thermoanaerobacter ethanolicus strain 39E maltose ABC transport system; Jones CR et al.; Thermoanaerobacter ethanolicus strain 39E is a Gram-positive thermophile that converts sugars resulting from plant carbohydrate polymer degradation into ethanol . A putative maltose ATP-binding cassette (ABC) transport operon was isolated with genes encoding for the integral membrane components (malF and malG); the ATP-binding protein (malK); and a partial gene for the maltose-binding protein (malE) . This operon is unlike most other maltose transport operons, which do not contain a contiguous malK gene . Sequence analysis showed that the individual genes in the putative operon possessed a considerable range of similarities to their respective homologs in other eubacteria and archaea . MalK had 52% amino-acid identity and over 70% similarity with its homolog from the archaeon Thermococcus litoralis, while the membrane components and binding protein exhibited much less similarity with a range of other thermophilic eubacteria . Transcript was not detected in maltose-, glucose-, or xylose-grown cells using Northern blotting, but RT-PCR showed that malFGK were expressed in cells grown on maltose or xylose . Based on these results, the strain 39E maltose operon may be subject to glucose catabolite repression.

Extremophiles, 2002 Aug, 6(4), 283 - 9 Epub 2002 Mar 26.
Cloning, expression, and characterization of the gsdA gene encoding thermophilic glucose-6-phosphate dehydrogenase from Aquifex aeolicus; Iyer RB et al.; The gsdA gene of the extreme thermophilic bacterium Aquifex aeolicus, encoding glucose-6-phosphate dehydrogenase (G6PDH), was cloned into a high-expression vector and overexpressed as a fusion protein in Escherichia coli . Here we report the characterization of this recombinant thermostable G6PDH . G6PDH was purified to homogeneity by heat precipitation followed by immobilized metal affinity chromatography on a nickel-chelate column . The data obtained indicate that the enzyme is a homodimer with a subunit molecular weight of 55 kDa . G6PDH followed Michaelis-Menten kinetics with a K(M) of 63 micro M for glucose-6-phosphate at 70 degrees C with NADP as the cofactor . The enzyme exhibited dual coenzyme specificity, although it showed a preference in terms of k(cat)/ K(M) of 20.4-fold for NADP over NAD at 40 degrees C and 5.7-fold at 70 degrees C . The enzyme showed optimum catalytic activity at 90 degrees C . Modeling of the dimer interface suggested the presence of cysteine residues that may form disulfide bonds between the two subunits, thereby preserving the oligomeric integrity of the enzyme . Interestingly, addition of dithiothreitol or mercaptoethanol did not affect the activity of the enzyme . With a half-life of 24 h at 90 degrees C and 12 h at 100 degrees C, this is the most thermostable G6PDH described.

J Mol Biol, 2002 Sep 6, 322(1), 79 - 91
Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose; Hidaka M et al.; The beta-galactosidase from an extreme thermophile, Thermus thermophilus A4 (A4-beta-Gal), is thermostable and belongs to the glycoside hydrolase family 42 (GH-42) . As the first known structures of a GH-42 enzyme, we determined the crystal structures of free and galactose-bound A4-beta-Gal at 1.6A and 2.2A resolution, respectively . A4-beta-Gal forms a homotrimeric structure resembling a flowerpot . Each monomer has an active site located inside a large central tunnel . The N-terminal domain of A4-beta-Gal has a TIM barrel fold, as predicted from hydrophobic cluster analysis . The putative catalytic residues of A4-beta-Gal (Glu141 and Glu312) superimpose well with the catalytic residues of Escherichia coli beta-galactosidase . The environment around the catalytic nucleophile (Glu312) is similar to that in the case of E.coli beta-galactosidase, but the recognition mechanism for a substrate is different . Trp182 of the next subunit of the trimer constitutes a part of the active-site pocket, indicating that the trimeric structure is essential for the enzyme activity . Structural comparison with other glycoside hydrolases revealed that many features of the 4/7 superfamily are conserved in the A4-beta-Gal structure . On the basis of the results of 1H NMR spectroscopy, A4-beta-Gal was determined to be a "retaining" enzyme . Interestingly, the active site was similar with those of retaining enzymes, but the overall fold of the TIM barrel domain was very similar to that of an inverting enzyme, beta-amylase.

Eur J Clin Nutr, 2002 Sep, 56(9), 843 - 9
Long-term consumption of fermented dairy products over 6 months increases HDL cholesterol; Kiessling G et al.; OBJECTIVE: Assessment of the hypocholesterolaemic effect of yoghurt supplemented with Lactobacillus acidophilus 145 and Bifidobacterium longum 913 in women . DESIGN: The cross-over study consisted of three periods (7 weeks each): first period, control yoghurt for all 29 women; second period, probiotic yoghurt for 18 women, control yoghurt for 11 women; third period, the reverse of that in the second period . SETTING: Department of Nutritional Physiology, Institute of Nutritional Science, Friedrich Schiller University, Jena . SUBJECTS: Twenty-nine healthy women, aged 19-56 y . Fifteen of these were normocholesterolaemic and 14 women were hypercholesterolaemic . INTERVENTION: Yoghurt (300 g) daily containing 3.5% fat and starter cultures of Streptococcus thermophilus and L . lactis . Probiotic yoghurt was the control yoghurt enriched with L . acidophilus 145, B . longum 913 and 1% oligofructose (synbiotic) . RESULTS: The mean serum concentration of total cholesterol and the LDL cholesterol was not influenced by the synbiotic (P>0.05) . The HDL concentration increased significantly by 0.3 mmol/l (P=0.002) . The ratio of LDL/HDL cholesterol decreased from 3.24 to 2.48 (P=0.001) . CONCLUSIONS: The long-term daily consumption of 300 g yoghurt over a period of 21 weeks (control and synbiotic) increased the serum concentration of HDL cholesterol and lead to the desired improvement of the LDL/HDL cholesterol ratio.

Regul Toxicol Pharmacol, 2002 Jun, 35(3), 296 - 307
Toxicological studies on Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae; Brinch DS et al.; The bioindustrially produced enzyme laccase can be used in different technical and food applications to facilitate processes . It can be added to different oral care products such as mouthwash, toothpaste, mints, and gums to prevent halitosis . Laccase, produced by submerged fermentation of Aspergillus oryzae, containing a gene originating from Myceliophthora thermophila, was subject to a series of toxicological tests to document its safety in use . It was not found to be mutagenic in the Salmonella typhimurium reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes . No evidence of inhalation toxicity or skin and eye irritation was found . There was no evidence of possible skin sensitization in a human skin sensitization test when Laccase was tested at 10% (w/v): thus Laccase would appear to have a low skin sensitization potential . Oral administration to rats of up to 10.0 mL/kg body wt/day (equivalent to a total organic solids dosage of 1.72 g/kg body wt/day) for 13 weeks did not cause any adverse effect .

J Dairy Sci, 2002 Jul, 85(7), 1705 - 8
Direct observation of bacterial exopolysaccharides in dairy products using confocal scanning laser microscopy; Hassan AN et al.; The objective of this work was to develop a methodology for direct visualization of bacterial exopolysaccharides (EPS) in fully hydrated dairy products . The new method involved staining EPS with wheat germ agglutinin labeled with Alexa fluor 488 or staining with concanavalin A 488 . Samples were observed using confocal scanning laser microscopy . Distribution of EPS produced by Lactococcus lactis (CHCC 3367), a combination of Streptococcus thermophilus (CHCC 3534) and Lactobacillus delbrueckii ssp . bulgaricus (CHCC 769) and Lactobacillus delbrueckii ssp . bulgaricus RR in milk was compared in stirred and unstirred fermented milk . The EPS and proteins were observed as distinct entities, with EPS present in the protein network pores . EPS was observed in greater amounts in milk fermented by the ropy L . lactis culture than in milk fermented by the less ropy strain of S . thermophilus . Stirring the fermented milk caused aggregation of EPS into more extended structures . The more ropy the culture, the longer and larger the strands formed during stirring . The method was also applied to Feta cheese made with an EPS-producing strain of S . thermophilus . EPS was observed in the cheese as thick sheets filling pores in the protein network.

Water Sci Technol, 2002, 45(12), 335 - 8
Biological and chemical treatment of solid waste from soy sauce manufacture; Nagai H et al.; To reduce the solid waste in soy sauce refuse (SSR) that is a by-product of soy sauce fermentation, both biological and chemical treatment were examined . When anaerobic digestion of SSR was carried out at the amounts of 25 g/l under 30% (v/v) inocula of thermophilic methanogenic sludge at 50 degrees C, 120 mM of CH4 was produced and 50% (w/v) of mixed-liquor suspended solids in SSR was decreased after 35 days . Although in this culture 30 days' cultivation was required to get 100 mM of CH4, when the same amounts of SSR (25 g/l) were repeatedly added to the culture, the time requirement to get 100 mM of CH4 could be reduced to 20 days and 15 days at second and third batch cultures, respectively . When SSR was treated with 5% (w/v) NaOH for 24 h, the supernatant contained 98% (w/v) of protein that were alkaline-solubilized and insoluble in the intact refuse, and the residual pellet contained insoluble fiber.

Water Sci Technol, 2002, 45(12), 143 - 50
High-rate methane fermentation of lipid-rich food wastes by a high-solids co-digestion process; Li YY et al.; This paper presents an experimental study on anaerobic degradation of lipids-rich food wastes by using the high solids co-digestion process . The experiments were conducted under mesophilic (35 degrees C) and thermophilic (55 degrees C) condition, respectively, by using a semi-continuous flow completely mixed reactor . The influent TS level was controlled at around 10%, while the hydraulic retention time (HRT) was changed from 15 days to 7.5 days . The lipids (fats or oil and grease) content in the influent TS was changed from 8% to 40% by adding salad oil (vegetable) and lard (animal) to the food wastes . The result of this study showed that the food wastes containing high lipids content was effectively degraded by the high solids co-digestion process and over 85% of lipid was degraded to biogas with 60-65% of methane . In addition, thermophilic methane fermentation was more effective for reducing lipids and had more higher loading capacity compared with mesophilic condition.

Water Sci Technol, 2002, 45(12), 127 - 34
A strategy in wastewater treatment process for significant reduction of excess sludge production; Shiota N et al.; A novel wastewater treatment process (S-TE PROCESS) with significantly reduced production of excess sludge has been developed . The process consists of two different stages, one for a biological wastewater treatment and the other for a thermophilic aerobic digestion of the resulting sludge . A portion of return sludge from the wastewater treatment step is injected into a thermophilic aerobic sludge digester (TASD), in which the injected sludge is solubilized by the action of thermophilic aerobic bacteria . The solubilized sludge is returned to the aeration tank in the wastewater treatment step for its further degradation . Pilot-scale facilities of the S-TE process and the conventional activated sludge process as a control, both treating the same industrial wastewater, were comparatively operated for totally 270 days . As a result, 93% reduction in overall excess sludge production was achieved in the S-TE operation . The SS solubilization rate in TASD was stable at around 30% . Only a slight increase in the effluent SS and TOC concentrations was observed compared with those of the control facility . Otherwise the removal efficiency of TOC was approximately 95% for both plants . A full-scale plant treating domestic sewage was operated for three years, showing 75% reduction of overall excess sludge production . It was concluded that the new process was feasible.

