Polyamine analysis for chemotaxonomy of thermophilic eubacteria: Polyamine distribution profiles within the orders Aquificales, Thermotogales, Thermodesulfobacteriales, Thermales, Thermoanaerobacteriales, Clostridiales and Bacillales.
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Cellular polyamines of 45 thermophilic and 8 related mesophilic eubacteria were investigated by HPLC and GC analyses for the thermophilic and chemotaxonomic significance of polyamine distribution profiles. Spermidine and a quaternary branched penta-amine, N4-bis(aminopropyl)norspermidine, were the major polyamine in Thermocrinis, Hydrogenobacter, Hydrogenobaculum, Aquifex, Persephonella, Sulfurihydrogenibium, Hydrogenothermus, Balnearium and Thermovibrio, located in the order Aquificales. Thermodesulfobacterium and Thermodesulfatator belonging to the order Thermodesulfobacteriales contained another quaternary penta-amine, N4-bis(aminopropyl)spermidine. In the order Thermotogales, Thermotoga contained spermidine, norspermidine, caldopentamine and homocaldopentamine. The latter two linear penta-amines were not found in Marinitoga and Petrotoga. In the order Thermales, Thermus and Marinithermus contained homospermidine, norspermine and the linear penta-amines. Meiothermus lacked penta-amines. Vulcanithermus contained linear penta-amines and hexa-amines but not homospermidine. Oceanithermus contained spermine alone. Within the order Thermoanaerobacteriales, the two quaternary branched penta-amines were found in Thermanaeromonas and Thermoanaerobacter. Caldanaerobacter contained N4-bis(aminopropyl)spermidine. Thermoanaerobacterium lacked penta-amines. Thermaerobacter of the order Clostridiales contained N4-bis(aminopropyl)spermidine and agmatine. Thermosyntropha, Thermanaerovibrio, Thermobrachium ( the order Clostridiales), Sulfobacillus, Alicyclobacillus, Anoxybacillus, Ureibacillus, Thermicanus ( the order Bacillales), Desulfotomaculum, Desulfitobacterium and Pelotomaculum (the family Peptococcaceae) ubiquitously contained spermine. Some thermophiles of Bacillales added linear and branched penta-amines. (+info)
Thermoglobin, oxygen-avid hemoglobin in a bacterial hyperthermophile.
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The hemoglobin family of proteins, ubiquitous in all domains of life, evolved from an ancestral protein of primordial function to extant hemoglobins that perform a myriad of functions with diverged biochemical properties. Study of homologs in bacterial hyperthermophiles may shed light on both mechanisms of adaptation to extreme conditions and the nature of the ancestral protein. A hemoglobin was identified in Aquifex aeolicus, cloned, recombinantly expressed, purified, and characterized. This hemoglobin is monomeric, resistant to thermal and chemical denaturation, pentacoordinate in the ferrous deoxygenated state, and oxygen-avid. The oxygen equilibrium dissociation constant is approximately 1 nm at room temperature, due in part to a hydrogen bond between the bound ligand and a tyrosine residue in the distal pocket. These biochemical properties of A. aeolicus thermoglobin, AaTgb, may have been shared by the ancestral hemoglobin, thus suggesting possible primordial functions and providing a starting point for consequent evolution of the hemoglobin family. (+info)
Enzyme-coupled assay for beta-xylosidase hydrolysis of natural substrates.
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We describe here a new enzyme-coupled assay for the quantitation of d-xylose using readily available enzymes that allows kinetic evaluation of hemicellulolytic enzymes using natural xylooligosaccharide substrates. Hydrogen peroxide is generated as an intermediary analyte, which allows flexibility in the choice of the chromophore or fluorophore used as the final reporter. Thus, we present d-xylose quantitation results for solution-phase assays performed with both the fluorescent reporter resorufin, generated from N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), whose corresponding radical cation has an absorbance maximum at approximately 400 nm. We also describe a useful solid-phase variation of the assay performed with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride, which produces an insoluble brown precipitate. In addition, kinetic parameters for hydrolysis of the natural substrates xylobiose and xylotriose were obtained using this assay for a glycosyl hydrolase family 39 beta-xylosidase from Thermoanaerobacterium sp. strain JW/SL YS485 (Swiss-Prot accession no. O30360). At higher xylobiose substrate concentrations the enzyme showed an increase in the rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentrations k(cat) was 2.7 +/- 0.4 s(-1), K(m) was 3.3 +/- 0.7 mM, and k(cat)/K(m) was 0.82 +/- 0.21 mM(-1) . s(-1). Nonlinear curve fitting to a substrate inhibition model showed that for xylotriose K(i) was 1.7 +/- 0.1 mM, k(cat) was 2.0 +/- 0.1 s(-1), K(m) was 0.144 +/- 0.011 mM, and k(cat)/K(m) was 14 +/- 1.3 mM(-1) . s(-1). (+info)
Role of spontaneous current oscillations during high-efficiency electrotransformation of thermophilic anaerobes.
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Current oscillations at about 24 MHz were observed during electrotransformation (ET) of the thermophilic anaerobes Clostridium thermocellum ATCC 27405, C. thermocellum DSM 1313, and Thermoanaerobacterium saccharolyticum YS 485, using a pulse gated by a square signal generated by a custom generator. In experiments in which only