Appl Environ Microbiol, 2002 Sep, 68(9), 4266 - 73
Albidovulum inexpectatum gen . nov., sp . nov., a nonphotosynthetic and slightly thermophilic bacterium from a marine hot spring that is very closely related to members of the photosynthetic genus Rhodovulum; Albuquerque L et al.; Several bacterial isolates, with an optimum growth temperature of about 50 degrees C, were recovered from the marine hot spring at Ferraria on the island of Sao Miguel in the Azores . The geothermal water emerged from a porous lava flow and rapidly cooled in contact with seawater except at low tide . The bacterial species represented by strains FRR-10(T) and FRR-11 was nonpigmented, strictly aerobic, and organotrophic . Several genes, bchZ, pufB, pufA, pufL, or pufM, encoding the photosynthetic reaction center proteins and the core light-harvesting complexes were not detected in these strains . The organism oxidized thiosulfate to sulfate with enhancement of growth . The organism did not require additional NaCl in the culture medium for growth, but NaCl at 1.0% enhanced growth . Phylogenetic analyses using the 16S rRNA gene sequence of strain FRR-10(T) indicated that the new organism represented a new species of the alpha-3 subclass of the Proteobacteria and that it branches within the species of the genus Rhodovulum . The contradiction of classifying an organism which branches within the radiation of the genus Rhodovulum but does not possess the hallmark characteristics of this genus is discussed . However, the absence of several of these characteristics, namely, the lack of photosynthesis and pigmentation, which could be related to colonization of dark environments, and growth at high temperatures, leads to our proposal that strains FRR-10(T) and FRR-11 should be classified as a new species of a novel genus, Albidovulum inexpectatum, representing, at present, the most thermophilic organism within the alpha-3 subclass of the Proteobacteria.

Acta Crystallogr D Biol Crystallogr, 2002 Sep, 58(Pt 9), 1497 - 500 Epub 2002 Aug 23.
Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus; Vassylyeva MN et al.; RNA polymerase holoenzyme from Thermus thermophilus, consisting of six protein subunits (alpha(2), beta, beta', omega and sigma(70)) and having a total molecular mass of about 450 kDa, was purified and crystallized by the hanging-drop vapour-diffusion technique under mild near-physiological conditions . The crystals diffract beyond 3 A resolution . Careful analysis of diffraction data revealed that the crystals belong to space group P3(2), with unit-cell parameters a = b = 236.35, c = 249.04 A, and have perfect twinning along the threefold axis . A complete data set at 3 A resolution was collected and an unambiguous molecular-replacement solution was found using the structure of T . aquaticus RNA polymerase core enzyme as a search model . The refinement of structure and model building of the sigma(70) subunit is now in progress.

Biochem Soc Trans, 2002 Aug, 30(4), 685 - 7
Sulfolobus aconitase, a regulator of iron metabolism?
Uhrigshardt H, Zoske S, Anemuller S.
The aconitase of Sulfolobus solfataricus, a hyperthermophilic crenarchaeon, was cloned and heterologously expressed in Escherichia coli . Enzymic analyses and EPR measurements indicated clearly that the iron-sulphur cluster of the thermophilic aconitase was already inserted in the mesophilic host . The enzyme was purified to a specific activity of approx . 44 units/mg and to 90% homogeneity . The enzymic parameters of the recombinant aconitase turned out to be in the same range as the respective values for the previously characterized native enzyme from the closely related S . acidocaldarius . Based on its primary sequence, the recombinant aconitase is closely related to bacterial A-like and to eukaryotic iron regulatory protein-like proteins . Specific aconitase activities in cytosolic extracts of S . acidocaldarius were found to be decreased markedly in iron-starved compared with iron-repleted cells . However, no differences in aconitase levels between iron-starved and iron-supplemented cells could be detected by immunostaining.

Proc Natl Acad Sci U S A, 2002 Sep 3, 99(18), 11664 - 9 Epub 2002 Aug 22.
Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline; Lesley SA et al.; Structural genomics is emerging as a principal approach to define protein structure-function relationships . To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures . We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima . By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T . maritima proteome . Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Arch Microbiol, 2002 Sep, 178(3), 229 - 37 Epub 2002 Jun 27.
Alkane-1,2-diol-based glycosides and fatty glycosides and wax esters in Roseiflexus castenholzii and hot spring microbial mats; van ver Meer MT et al.; The lipid composition of Roseiflexus castenholzii, a thermophilic filamentous phototrophic bacterium related to uncultivated filamentous phototrophic bacteria that predominate in hot spring microbial mats, is reported . R . castenholzii lipid extracts were dominated by components characterized by alkane-1-ol-2-alkanoate moieties glycosidically bonded to a C(6) sugar . Similar fatty glycosides, with an additional fatty acid esterified, were detected by HPLC-MS . R . castenholzii also produces a suite of wax esters ranging from 37 to 40 carbon atoms in length . In lipid extracts from two nonsulfidic hot spring microbial mats, similar alkane-1,2-diol-based lipids were detected in minor amounts . R . castenholzii lipids are compared to lipids of mats and other thermophilic mat isolates.

Curr Protein Pept Sci, 2002 Apr, 3(2), 223 - 30
Cold adaptation of archaeal elongation factor 2 (EF-2) proteins; Thomas T et al.; Cell growth at low temperature is dependent on the ability of cells to perform protein synthesis . Cold adapted micro-organisms (psychrophilic or psychrotolerant) have a superior ability to perform translation at low temperature . This review addresses cold adaptation of protein synthesis in Archaea by examining what is presently known about thermal adaptation of elongation factor 2 (EF-2) proteins from Archaea . Despite the knowledge that Archaea are abundant in cold environments (e.g . the ocean), few cold adapted species have been isolated and studied . As a result this review is largely confined to comparative analyses of EF-2 proteins from psychrotolerant (Methanococcoides burtonii) and thermophilic (Methanosarcina thermophila) methanogens . A key finding from these studies is that in addition to inherent properties of the EF-2 proteins, intracellular factors (e.g . ribosomes and intracellular solutes) play a central role in thermal adaptation.

Water Sci Technol, 2002, 45(10), 93 - 8
Modelling the competition between sulphate reducers and methanogens in a thermophilic methanol-fed bioreactor; Spanjer H et al.; Sulphate can be removed from wastewater by means of biological anaerobic reduction to sulphide . The reduction requires the presence of a substrate that can serve as an electron donor . Methanol a suitable electron donor for sulphate reduction under thermophilic conditions . In an anaerobic system containing methanol and sulphate, acetogenic bacteria (AB) and methanogenic archaea (MA) compete with sulphate reducing bacteria (SRB) for methanol or its degradation intermediates . Previously obtained results indicate that at 65 degrees C SRB and MA mainly compete for the intermediate hydrogen instead of methanol . For efficient use of methanol as electron donor for sulphate reduction it is important that for the treatment of sulphate wastewater in an anaerobic reactor SRB out-compete MA . The mechanisms that determine the outcome of the competition are, however, not well understood . This paper describes a model based on growth kinetics of methanol-oxidising AB, and hydrogen-consuming SRB and MA, that can describe the competition between SRB and MA in a methanol-fed bioreactor . We present the model and its calibration using experimental data, and we discuss its shortcomings and suggest possible improvements.

Water Sci Technol, 2002, 45(10), 293 - 8
State of the art and future perspectives of thermophilic anaerobic digestion; Ahring BK et al.; The state of the art of thermophilic digestion is discussed . Thermophilic digestion is a well established technology in Europe for treatment of mixtures of waste in common large scale biogas plants or for treatment of the organic fraction of municipal solid waste . Due to a large number of failures over time with thermophilic digestion of sewage sludge this process has lost its appeal in the USA . New demands on sanitation of biosolids before land use will, however, bring the attention back to the use of elevated temperatures during sludge stabilization . In the paper we show how the use of a start-up strategy based on the actual activity of key microbes can be used to ensure proper and fast transfer of mesophilic digesters into thermophilic operation . Extreme thermophilic temperatures of 65 degrees C or more may be necessary in the future to meet the demands for full sanitation of the waste material before final disposal . We show data of anaerobic digestion at extreme thermophilic temperatures.

Water Sci Technol, 2002, 45(10), 27 - 33
Evaluation of thermophilic anaerobic microbial consortia using fluorescence in situ hybridization (FISH); Domingues MR et al.; In this study we investigated the development of anaerobic biofilms in differential reactors and suspension cultures in batch reactors under thermophilic (55 degrees C) conditions . FISH, SEM, chemical and chromatographic analysis were used . The differential reactors reached 99.6%, 92.3% and 6.7% of acetic acid, COD and sulfate removal efficiencies, respectively, after 166 h of incubation time . The batch reactor reached 95.6% and 31.8% of acetic acid and sulfate removal efficiencies after 675 h, respectively . FISH results showed that bacterial cells rather than archaeal cells dominated biofilms . These cells, detected with the Bacteria specific probe (EUB338), accounted for 61.1% (+/-3.6) of the DAPI-stained cells and resembled acetate-oxidizing rods and Desulfotomaculum morphologies . Archaeal cells, which hybridized to the Archaea specific probe (ARC915), were also detected in biofilm but they accounted for 36.7% (+/-2.9) of the DAPI-stained cells . These cells were similar to Methanosaeta-like and hydrogenotrophic methanogen rods . In the suspension culture, archaeal cells (58.0%+/-3.8) morphologically similar to Methanosarcina and hydrogenotrophic methanogen rods were predominant over bacterial cells (41.0%+/-4.5), which resembled acetate-oxidizing rods and Desulfotomaculum morphologies . The percentage of sulfate-reducing bacteria cells (SRB) ranged from 12.2% (+/-2.5) to 21.7% (+/-2.8) in the biofilms and from 13.3% (+/-3.6) to 21.7% (+/-4.3) of the DAPI stained cells in suspension culture.

Water Sci Technol, 2002, 45(10), 231 - 5
Mesophilic and thermophilic anaerobic digestion of sulphate-containing wastewaters; Colleran E et al.; The effect of sulphate at an influent chemical oxygen demand (COD):sulphate ratio of 4 on the operational performance of anaerobic hybrid reactors treating molasses wastewater was investigated under mesophilic and thermophilic conditions in a long-term laboratory-scale study over a 1,081 day period . The presence of sulphate reduced the COD removal efficiency under both mesophilic and thermophilic conditions . At 55 degrees C, effluent acetate levels were consistently greater than 4000 mg l(-1) indicating that thermophilic acetate-utilising methane-producing bacteria (MPB) or sulphate-reducing bacteria (SRB) had not developed in the reactor under the conditions applied . At 37 degrees C, acetate was exclusively utilised by acetoclastic methanogens, whereas H2-utilising SRB predominated over H2-utilising MPB in the competition for hydrogen . By contrast, hydrogenotrophic MPB were shown to outcompete H2-utilising SRB during long-term thermophilic operation . 16SrDNA analysis of the seed sludge and reactor biomass on conclusion of the 37 degrees C and 55 degrees C trials illustrated that the dominant methanogen present on conclusion of the thermophilic trial in the absence of influent sulphate was related to Methanocorpusculum parvuum, and was capable of growth on both acetate and hydrogen . By contrast, an organism closely related to Methanobacterium thermoautotrophicum was the dominant methanogen present in the sulphate-fed reactor on completion of the thermophilic trial.

Water Sci Technol, 2002, 45(10), 225 - 30
Pilot-plant study on anaerobic treatment of a lipid- and protein-rich food industrial wastewater by a thermophilic multi-staged UASB reactor; Tagawa T et al.; An on-site pilot-scale experiment was conducted to investigate the performance of a multi-staged UASB (MS-UASB) reactor by feeding with a food processing wastewater containing high strength of lipid and protein . The reactor was operated at a thermophilic condition (55 degrees C) for a period of 600 days . The reactor finally achieved 50 kgCOD.m(-3) d(-1) with a soluble COD removal of 90% (based on the influent total COD versus the effluent filtered COD), while the overall COD removal (based on the effluent COD-total) as considerably unsatisfactory at around only 60-70% . The presence of high strength of lipid and protein along with high concentration of Mg and Ca ions in the raw wastewater caused a severe scum and/or insolubilized substance formation within the UASB sludge bed, resulting in hindering the contact efficiency between substrate and sludge . The replacement of active microbial granules in the sludge bed with the insolubilized protein and lipid brought about deterioration of sludge methanogenic activity.

Water Sci Technol, 2002, 45(10), 19 - 25
Molecular and conventional analyses of microbial diversity in mesophilic and thermophilic upflow anaerobic sludge blanket granular sludges; Sekiguchi Y et al.; The microbial community structure of mesophilic (35 degrees C) and thermophilic (55 degrees C) methanogenic granular sludges was surveyed by using both cultivation-independent molecular approach and conventional cultivation technique in order to address the fundamental questions on the microbial populations, i.e . who are present, where they are located, and what they are doing there . To elucidate the microbial constituents within both sludges, we first constructed 16S ribosomal DNA clone libraries, and partial sequencing of the clones was conducted for phylogenetic analysis . In this experiment, we found a number of unidentifiable clones within the domain Bacteria as well as clones that were closely related with 16S rDNAs of cultured microbes . The unidentifiable clones accounted for approximately 60-70% of the total clones in both mesophilic and thermophilic libraries . 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was subsequently employed to examine where the uncultured populations were located within sludge granules . Spatial organization of uncultured microbes was visualized in thin-sections of both types of granules using fluorescent oligonucleotide probes, which were designed based on the clone sequences of certain novel clusters . This resulted in the detection of two types of uncultured cells in specific locations inside the granules . Finally, the goal-directed conventional cultivation technique was employed to recover such uncultured anaerobes and uncover their physiology and functions . In this approach, a total of five new species of thermophilic microorganisms were isolated, including several types of syntrophs and a novel sugar-fermenting bacterium . In the previous molecular approaches, all of these isolates were suggested to be significant populations within thermophilic granular sludge, hence obtaining these isolates in pure culture decreased the fraction of unknown clones in the previous thermophilic clone library from 70% to 40% . In conclusion, these approaches successfully revealed biodiversity and spatial organization of microbes of interest in sludge granules, and enlarged the fundamental knowledge of microbial constituents functioning as significant populations in the UASB processes.

Water Sci Technol, 2002, 45(10), 145 - 50
Start-up of a thermophilic methanol-fed UASB reactor: change in sludge characteristics; Paulo PL et al.; Experiments were performed to study the change in sludge characteristics and sludge granulation during the start-up of a thermophilic methanol-fed upflow anaerobic sludge bed (UASB) reactor . The laboratory scale reactor, was inoculated with thermophilic granular sludge and operated at 55 degrees C over 130 days at organic loading rates (OLR) varying from 2.7 to 47 gCOD.L(-1).d(-1) . Physical characterisation was performed for both the seed and the cultivated sludge . Results demonstrated that a good quality, well settleable granular sludge was cultivated and retained in the reactor, allowing an OLR of 47 gCOD.L.d(-1) with 93% of methanol removal, where 79% was converted into methane . Using a community analysis of the cultivated consortia, high numbers of rod-shaped hydrogenotrophic methanogens were enumerated . Biomass washout coincided with a high specific gas load, but was not detrimental to the system in the conditions tested.

Water Sci Technol, 2002, 45(10), 121 - 6
Effect of high salinity on the fate of methanol during the start-up of thermophilic (55 degrees C) sulfate reducing reactors; Vallero MV et al.; Two 6.5 L lab-scale upflow anaerobic sludge bed (UASB) reactors were operated at 55 degrees C fed with methanol as the sole electron and carbon source and in excess of sulfate (COD/SO4(2-) of 0.5) in order to investigate the effect of high wastewater salinity on the start-up period . The first reactor (UASB I) was operated without NaCl addition, while the second reactor (UASB II) was fed with 25 g x L(-1) of NaCl in the first 13 days of operation . Successful start-up of UASB I was achieved, with full methanol conversion (100% elimination) to methane gas (methane production rate up to 3.66 gCOD.L(-1).day(-1)) . Despite the detection of sulfide from day 15 onwards in UASB I, methane was the main mineralization product when operating at an organic loading rate (OLR) of 5 gCOD.L(-1).day(-1) and a hydraulic retention time (HRT) of 10 hours . Sulfide and acetate started to be produced after salt omission from the influent in UASB II at day 13, with no detection of methane . Acetate was the main product when operating at an OLR of 10 gCOD.L(-1).day(-1) and HRT of 6.5 hours in both reactors . Apparently, the methane producing bacteria (MPB) are the trophic group most sensible to the NaCl shock.

Water Sci Technol, 2002, 45(10), 113 - 20
Ammonia inhibition on thermophilic aceticlastic methanogens; Liu T et al.; The inhibition effects of total ammonia nitrogen (TAN) on aceticlastic methanogenic activity using biomass from thermophilic anaerobic reactors were investigated in this study . Anaerobic Toxicity Assays (ATA) were conducted after the biomass was acclimated to different levels of TAN . The TAN background concentrations in the reactors were 400, 1,200, and 3,050 mg/L . The results from ATA showed: 1) high TAN concentrations could cause inhibition of aceticlastic methanogens; 2) biomass acclimated to higher TAN concentrations could alleviate the inhibition effect due to the increase of TAN concentration; 3) the lethal TAN concentration for methanogens was approximately 10,000 mg/L regardless of the background TAN concentration; 4) ATA results also revealed the role played by pH . At a given TAN concentration, methanogenic activity varied with the pH values . The highest methanogenic activity was always observed at a pH of 7.0-7.5 . 5) It was also observed that acclimation could increase the pH tolerance range, which made methanogens less sensitive to pH changes.

Water Res, 2002 Jul, 36(13), 3203 - 10
Microbial community dynamics during start-up of acidogenic anaerobic reactors; Liu WT et al.; Start-up of two acidogenic reactors under mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions was carried out with methanogenic granular sludge as an inoculum and dairy wastewater as feed . During these 71 days of the start-up period, microbial community dynamics in these two acidogenic reactors, as monitored by denaturing gradient gel electrophoresis (DGGE) and dot-blot hybridization with group-specific oligonucleotide probes, was correlated to reactor performance . Due to pH drop to 5.5, DGGE community fingerprints for domains Bacteria and Archaea populations showed significant shifts after 13 days of operation, and this change was accompanied with an increase in volatile fatty acid production, a decrease in methane formation, and rapid sludge disintegration . Dot-blot hybridization results further indicated that the decrease in methane production was related to the decrease in Archaea population in particular with methanogens from 34.1% of total 16S-rRNA in the seed sludge to 8% within the first 13 days, and to 2-5% at day 71 . Among the methanogens monitored, the class Methanomicrobiales was the most abundant followed up by Methanobacteriales and Methanococcales . Due to an elevated temperature, the microbial community change was more significant and rapid in the thermophilic reactor than in the mesophilic reactor . Significant microbial population changes took place at the first 13 days for both reactors, but a longer period up to 71 days was required to establish a microbial community with a stable metabolic activity.

Genome Biol . 2002 Jul 19;3(8):PREPRINT0006 . Epub 2002 Jul 19.
Preferred codons and amino acid couples in hyperthermophiles; De Farias ST et al.; BACKGROUND: 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 . What makes these cells so resistant to heat? Their biomolecules must be sufficiently stable, especially proteins, to work under these extreme conditions, but the bases for thermostability remains elusive . RESULTS: The preferential usage of certain couples of amino acids and codons in thermal adaptation was investigated, by comparative proteome analysis, using 28 complete genomes from 18 mesophiles, 4 thermophiles, and 6 hyperthermophiles . In the hyperthermophiles proteomes, whenever the percent of Glu (E) and Lys (K) Increased, the percent of Gln (Q) and His (H) decreased, so that the E+K/Q+H ratio was > 4,5; in the mesophiles proteomes, it was < 2,5 and in the thermophiles an intermediary value was observed . The E+K/Q+H ratios for chaperonins, potentially thermostable proteins, were higher than their proteome ratios whereas, for DNA ligases, not necessarily thermostable, they followed the proteome ones . Analysis of codon usage revealed that hyperthermophiles preferred AGR codons for Arg in detriment of CGN codons, which were preferred by mesophiles . CONCLUSIONS: The results suggested that the E+K/Q+H ratio may provide a useful mark for distinguishing hyperthermophilic, thermophilic and mesophilic prokaryotes and that the high percent of the amino acid couple E+K, consistently associated to the low percent of the pair Q+H, could contribute to protein thermostability . Second, the preference for AGR codons for Arg was a signature of all hyperthermophilics so far analyzed.

Biochim Biophys Acta, 2002 Aug 19, 1591(1-3), 119 - 128
A cyclin-dependent protein kinase homologue associated with the basal body domains in the ciliate Tetrahymena thermophila; Zhang H et al.; The tight coupling between cell cycle progression and morphogenetic development in the unicellular ciliates presents a unique model system for examination of the roles of Cdks in developmental processes . We here describe the isolation and characterization of the first cyclin-dependent kinase (Cdk) homologue, TtCdk1, from Tetrahymena thermophila . TtCdk1 corresponds to the larger of the two polypeptides recognized by anti-PSTAIRE antibody in a whole cell lysate, which differ from each other in their affinity for yeast p13(suc1) protein . In contrast to the constant protein expression levels of typical eukaryotic Cdks, the TtCdk1 protein level fluctuates periodically over the vegetative cell cycle, reaching a maximum at the end of the cell cycle, correlating with its histone H1 kinase activity . Its association with the membrane-skeletal domains that surround mature, but not nascent, basal bodies in the cell cortex suggests that TtCdk1 plays a role in the regulation of cortical morphogenesis in T . thermophila . A partial TtCDK1 knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei . Unlike the situations in higher eukaryotic cells, no apparent effect on basal body duplication was found upon disruption of the TtCDK1 gene.

J Food Prot, 2002 Aug, 65(8), 1326 - 8
Prevalence of thermophilic Campylobacter spp . in ready-to-eat foods and raw poultry in Northern Ireland; Moore JE et al.; Although there have been numerous studies investigating the prevalence of campylobacters in animals and raw meats, there are limited data on the persistence of these organisms in ready-to-eat (RTE) foodstuffs . Although poultry is now well established as a major reservoir of thermophilic campylobacters, it is widely assumed that hazard analysis critical control point (HACCP) controls in commercial and industrial settings are effective in eliminating this hazard through thorough cooking of RTE products . Therefore, it was the primary aim of this study to investigate the effectiveness of HACCP controls in eliminating campylobacters in such cooked RTE foods by attempting to isolate viable organisms from product . Concurrently, the results of this study demonstrate that local poultry is highly contaminated with campylobacters . Commercially available RTE foodstuffs (n = 2,030) consisting of 1,061 poultry-related cooked products and 969 other products were analyzed and were not found to contain thermophilic Campylobacter spp . In addition, 107 raw chickens (63 fresh birds and 44 frozen birds) were sampled, and 94% of the fresh birds and 77% of the frozen birds examined were demonstrated to be contaminated with campylobacters, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari accounting for 69, 30, and 1% of the contaminating organisms, respectively . In general, commercially available RTE foodstuffs, including cooked poultry, are not commonly contaminated with campylobacters and thus do not appear to represent a significant cause of clinical infection of Campylobacter spp . in Northern Ireland . However, raw poultry produce, including fresh and frozen chicken, frequently tested positive for campylobacters . Implementation of HACCP systems by food processors will help to minimize and/or eliminate the risk posed by this organism to the consumer.

J Food Prot, 2002 Aug, 65(8), 1233 - 9
Occurrence and distribution of Arcobacter species in poultry processing; Houf K et al.; A total of 16 broiler flocks slaughtered in the morning in eight Belgian poultry slaughterhouses were examined for the presence of Campylobacteraceae . In samples collected before and after chilling, the prevalence of arcobacters was found to be higher than the prevalence of thermophilic campylobacters, with the slaughter procedure used having no clear effect . Two slaughterhouses were selected for a detailed investigation of the occurrence and distribution of arcobacters . Sampling carried out before slaughter revealed that both Arcobacter butzleri and Arcobacter cryaerophilus were commonly present on the slaughter equipment in both plants . These findings indicate inadequate decontamination of the slaughterhouse environment and suggest potential Arcobacter contamination of broiler carcasses through the slaughter equipment . Even before evisceration, contamination levels of hundreds to several thousands of arcobacters per gram of neck skin were detected . It appears unlikely that contamination through slaughter equipment alone explains the high contamination levels found for poultry products . Arcobacters were not isolated from the 30 intestinal tracts sampled for each broiler flock examined . A . cryaerophilus was the only Arcobacter species recovered from the transport crate samples collected before and after washing . Arcobacter contamination during slaughter, either direct (from chicken intestinal content or feces) or indirect (from equipment), was not confirmed . The origin and the precise routes of contamination remain to be determined.

Lett Appl Microbiol, 2002, 35(3), 185 - 9
flaA-like sequences containing internal termination codons (TAG) in urease-positive thermophilic Campylobacter isolated in Japan; Sekizuka T et al.; AIMS: To demonstrate two flaA-like sequences containing two internal termination codons (TAG) in two Japanese strains of urease-positive thermophilic Campylobacter (UPTC) . METHODS AND RESULTS: A primer pair of A1 and A2, which ought to generate a product of approx . 1700 bp of the flaA gene for Campylobacter jejuni, was used to amplify products of approx . 1450 bp for two Japanese strains of UPTC, CF89-12 and CF89-14 . After molecular cloning and sequencing, the nucleotide sequences of the amplicons from the two strains were found to be 1461 bp in length and to have nucleotide sequence differences in relation to each other at four nucleotide positions, respectively . CONCLUSIONS: Nucleotide and amino acid sequence alignment and homology analysis demonstrated that the polymerase chain reaction (PCR) amplicons from the two Japanese strains have approx . 83% nucleotide and 80% amino acid sequence homology to the possible open reading frame of the flaA gene of UPTC NCTC 12892 . SIGNIFICANCE AND IMPACT OF THE STUDY: Surprisingly, both PCR amplicons from the Japanese UPTC have two internal termination codons (TAG) at nucleotide positions from 775 to 777 and 817 to 819, respectively.

J AOAC Int, 2002 Jul-Aug, 85(4), 996 - 9
Rapid methods for detection and enumeration of Campylobacter spp . in foods; Wang H; Campylobacter spp . are the most commonly reported bacterial cause of acute diarrheal disease in humans throughout the world . Traditional cultural methods for the detection and quantitation of Campylobacterspp . are slow and tedious; therefore, specific, sensitive, and rapid methods for campylobacters are needed to collect sufficient data for risk assessment and food safety policy development . We developed several rapid methods based on polymerase chain reaction (PCR), DNA hybridization, hydrophobic grid membrane filters (HGMFs), and enzyme immunoassays (EIAs) . A PCR assay targeting C . jejuni, combined with a simple sample preparation procedure, detects as few as 0.3 most probable number (MPN)/mL C . jejuni in naturally contaminated chicken rinses after 20-24 h enrichment . An HGMF-EIA method using a commercial polyclonal antibody for Campylobacter detects and enumerates thermophilic Campylobacter spp . from spiked chicken rinse and milk, and naturally contaminated chicken rinses . A C . jejuni-specific probe in an HGMF-DNA hybridization protocol specifically detects and quantitates C . jejuni in food samples . A dot-blot EIA combined with an MPN procedure quantitates thermophilic campylobacters from samples that might be difficult to filter through HGMFs.

Nucleic Acids Res, 2002 Aug 15, 30(16), 3574 - 82
RNA molecules with structure dependent functions are uniquely folded; Le SY et al.; Cis-acting elements in post-transcriptional regulation of gene expression are often correlated with distinct local RNA secondary structure . These structures are expected to be significantly more ordered than those anticipated at random because of evolutionary constraints and intrinsic structural properties . In this study, we introduce a computing method to calculate two quantitative measures, NRd and Stscr, for estimating the uniqueness of an RNA secondary structure . NRd is a normalized score based on evaluating how different a natural RNA structure is from those predicted for its randomly shuffled variants . The lower the score NRd the more well ordered is the natural RNA structure . The statistical significance of NRd compared with that computed from structural comparisons among large numbers of randomly permuted sequences is represented by a standardized score, STSCR: We tested the method on the trans-activation response element and Rev response element of HIV-1 mRNA, internal ribosome entry sequence of hepatitis C virus, Tetrahymena thermophila rRNA intron, 100 tRNAs and 14 RNase P RNAs . Our data indicate that functional RNA structures have high Stscr, while other structures have low Stscr . We conclude that RNA functional molecules and/or cis-acting elements with structure dependent functions possess well ordered conformations and they are uniquely folded as measured by this technique.

J Biol Chem, 2002 Oct 18, 277(42), 39128 - 35 Epub 2002 Aug 12.
Conserved bases in the TPsi C loop of tRNA are determinants for thermophile-specific 2-thiouridylation at position 54; Shigi N et al.; 2-Thioribothymidine (s(2)T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs . The 2-thiocarbonyl group of s(2)T54 is known to be responsible for the thermostability of tRNA . The s(2)T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus . Little is known about the biosynthesis of s(2)T, including the sulfur donor, modification enzyme, and the tRNA structural requirements . To characterize 2-thiolation at position 54 in tRNA, we constructed an in vivo expression system using tRNA(Asp) with an altered sequence and a host-vector for T . thermophilus . We were able to detect in vivo activity of s(2)T54 thiolase using phenyl mercuric gel electrophoresis followed by Northern hybridization . 2-Thiolation at position 54 was identified in the precursor form of the tRNA, indicating that 2-thiolation precedes tRNA processing . To ascertain the elements that determine 2-thiolation in tRNA, systematic site-directed mutagenesis was carried out using the tRNA(Asp) gene . Conserved residues C56 and A58 were identified as major determinants of 2-thiolation, whereas tertiary interaction between the T and D loops and non-conserved nucleosides in the T loop were revealed not to be important for the reaction.

Schweiz Arch Tierheilkd, 2002 Jul, 144(7), 348 - 55
{Effects of housing, feeding and use on equine health with emphasis on respiratory and gastrointestinal diseases}; Feige K et al.; In a random population of Swiss horses 54% suffered from a subclinical to moderate COPD . Cause of a COPD is a hypersensitivity of the respiratory tract to spores of fungi and thermophil actinomyces . Teeth problems are strongly associated with the type of diet and the feeding regime . Problems of the teeth belong to the most often treated equine diseases by large animal practitioners . Racehorses are the population of horses most often affected by gastric ulcers with an ulcer prevalence between 63 and 90% . In contrast, a much lower prevalence (37%) of stomach ulcers is seen in pleasure horses and the degree of ulceration is less severe . Large amounts of concentrated high-energy feeds, small rations of forage and a low feeding frequency per day as well as the use of spoiled food can contribute to the development of colics.

J Agric Food Chem, 2002 Aug 14, 50(17), 4934 - 40
Olea europaea L . leaf extract and derivatives: antioxidant properties; Briante R et al.; This paper reports a very simple and fast method to collect eluates with high amounts of hydroxytyrosol, biotransforming Olea europaea L . leaf extract by a thermophilic beta-glycosidase immobilized on chitosan . Some phenolic compounds in the leaf tissue and in the eluates obtained by biotransformation are identified . To propose the eluates as natural substances from a vegetal source, their antioxidant properties have been compared with those of the leaf extract from which they are originated . The eluates possess a higher concentration of simple phenols, characterized by a stronger antioxidant capacity, than those available in extra virgin olive oils and in many tablets of olive leaf extracts, commercially found as dietetic products and food integrators.

FEBS Lett . 2002 Aug 14;525(1-3):88.
Ribosomal protein S1 from Thermus thermophilus: its detection, identification and overproduction; Shiryaev VM et al.; Ribosomal protein S1 has been identified in Thermus thermophilus ribosomes . The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli . A procedure for purification of the protein has been developed.

J Ind Microbiol Biotechnol, 2002 Aug, 29(2), 70 - 4
Amylase hyperproduction by deregulated mutants of the thermophilic fungus Thermomyces lanuginosus; Rubinder K et al.; Thermomyces lanuginosus was subjected to three cycles of mutagenesis (UV/NTG) and a selection procedure to develop amylase-hyperproducing, catabolite-repression-resistant and partially constitutive strains . One of the selected derepressed mutant strain III(51), produced approximately 7- and 3-fold higher specific activity of alpha-amylase (190 U/mg protein) and glucoamylase (105 U/mg protein), respectively, compared to a wild-type parental strain . Further, the effect of production parameters on mutant strain III(51) was studied using a Box-Behnken design . The regression models computed showed significantly high R(2) values of 96 and 97% for alpha-amylase and glucoamylase activities, respectively, indicating that they are appropriate for predicting relationships between corn flour, soybean meal and pH with alpha-amylase and glucoamylase production.

Biochem J, 2002 Nov 1, 367(Pt 3), 857 - 63
Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases; Del Vecchio P et al.; The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements . Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins . Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale . The present results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for the stability of the two esterases.

J Biochem (Tokyo), 2002 Aug, 132(2), 189 - 95
Importance of hydrophobic interaction between a SoxB-type cytochrome c oxidase with its natural substrate cytochrome c-551 and its mutants; Kagekawa S et al.; Cytochrome c-551, the electron donor of SoxB-type cytochrome c oxidase in thermophilic bacilli, can be over-expressed in Bacillus thermodenitrificans cells by tranformation with pSTEc551 . Several mutant cytochromes c-551 were prepared by site-directed mutagenesis to this expression plasmid . Among them, several Lys residues were changed to Ala/Ser, and we found that these mutant cytochromes retained their activity as substrates, although their K(m) values were 0.04-0.12 microM, depending on the site replaced . In contrast, the C19A mutant cytochrome, which was produced in Brevibacillus choshinensis as a secretion protein, lost its activity as a substrate, suggesting that the fatty acyl-glyceryl residue covalently bound to the cysteine residue of the wild-type c-551 plays a very important role in the activity . The importance of the hydrophobic fatty acid residue for the binding of cytochrome c-551 to the oxidase was also shown by the loss of substrate activity in deacylated cytochrome c-551 . These results show the importance of the hydrophobic interaction between this cytochrome and SoxB-type oxidase, despite the fact that the importance of an electrostatic interaction between cytochrome c and mitochondrial cytochrome aa(3) oxidase has already been established.

Environ Microbiol, 2002 Aug, 4(8), 487 - 93
Microbial diversity in ikaite tufa columns: an alkaline, cold ecological niche in Greenland; Stougaard P et al.; Ikaite tufa columns from the Ikka Fjord in south-western Greenland constitute a natural, stable environment at low temperature and with a pH ranging from neutral at the exterior to very alkaline (pH 10.4) at the interior of the column . Phylogenetic analysis of culturable organisms revealed ten different isolates representing three of the major bacterial divisions . Nine of the isolates showed 94-99% similarity to known sequences, whereas one isolate displayed a low degree of similarity (less than 90%) to a Cyclobacterium species . Seven of the isolates were shown to be cold active alkaliphiles, whereas three isolates showed optimal growth at neutral pH . Phylogenetic analysis of DNA isolated directly from the ikaite material demonstrated the presence of a microbial flora more diverse than the culturable isolates . Whereas approximately half of the phylotypes showed 90-99% similarity to known meso- or thermophilic alkaliphiles, the rest of the sequences displayed less than 90% similarity when compared to known 16S rRNA gene sequences in databases . Thus, in the present paper, we demonstrate that ikaite columns that host a specialized macroscopic flora and fauna also contain a unique, cold active, alkaliphilic microflora.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10359 - 63 Epub 2002 Jul 29.
Experimental evaluation of topological parameters determining protein-folding rates; Miller EJ et al.; Recent work suggests that structural topology plays a key role in determining protein-folding rates and pathways . The refolding rates of small proteins that fold without intermediates are found to correlate with simple structural parameters such as relative contact order, long-range order, or the fraction of short-range contacts . To test and evaluate the role of structural topology experimentally, a set of circular permutants of the ribosomal protein S6 from Thermus thermophilus was analyzed . Despite a wide range of relative contact order, the permuted proteins all fold with similar rates . These results suggest that alternative topological parameters may better describe the role of topology in protein-folding rates.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1361 - 8
Thermotoga lettingae sp . nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor; Balk M et al.; A novel, anaerobic, non-spore-forming, mobile, Gram-negative, thermophilic bacterium, strain TMOT, was isolated from a thermophilic sulfate-reducing bioreactor operated at 65 C with methanol as the sole substrate . The G+C content of the DNA of strain TMOT was 39.2 mol% . The optimum pH, NaCl concentration, and temperature for growth were 7.0, 1.0%, and 65 degrees C, respectively . Strain TMOT was able to degrade methanol to CO2 and H2 in syntrophic culture with Methanothermobacter thermautotrophicus AH or Thermodesulfovibrio yellowstonii . Thiosulfate, elemental sulfur, Fe(III) and anthraquinone-2,6-disulfonate were able to serve as electron acceptors during methanol degradation . In the presence of thiosulfate or elemental sulfur, methanol was converted to CO2 and partly to alanine . In pure culture, strain TMOT was also able to ferment methanol to acetate, CO2 and H2 . However, this degradation occurred slower than in syntrophic cultures or in the presence of electron acceptors . Yeast extract was required for growth . Besides growing on methanol, strain TMOT grew by fermentation on a variety of carbohydrates including monomeric and oligomeric sugars, starch and xylan . Acetate, alanine, CO2, H2, and traces of ethanol, lactate and alpha-aminobutyrate were produced during glucose fermentation . Comparison of 16S rDNA genes revealed that strain TMOT is related to Thermotoga subterranea (98%) and Thermotoga elfii (98%) . The type strain is TMOT (= DSM 14385T = ATCC BAA-301T) . On the basis of the fact that these organisms differ physiologically from strain TMOT, it is proposed that strain TMOT be classified as a new species, within the genus Thermotoga, as Thermotoga lettingae.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1349 - 59
Persephonella marina gen . nov., sp . nov . and Persephonella guaymasensis sp . nov., two novel, thermophilic, hydrogen-oxidizing microaerophiles from deep-sea hydrothermal vents; Gotz D et al.; Two thermophilic, strictly chemolithoautotrophic, microaerophilic, hydrogen-oxidizing members of the Bacteria designated strain EX-H1T and strain EX-H2T were isolated from two separate deep-sea hydrothermal vent sites at 9 degrees N 104 degrees W in the Pacific Ocean and Guaymas Basin . The motile 2-4-microm-long rods were Gram-negative and non-sporulating . The temperature range for growth was between 55 and 80 degrees C for EX- H1T (optimum at 73 degrees C) and 55-75 C for EX-H2T (optimum at 70 C) . Both strains grew fastest at 2.5% (w/v) NaCl and at pH 6, although growth was observed from pH 4.7 to pH 7.5 . EX-H1T and EX-H2T were able to use elemental sulfur, thiosulfate or hydrogen as an electron donor, and oxygen (2-3%, v/v) or nitrate as an electron acceptor . EX-H1T was also able to use elemental sulfur as an electron acceptor . EX-H1T and EX-H2T further differed in their genomic G+C content (38.5 and 37.4 mol%, respectively) and 16S rRNA sequences (4% difference) . Maximum-likelihood analysis of the 16S rRNA phylogeny placed both isolates within the Aquificales as a distinct lineage and showed them to be only about 85% similar to Aquifex pyrophilus . On the basis of phenotypic and phylogenetic characteristics, it is proposed that EX-H1T and EX-H2T belong to a new genus within the Aquificales, namely Persephonella gen . nov . It is further proposed that EX-H1T be named Persephonella marina sp . nov., the type species of the genus, and that EX-H2T be named Persephonella guaymasensis sp . nov., a second species in this genus.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1331 - 9
Marinitoga piezophila sp . nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent; Alain K et al.; A thermophilic, anaerobic, piezophilic, chemo-organotrophic sulfur-reducing bacterium, designated as KA3T, was isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2630 m on the East-Pacific Rise (13 degrees N) . When grown under elevated hydrostatic pressure, the cells are rod-shaped with a sheath-like outer structure, motile, have a mean length of 1-1.5 microm and stain Gram-negative . They appear singly or in short chains . When grown at lower, or atmospheric, pressures, the cells elongate and become twisted . Growth is enhanced by hydrostatic pressure; the optimal pressure for growth is 40 MPa (26 MPa pressure at sampling site) . The temperature range for growth is 45-70 degrees C, the optimum being around 65 degrees C (doubling time is approximately 20 min at 40 MPa) . Growth is observed from pH 5 to pH 8, the optimum being at pH 6 . The salinity range for growth is 10-50 g NaCl l(-1), the optimum being at 30 g l(-1) . The isolate is able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates, and growth is stimulated by L-cystine and elemental sulfur . The G+C content of the genomic DNA is 29 +/- 1 mol% . According to phylogenetic analysis of the 16S rDNA gene, the strain is placed within the order Thermotogales, in the bacterial domain . On the basis of 16S rDNA sequence comparisons and morphological, physiological and genotypic characteristics, it is proposed that the isolate be described as a novel species of the genus Marinitoga, with Marinitoga piezophila sp . nov . as the type species . The type strain is KA3T (= DSM 14283T = JCM 11233T).

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1317 - 23
Caminibacter hydrogeniphilus gen . nov., sp . nov., a novel thermophilic, hydrogen-oxidizing bacterium isolated from an East Pacific Rise hydrothermal vent; Alain K et al.; A novel thermophilic, anaerobic, hydrogen-oxidizing bacterium, designated strain AM1116T, was isolated from an East Pacific Rise hydrothermal vent sample . The cells were rod-shaped (1.01-5 x 0.5 microm), motile with polar flagella . They grew at temperatures between 50 and 70 degrees C (optimum 60 degrees C; doubling time approximately 1.5 h), at between pH 5.0 and 7.5 (optimum around pH 5.5-6.0) and in between 10 and 40 g NaCl l(-1) (optimum 20-25 g l(-1)) . Cells grew chemolithoautotrophically in a H2/CO2 atmosphere (80:20; 200 kPa) . Poor heterotrophic growth was observed on complex organic substrates . Elemental sulphur and nitrate served as electron acceptors, respectively yielding hydrogen sulphide and ammonia (doubling times were equal with the two electron acceptors) . In contrast, when cystine was used as electron acceptor, growth was poor . The G+C content of the genomic DNA was 29 +/- 1 mol % . Phylogenetic analyses of the 16S rRNA gene located the strain within the epsilon-Proteobacteria, in the bacterial domain . On the basis of 16S rDNA sequence comparisons, physiological and biochemical characteristics, it is proposed that the isolate should be described as the type species of a new genus, Caminibacter gen . nov., as Caminibacter hydrogeniphilus sp . nov . The type strain is strain AM1116T (= DSM 14510T = CIP 107140T).

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1299 - 304
Nautilia lithotrophica gen . nov., sp . nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent; Miroshnichenko ML et al.; A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525T, was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13 degrees N) . This organism grew in the temperature range 37-68 degrees C, the optimum being 53 degrees C, and in the pH range 6.4-7.4, the optimum being 6.8-7.0 . The NaCl range for growth was 0.8-5.0%, the optimum being 3.0% . Strain 525T grew lithoautotrophically with H2 as energy source, S0 as electron acceptor and CO2 as carbon source . Alternatively, strain 525T was able to use formate as an energy source . The G+C content of the genomic DNA was 34.7 mol% . Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525T in the epsilon-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives . On the basis of phenotypic and phylogenetic differences between strain 525T and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen . nov., sp . nov . is proposed . The type strain is strain 525T (= DSM 13520T).

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1225 - 8
Streptomyces thermospinisporus sp . nov., a moderately thermophilic carboxydotrophic streptomycete isolated from soil; Kim SB et al.; A carboxydotrophic actinomycete strain, AT10T (= DSM 41779T = KCTC 9909T), was the subject of a polyphasic study . The morphological and chemical properties of the strain were found to be consistent with its assignment to the genus Streptomyces . The organism formed a distinct phyletic line within the 16S rDNA Streptomyces tree, and DNA-DNA relatedness experiments further confirmed that it formed a distinct genomic species . The strain was also distinguished from related species using phenotypic properties . Strain AT10T, therefore, merits species status within the genus Streptomyces; the name Streptomyces thermospinisporus sp . nov . is proposed for this new taxon.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1217 - 23
Sporanaerobacter acetigenes gen . nov., sp . nov., a novel acetogenic, facultatively sulfur-reducing bacterium; Hernandez-Eugenio G et al.; A strictly anaerobic, moderately thermophilic, sporulating rod, designated strain Lup 33T, was isolated from an upflow anaerobic sludge blanket (UASB) reactor in Mexico . Strain Lup 33T possessed a few laterally inserted flagella, had a DNA G+C content of 32.2 mol % and grew optimally at pH 7.4 and 40 degrees C . Growth was observed at temperatures of up to 50 degrees C and was inhibited in the presence of 5% NaCl . Strain Lup 33T is heterotrophic and utilized some sugars, peptides and various single amino acids . Gelatin and casein were not used as energy sources . It performed the Stickland reaction and reduced elemental sulfur to sulfide . Acetate was the only fatty acid detected from glucose fermentation, whereas acetate together with isobutyrate and isovalerate were found as end products from peptone fermentation . Phylogenetically, strain Lup 33T branched with members of cluster XII of the order Clostridiales, with Clostridium hastiforme as the closest relative (similarity of 93%) . On the basis of the phenotypic, genotypic and phylogenetic characteristics of the isolate, it is proposed as a novel species of a new genus, Sporanaerobacter acetigenes gen . nov., sp . nov . The type strain is strain Lup 33T (= DSM 13106T = CIP 106730T).

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1177 - 84
Caloramator viterbensis sp . nov., a novel thermophilic, glycerol-fermenting bacterium isolated from a hot spring in Italy; Seyfried M et al.; A moderately thermophilic, anaerobic bacterium, strain JW/MS-VS5T, was isolated from a mixed sediment/water sample of a hot spring at Bagnaccio (near Viterbo, Italy) . The cells of this organism were straight to slightly curved rods, 0.4-0.6 x 2.03.0 microm in dimension . Cells occurred singly and stained Gram-positive . The temperature range for growth at pH(25C) 6.0 was 33-64 degrees C, the optimum being 58 degrees C . The pH(25C) range for growth was from 5.0 to 7.8, the optimum being 6.0-6.5 . The substrates utilized included glycerol, glucose, fructose, mannose, galactose, sucrose, cellobiose, lactose, starch and yeast extract . Acetate and 1,3-propanediol were the only detectable organic products of glycerol fermentation; significant amounts of H2 were produced during growth . The strain was unable to grow autotrophically in the presence of H2 and CO2 . The main products of glucose fermentation were CO2, H2, acetate and ethanol . Single amino acids, including serine, glutamine, threonine, leucine, methionine, aspartate, valine and histidine (but not arginine), served as carbon sources . Growth was completely inhibited by ampicillin, chloramphenicol, erythromycin, rifampicin and kanamycin at 100 microg ml(-1) and was retarded by streptomycin and tetracycline . The G+C content of the DNA was 32 mol% (HPLC) . According to 16S rDNA sequence analysis, the isolate is located within the Gram-type positive Bacillus-Clostridium branch of the phylogenetic tree . On the basis of physiological properties and phylogenetic analysis, it is proposed that strain JW/MS-VS5T (the only, and type, strain) (= DSM 13723T = ATCC PTA 584T), constitutes the new species Caloramator viterbensis.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1089 - 95
Methanothermococcus okinawensis sp . nov., a thermophilic, methane-producing archaeon isolated from a Western Pacific deep-sea hydrothermal vent system; Takai K et al.; A novel thermophilic, methane-producing archaeon was isolated from a deep-sea hydrothermal vent chimney at the Iheya Ridge, in the Okinawa Trough, Japan . The cells were highly motile, irregular cocci, with a polar bundle of flagella . Growth was observed between 40 and 75 degrees C (optimum 60-65 degrees C; 30 min doubling time) and between pH 4.5 and 8.5 (optimum pH 6.7) . The isolate was a strictly anaerobic autotroph capable of using hydrogen and carbon dioxide as sole sources of energy and carbon . Formate can serve as an alternative energy source . The G+C content of the genomic DNA was 33.5 mol% . Phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization analysis indicated that the isolate was closely related to members of the genera Methanococcus and Methanothermococcus . This isolate, however, could be differentiated from the previously described species of these genera on the basis of its physiological and molecular properties . The name Methanothermococcus okinawensis sp . nov is proposed, with the type strain IH1T (=JCM 11175T=DSM 14208T).

Appl Environ Microbiol, 2002 Aug, 68(8), 3708 - 15
Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-beta-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155; Graninger M et al.; The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of L-rhamnose- and D-glycero-D-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages . In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-L-rhamnose (dTDP-L-rhamnose) and their specific properties . Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-L-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD) . The rhamnose biosynthesis operon from A . thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli . Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis . The closely related strain A . thermoaerophilus L420-91(T) produces D-rhamnose- and 3-acetamido-3,6-dideoxy-D-galactose-containing S-layer glycan chains . Comparison of the enzyme activity patterns in A . thermoaerophilus strains DSM 10155 and L420-91(T) for L-rhamnose and D-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A . thermoaerophilus L420-91(T) is not able to synthesize dTDP-L-rhamnose . These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.

Biochem Biophys Res Commun, 2002 Aug 9, 296(1), 161 - 6
Atomic resolution structure of the major endoglucanase from Thermoascus aurantiacus; Van Petegem F et al.; The crystal structure of the major endoglucanase from the thermophilic fungus Thermoascus aurantiacus was determined by single isomorphous replacement at 1.12A resolution . The full sequence supports the classification of the protein in a subgroup of glycoside hydrolase family 5 for which no structural data are available yet . The active site shows eight critical residues, strictly conserved within family 5 . In addition, aromatic residues that line the substrate-binding cleft and that are possibly involved in substrate-binding are identified . A number of residues seem to be conserved among members of the subtype, including a disulphide bridge between Cys212 and Cys249.

Biochem Biophys Res Commun, 2002 Aug 9, 296(1), 8 - 12
Stabilization of FtsH-unfolded protein complex by binding of ATP and blocking of protease; Makyio H et al.; The function of an ATP-dependent membrane protease FtsH was investigated using the enzyme from Thermus thermophilus HB8 . An FtsH mutant with replacement of Glu-419 in the zinc-binding motif by Cys lost the activity to digest casein, a model unfolded protein, and the small ATPase activity of this mutant was no longer stimulated by casein . In the presence of ATP or ATPgammaS, but not ADP, a mutant FtsH-unfolded protein complex was isolated, indicating that ATP binding, but not ATP hydrolysis, is required for FtsH to form a stable complex with an unfolded protein . The FtsH without mutation at Glu-419 did not produce a stable complex with casein in the presence of any nucleotides tested and therefore it appears that blocking proteolysis also contributes to stabilization of the complex.

J Appl Microbiol, 2002, 93(2), 278 - 87
Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products; Mora D et al.; AIMS: To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products . Methods METHODS AND RESULTS: Strains were analysed for some relevant technological properties, i.e . exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation . The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains . Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea . A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep . thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters . A total of 11 strains were able to produce acid in presence of galactose . Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes . CONCLUSIONS: Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes . SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological characterization carried out on several Strep . thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.

Bioresour Technol, 2002 Oct, 85(1), 57 - 61
Biodegradation of potato slops from a rural distillery by thermophilic aerobic bacteria; Cibis E et al.; A study has been made of thermophilic aerobic biodegradation of the liquid fraction of potato slops (distillation residue) from a rural distillery . The COD of this fraction ranged from 49 to 104 g O2/l, the main contributions to the COD coming from organic acids, reducing substances, and glycerol . It was found that biodegradation could be divided into the following stages: organic acids were removed first, followed by reducing substances and glycerol . The extent of removal varied according to the process temperature . At 50 degrees C, acetic and malic acids were removed completely, but the amount of isobutyric acid increased . At 60 degrees C, organic acid removal ranged from 51.2% (isobutyric acid) to 99.6% (lactic acid) . Removals of glycerol and reducing substances were 86.2% and 87.4%, respectively . COD reduction was also temperature dependent, the highest removal efficiency (76.7%) being achieved at 60 degrees C . Dissolved oxygen may have limited the biodegradation process, as indicated by the DOT-versus-time profile.

Bioresour Technol, 2002 Oct, 85(1), 1 - 8
Bio-degradation of olive mill wastewater sludge by its co-composting with agricultural wastes; Paredes C et al.; The use of maize straw (MS) or cotton waste (CW) as bulking agents in the composting of olive mill wastewater (OMW) sludge was compared by studying the organic matter (OM) mineralisation and humification processes during composting and the characteristics of the end products . Both composts were prepared in a pilot-plant using the Rutgers static-pile system . The use of CW instead of MS to compost OMW sludge extended both the thermophilic and bio-oxidative phases of the process, with higher degradation of polymers (mainly lignin and cellulose), a greater formation of nitrates, higher total nitrogen losses and a lower biological nitrogen fixation . The CW produced a compost with a more stabilised OM and more highly polymerised humic-like substances . In the pile with CW and OMW sludge, OM losses followed a first-order kinetic equation, due to OM degradation being greater at the beginning of the composting and remaining almost constant until the end of the process . However, in the pile with MS and OMW sludge this parameter followed a zero-order kinetic equation, since OM degraded throughout the process . The germination index indicated the reduction of phytotoxicity during composting.

Chemosphere, 2002 Jul, 48(3), 287 - 97
Fate of methidathion residues in biological waste during anaerobic digestion; Vorkamp K et al.; The aim of this study was to examine the fate of the organothiophosphorus pesticide methidathion during anaerobic digestion of biological waste . Three reactor experiments were conducted under various conditions of temperature, pH and retention time . The influence of pH and temperature as well as the partitioning between solid and aqueous phase were studied in batch experiments . The mesophilic (25, 35 degrees C) reactor experiments showed a decline to about 10% of the maximum methidathion concentration within 30-80 d . In the thermophilic (55 degrees C) reactor experiment, methidathion disappeared within 20 d . The batch experiments showed an abiotic hydrolysis of methidathion over the experiment period of 4 d, accelerated by alkaline conditions (pH 10.5 and 12.8) and high temperatures (55 degrees C) . The hydrolysis was also noticeable at a neutral pH, while methidathion was most stable at weakly acid pH values . Methidathion bonded strongly to the biological waste, and the amount released into the water phase was below the maximum aqueous solubility . About 10% of methidathion remained non-extractable . High concentrations of dissolved organic carbon and yeast extract as a model substance for disintegrated cells further reduced the content of methidathion in the water phase, possibly caused by co-sorption to the solid organic matter.

J Dairy Sci, 2002 Jun, 85(6), 1390 - 7
Microbial community dynamics during the Scamorza Altamurana cheese natural fermentation; Baruzzi F et al.; The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests . Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening . RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus . The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic . Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp . bulgaricus B15Z dominated all through the fermentation process . Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo . The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products.

Annu Rev Microbiol, 2002, 56, 65 - 91 Epub 2002 Jan 30.
Heavy metal mining using microbes; Rawlings DE; The use of acidiphilic, chemolithotrophic iron- and sulfur-oxidizing microbes in processes to recover metals from certain types of copper, uranium, and gold-bearing minerals or mineral concentrates is now well established . During these processes insoluble metal sulfides are oxidized to soluble metal sulfates . Mineral decomposition is believed to be mostly due to chemical attack by ferric iron, with the main role of the microorganisms being to reoxidize the resultant ferrous iron back to ferric iron . Currently operating industrial biomining processes have used bacteria that grow optimally from ambient to 50 degrees C, but thermophilic microbes have been isolated that have the potential to enable mineral biooxidation to be carried out at temperatures of 80 degrees C or higher . The development of higher-temperature processes will extend the variety of minerals that can be commercially processed.

J Bacteriol, 2002 Aug, 184(16), 4442 - 8
Purification, overproduction, and partial characterization of beta-RFAP synthase, a key enzyme in the methanopterin biosynthesis pathway; Scott JW et al.; Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria . Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway . The enzyme beta-ribofuranosylaminobenzene 5'-phosphate synthase (beta-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form beta-RFAP, CO2, and inorganic pyrophosphate . The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding beta-RFAP synthase has not yet been identified . In the present work, we report on the purification to homogeneity of beta-RFAP synthase . The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus . The putative beta-RFAP synthase gene from A . fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified . A BLAST search using the deduced amino acid sequence of the beta-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens . The identification of a gene encoding a potential beta-RFAP synthase in M . extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.

Mikrobiologiia, 2002 May-Jun, 71(3), 391 - 8
{Geobacillus uralicus, a new species of thermophilic bacteria}; Popova NA et al.; The K2T strain of thermophilic spore-forming bacteria was isolated from a biofilm on the surface of a corroded pipeline in an extremely deep well (4680 m, 40-72 degrees C) in the Ural . The cells are rod-shaped, motile, gram-variable . They grow on a complex medium with tryptone and yeast extract and on a synthetic medium with glucose and mineral salts without additional growth factors . The cells use a wide range of organic substances as carbon and energy sources . They exhibit a respiratory metabolism but are also capable of anaerobic growth on a nitrate-containing medium and of fermentation . Growth occurs within the 40-75 degrees C temperature range (with an optimum of 65 degrees C) and at pH 5-9 . The minimum generation time (15 min) was observed at pH 7.5 . Ammonium salts and nitrates are used as nitrogen sources . The G + C content of the DNA is 54.5 mol% . From the morphological, physiological, and biochemical properties and the nucleotide sequence of the 16S rRNA gene, it was concluded that the isolate K2T represents a new species of the genus Geobacillus, Geobacillus uralicus.

Acta Crystallogr D Biol Crystallogr, 2002 Aug, 58(Pt 8), 1376 - 8 Epub 2002 Jul 20.
Crystallization of the 43 kDa ATPase domain of Thermus thermophilus gyrase B in complex with novobiocin; Lamour V et al.; The 43 kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks . Crystals of the 43 kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained . Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent . The plate-shaped crystals, which were less than 10 microm in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3 A allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin.

Acta Crystallogr D Biol Crystallogr, 2002 Aug, 58(Pt 8), 1365 - 7 Epub 2002 Jul 20.
Overproduction, crystallization and preliminary X-ray diffraction analysis of a quinone oxidoreductase from Thermus thermophilus HB8; Shimomura Y et al.; A probable quinone oxidoreductase (MW = 32.1 kDa) from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified . Gel-filtration chromatography suggested the protein to be in a dimeric state . This protein enhanced the reduction activity of quinones by NADPH . It was crystallized in the absence and the presence of NADPH by the hanging-drop vapour-diffusion method . Both crystals were hexagonal, space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 77.6, c = 236.7 A for the apo form and a = b = 77.6, c = 235.9 A for the complex with NADPH . They diffract to better than 2.3 A resolution with synchrotron radiation . The asymmetric unit has one protein subunit (V(M) = 3.2 A(3) Da(-1) and V(sol) = 0.62 for the apo form), indicating that the twofold axis of the dimeric protein and the crystallographic twofold axis coincide.

Eur J Biochem, 2002 Jul, 269(14), 3355 - 61
Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability; Sambongi Y et al.; Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution . Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation . Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed . Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c-552 and its mesophilic counterpart have provided substantial clues to the mechanism underlying protein stability at the amino-acid level . The molecular mechanisms underlying protein maturation, folding, and stability in bacterial cytochromes c are beginning to be understood.

J Biol Chem, 2002 Sep 20, 277(38), 34743 - 8 Epub 2002 Jul 18.
Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases; Ahel I et al.; Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation . Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids . Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes . A striking difference between these groups is the presence of an insertion domain (approximately 180 amino acids) in the bacterial-type ProRS . Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS . Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus . This non-cognate amino acid was efficiently acylated in vitro onto tRNA(Pro), and the misacylated Cys-tRNA(Pro) was not edited by ProRS . Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases.

J Chromatogr, 1992 Nov 13, 625(1), 21 - 31
Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus; Ritter S et al.; Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied . A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined . Among all the metal ions tested, only copper was able to bind the two protein complexes . For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II . All other gradients proved to be inappropriate for the separation of these two photosystems . Mechanisms of protein elution by these compounds are discussed . Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole . When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.

FEBS Lett, 2002 Jul 17, 523(1-3), 99 - 102
A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus; Zafra O et al.; A cytochrome c (NarC) is encoded as the first gene of the operon for nitrate respiration in Thermus thermophilus . NarC is required for anaerobic growth and for the synthesis of active nitrate reductase (NR) . The alpha and delta subunits (NarG, NarJ) of the NR were constitutively expressed in narC::kat mutants, but NarG appeared in the soluble fraction instead of associated with the membranes . Our data demonstrate for NarC an essential role in the synthesis of active enzyme and for the attachment to the membrane of the respiratory NR from T . thermophilus.

J Eukaryot Microbiol, 2002 May-Jun, 49(3), 227 - 38
Morphological, small subunit rRNA, and physiological characterization of Trimyema minutum (Kahl, 1931), an anaerobic ciliate from submarine hydrothermal vents growing from 28 degrees C to 52 degrees C; Baumgartner M et al.; A thermophilic strain of Trimyema minutum was isolated from the hydrothermally heated sea floor at Vulcano Island (Italy) and cultivated monoxenically on Marinobacter sp . and Methanococcus thermolithotrophicus . It can be propagated strictly anaerobically and is sensitive to oxygen: if exposed to air at 48 degrees C all cells die within 60 min . It grows from 0.45-7.2% (w/v) salt and at pH 6.0-8.0 . The isolate is the most extreme thermophilic ciliate which ever has been cultivated, exhibiting an optimal growth temperature of 48 degrees C (doubling time 6 h) . Growth occurs between 28 degrees C and 52 degrees C . Trimyema minutum is redescribed using live observation and silver impregnation . Its morphology and the small subunit ribosomal RNA sequence is distinctly different from that of T . compressum, but morphology is highly similar to that of T . shoalsia Nerad et al . 1995, which is thus probably a junior synonym of T . minutum . To stabilize the bewildering species taxonomy in Trimyema, we suggest to recognize our population as a neotype of T . minutum.

Eur J Oral Sci, 2002 Jun, 110(3), 218 - 24
Selection of dairy bacterial strains as probiotics for oral health; Comelli EM et al.; The aim of the present study was to select bacterial strains with potential properties as oral probiotics, namely for the prevention of dental caries . We examined 23 dairy microorganisms, out of which we identified two Streptococcus thermophilus and two Lactcoccus lactis strains that were able to adhere to saliva-coated hydroxyapatite beads to the same extent as Streptococcus sobrinus OMZ176 . Two of them, Strep . thermophilus NCC1561 and Lactoc . lactis ssp . lactis NCC2211, were further successfully incorporated into a biofilm mimicking the dental plaque . Furthermore, they could grow in such a biofilm together with five strains of oral bacterial species, representative of supragingival plaque . In this system, Lactoc . lactis NCC2211 was able to modulate the growth of the oral bacteria, and in particular to diminish the colonization of Streptococcus oralis OMZ607, Veillonella dispar OMZ493, Actinomyces naeslundii OMZ745 and of the cariogenic Strep . sobrinus OMZ176 . These findings encourage further research with selected non-pathogenic dairy bacterial strains with the aim to decrease the cariogenic potential of dental plaque.

Environ Technol, 2002 Jun, 23(6), 643 - 54
Reactor configuration--Part II . Comparative process stability and efficiency of thermophilic anaerobic digestion; Kim M et al.; Comparative process stability and efficiency of thermophilic anaerobic digestion (55 degrees C) has been evaluated for four different reactor configurations, which are: daily batch-fed single-stage continuously stirred tank reactor (CSIR) (TB), continuously-fed single-stage CSTR (TC), daily batch-fed two-phase CSTR (TTP), and daily batch-fed non-mixed single-stage reactor (TNMR) . The results are discussed for periods, 1) start-up until steady state (Days 0-200) and 2) OLR increase from 4% solids at steady state to reactor failure by increasing solids concentration in feed and decreasing the HRT (Days 201-) . During the start-up period, the TB showed the worst stability with a pH drop whenever the solids concentration in the feed was increased . Conversely the TNMR reached steady state with 4% feed solids in the shortest time with relatively stable pH and very low VFA . The superior performance of the TNMR confirms the importance of microbial consortia proximity especially for the removal of propionate . The cocktail of inorganic nutrients (Ca, Ni, Fe, and Co) was added daily into all reactors showing high VFA . In the case of the TNMR, complete removal of propionate occurred after supplementation of nutrients . The results indicated that adding nutrients stimulated gas production and facilitated removal of almost all VFA confirming the importance of inorganic nutrients bioavailability . During the long-term operation with OLR increases until reactor failure (pH below 5.5), the results show that TB failed at the lowest OLR while the TC and the TNMR reached the highest OLR . Compared with the daily batch fed reactors, the constant pH of the MC seems to be the reason why the MC reached the highest OLR . The superior performance of and the TNMR during both the start-up long-term period confirms the importance of microbial consortia proximity . Two-phase digestion showed little benefit over single stage during the start-up period and no benefit was observed during the long-term period . Additional experiments in which the reactor configuration was changed from CSTR to non-mixed reactor showed significant benefit with respect to gas production and VFA threshold concentration . This once again manifests the importance of microbial consortia proximity.

J Food Prot, 2002 Jul, 65(7), 1110 - 6
Survival of Campylobacter jejuni in biofilms isolated from chicken houses; Trachoo N et al.; Campylobacter jejuni is a thermophilic and microaerophilic enteric pathogen associated with poultry . Biofilms may be a source of C . jejuni in poultry house water systems since they can protect constituent microorganisms from environmental stress . In this study, the viability of C . jejuni in biofilms of gram-positive chicken house isolates (P1, Y1, and W1) and a Pseudomonas sp . was determined using a cultural method (modified brucella agar) and direct viable count (DVC) . Two-day biofilms grown on polyvinyl chloride (PVC) coupons in R2A broth at 12 and 23 degrees C were incubated with C . jejuni for a 6-h attachment period . Media were then refreshed every 24 h for 7 days to allow biofilm growth . Two-day biofilms of P1, Y1, and Pseudomonas spp . enhanced attachment (P < 0.01) of C . jejuni (4.74, 4.62, and 4.78 log cells/cm2, respectively) compared to W1 and controls without preexisting biofilm (4.31 and 4.22 log cells/cm2, respectively) . On day 7, isolates P1 and Y1 and Pseudomonas biofilms covered 5.4, 7.0, and 21.5% of the surface, respectively, compared to 4.9% by W1 . Viable C . jejuni on the surface decreased (P < 0.05) with time, with the greatest reduction occurring on surfaces without a preexisting biofilm . The number of viable C . jejuni determined by DVC was greater than that determined by the cultural method, indicating that C . jejuni may form a viable but nonculturable state within the biofilm . Both DVC and the cultural method indicate that biofilms enhance (P < 0.01) the survival of C . jejuni during incubation at 12 and 23 degrees C over a 7-day period.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 160 - 9 Epub 2002 May 01.
Thermophilic production of lactic acid using integrated membrane bioreactor systems coupled with monopolar electrodialysis; Danner H et al.; The thermophilic Bacillus strain BS119 was selected for this study to demonstrate the long term performance of lactic acid production and simultaneous pre-purification . Integrated continuous cell recycle cultivation using ultra-filtration membrane bioreactor (MBR) systems was investigated . The permeate from the MBR was routed to an on-line electrodialysis (ED) to recover, pre-purify and concentrate lactate . The cultivation and ED was operated at 60 degrees C for more than 1,000 h at a pH of 6.5 . At lower dilution rate (0.02 h(-1)), lactate concentration reached a maximum of 55 g l(-1) with clearly lower residual glucose levels . At 0.04 h(-1), lactate concentration was significantly lower at 35 g l(-1) . Maximal volumetric productivities of 1.38 g l(-1) h(-1) were achieved . Under stable conditions, lactic acid yield on consumed glucose appeared stable at around 80% . It could be demonstrated that the addition of supplements like yeast extract and peptone severely influences product formation . Integration of mono-polar ED with the MBR systems yields lactate solutions with concentrations of up to 115 g l(-1) . Because of the low substrate feed concentrations (less than 50 g l(-1)), lactate flux was rather poor, reaching a low maximum of 140 g m(-2) h(-1); nevertheless, stack energy consumption was positive with an average of 0.49 kWh kg(-1) lactate.

EMBO J, 2002 Jul 15, 21(14), 3829 - 40
Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition; Yaremchuk A et al.; Bacterial tyrosyl-tRNA synthetases (TyrRS) possess a flexibly linked C-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme . We have determined the structure of Thermus thermophilus TyrRS at 2.0 A resolution in a crystal form in which the C-terminal domain is ordered, and confirm that the fold is similar to part of the C-terminal domain of ribosomal protein S4 . We have also determined the structure at 2.9 A resolution of the complex of T.thermophilus TyrRS with cognate tRNA(tyr)(G Psi A) . In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA . The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Psi-35 are base-specifically recognized . The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem.

Can J Microbiol, 2002 May, 48(5), 473 - 8
Transposition of pGh9:ISS1 is random and efficient in Streptococcus thermophilus CNRZ368; Thibessard A et al.; Streptococcus thermophilus bacteria are used as a starter in the fermentation of yogurts and many cheeses . To construct mutants of S . thermophilus CNRZ368, the use of the plasmid pGh9:ISS1 was considered . This plasmid is known to be a good tool for insertional mutagenesis in gram-positive bacteria, owing to its ability to integrate in the genome by a mechanism of replicative transposition . However, the presence of three endogenous ISS1 copies in the genome of S . thermophilus CNRZ368 and the possible occurrence of homologous recombination could reduce the efficiency of pGh9:ISS1 as a tool for generating mutants . To address this question, the ability of pGh9:ISS1 to transpose randomly in the genome of strain CNRZ368 was investigated . The results of our experiments indicated that: (i) the frequency of transposition of ISS1 was high, approximately 2 x 10(-2), in S . thermophilus CNRZ368; (ii) the integration of multiple tandem copies of the plasmid was frequent; (iii) homologous recombination events between ISS1 were not predominant; and (iv) plasmid pGh9:ISS1 transposed randomly around the S . thermophilus CNRZ368 chromosome . In addition, we describe the strategy used to localize the pGh9:ISS1 insertion locus on the physical map of strain CNRZ368 and the method used to clone the regions flanking this insertion site, especially when multiple copies of the plasmid were integrated in tandem.

J Biol Chem, 2002 Sep 6, 277(36), 32867 - 74 Epub 2002 Jul 09.
Fourier transform infrared (FTIR) and step-scan time-resolved FTIR spectroscopies reveal a unique active site in cytochrome caa3 oxidase from Thermus thermophilus; Pinakoulaki E et al.; Fourier transform infrared (FTIR) and step-scan time-resolved FTIR difference spectra are reported for the {carbonmonoxy}cytochrome caa(3) from Thermus thermophilus . A major C-O mode of heme a(3) at 1958 cm(-1) and two minor modes at 1967 and 1975 cm(-1) (7:1:1) have been identified at room temperature and remained unchanged in H(2)O/D(2)O exchange . The observed C-O frequencies are 10 cm(-1) higher than those obtained previously at 21 K (Einarsdottir, O., Killough, P . M., Fee, J . A., and Woodruff, W . H . (1989) J . Biol . Chem . 264, 2405-2408) . The time-resolved FTIR data indicate that the transient Cu(B)(1+)-CO complex is formed at room temperature as revealed by the CO stretching mode at 2062 cm(-1) . Therefore, the caa(3) enzyme is the only documented member of the heme-copper superfamily whose binuclear center consists of an a(3)-type heme of a beta-form and a Cu(B) atom of an alpha-form . These results illustrate that the properties of the binuclear center in other oxidases resulting in the alpha-form are not required for enzymatic activity . Dissociation of the transient Cu(B)(1+)-CO complex is biphasic . The rate of decay is 2.3 x 10(4) s(-1) (fast phase, 35%) and 36.3 s(-1) (slow phase, 65%) . The observed rate of rebinding to heme a(3) is 34.1 s(-1) . The implications of these results with respect to the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases are discussed.

Mol Biochem Parasitol, 2002 Jul, 122(2), 119 - 26
The circumsporozoite protein of Plasmodium falciparum is expressed and localized to the cell surface in the free-living ciliate Tetrahymena thermophila; Peterson DS et al.; Heterologous expression is an important tool for characterization of protein function, structural studies, and production of antigen . While many different host systems have been utilized for the expression of Plasmodium falciparum proteins, the extreme AT-richness of its genome represents an obstacle to efficient expression . In addition, primary sequence motifs such as glycosyl phosphatidyl-inositol (GPI) cleavage/attachment sites of P . falciparum are not recognized in currently used expression hosts . Recently, DNA-mediated transformation has been used for expression of heterologous genes in the ciliated protozoan Tetrahymena thermophila . We report the stable expression of full-length P . falciparum circumsporozoite (CS) protein in T . thermophila . The expressed gene utilized the native CS protein N-terminal secretory signal sequence and the C-terminal GPI anchoring signal . Immunofluorescence imaging demonstrated that the CS protein was localized to the cell surface of Tetrahymena . Metabolic labeling with tritiated myristate resulted in incorporation of label into the recombinant CS protein, indicating that the protein was bound to the cell surface via a GPI anchor . This is the first report of the recognition of targeting and GPI anchoring signals of the P . falciparum CS protein in a heterologous expression host.

Mycoses, 2002 Jun, 45(5-6), 184 - 7
Influence of buffered propionic acid on the development of micro-organisms in hay; Reboux G et al.; We tested the benefit of using buffered propionic acid (BPA) as a means of preventing farmer's lung disease (FLD) . BPA, a new formulation of propionic acid, a hay preservative with no deleterious effect on farm machinery or cattle, reduces the development of micro-organisms in hay . Twenty pairs of round bales were analysed for concentration of micro-organisms measured in the winter following hay treatment . Each pair included one untreated bale and one bale treated with BPA during haymaking . Our results showed the following decreases in concentration in treated bales: total fungal species, 40% (P < 0.05); Eurotium amstelodami (the main species found), 65% (P < 0.01); and thermophilic actinomycetes, 60% (not significant), respectively . We conclude that BPA could be used to prevent FLD.

Waste Manag, 2002, 22(4), 453 - 9
The first commercial supercritical water oxidation sludge processing plant; Griffith JW et al.; Final disposal of sludge continues to be one of the more pressing problems for the wastewater treatment industry . Present regulations for municipal sludge have favored beneficial use, primarily in land application . However, several agencies and entities have warned of potential health risks associated with these methods . Hydrothermal oxidation provides an alternative method that addresses the health concerns associated with sludge disposal by completely converting all organic matter in the sludge to carbon dioxide, water, and other innocuous materials . A hydrothermal oxidation system using HydroProcessing, L.L.C.'s HydroSolids process has been installed at Harlingen, Texas to process up to 9.8 dry tons per day of sludge . Based on a literature review, this system is the largest hydrothermal oxidation system in the world, and the only one built specifically to process a sludge . Start up of Unit 1 of two units of the HTO system began in April 2001 . Early results have indicated COD conversion rates in excess of 99.9% . Harlingen Waterworks System estimates that the HydroSolids system will cost less than other alternatives such as autothermal thermophilic aerobic digestion and more traditional forms of digestion that still require dewatering and final disposal . The Waterworks intends to generate income from the sale of energy in the form of hot water and the use of carbon dioxide from the HydroSolids process for neutralization of high pH industrial effluent . The Waterworks also expects to generate income from the treatment of septage and grease trap wastes.

J Neurosci, 2002 Jul 1, 22(13), 5727 - 33
Thermotaxis in Caenorhabditis elegans analyzed by measuring responses to defined Thermal stimuli; Ryu WS et al.; In a spatial thermal gradient, Caenorhabditis elegans migrates toward and then isothermally tracks near its cultivation temperature . A current model for thermotactic behavior involves a thermophilic drive (involving the neurons AFD and AIY) and cryophilic drive (involving the neuron AIZ) that balance at the cultivation temperature . Here, we analyze the movements of individual worms responding to defined thermal gradients . We found evidence for a mechanism for migration down thermal gradients that is active at temperatures above the cultivation temperature, and a mechanism for isothermal tracking that is active near the cultivation temperature . However, we found no evidence for a mechanism for migration up thermal gradients at temperatures below the cultivation temperature that might have supported the model of opposing drives . The mechanisms for migration down gradients and isothermal tracking control the worm's movements in different manners . Migration down gradients works by shortening (lengthening) the duration of forward movement in response to positive (negative) temperature changes . Isothermal tracking works by orienting persistent forward movement to offset temperature changes . We believe preference for the cultivation temperature is not at the balance between two drives . Instead, the worm activates the mechanism for isothermal tracking near the cultivation temperature and inactivates the mechanism for migration down gradients near or below the cultivation temperature . Inactivation of the mechanism for migration down gradients near or below the cultivation temperature requires the neurons AFD and AIY.

J Biol Chem, 2002 Sep 6, 277(36), 32860 - 6 Epub 2002 Jul 03.
Observation of the equilibrium CuB-CO complex and functional implications of the transient heme a3 propionates in cytochrome ba3-CO from Thermus thermophilus . Fourier transform infrared (FTIR) and time-resolved step-scan FTIR studies; Koutsoupakis K et al.; We report the first evidence for the existence of the equilibrium Cu(B)1+-CO species of CO-bound reduced cytochrome ba(3) from Thermus thermophilus at room temperature . The frequency of the C-O stretching mode of Cu(B)1+-CO is located at 2053 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu(B) histidine ligands . The data and conclusions reported here are in contrast to the changes in protonation state of Cu(B)-His-290, reported recently (Das, T . K., Tomson, F . K., Gennis, R . B., Gordon, M., and Rousseau, D . L . (2001) Biophys . J . 80, 2039-2045 and Das, T . P., Gomes, C . M., Teixeira, M., and Rousseau, D . L . (1999) Proc . Natl . Acad . Sci . U . S . A . 96, 9591-9596) . The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex is 34.5 s(-1) and rebinding to heme a(3) occurs with k(2) = 28.6 s(-1) . The rate of decay of the transient Cu(B)1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a(3) propionates (COOH) . The nu(C-O) of the transient Cu(B)1+-CO species is the same as that of the equilibrium Cu(B)1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu(B) between these states . The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.

J Biochem (Tokyo), 2002 Jul, 132(1), 63 - 70
Effective structure of a leader open reading frame for enhancing the expression of GC-rich genes; Ishida M et al.; To overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus . When a leader ORF overlapped with the polA-initiation codon by 1 bp in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon . A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif . When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed . Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T . In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression . Using the most effective leader ORF obtained from these results, new expression vectors were constructed.

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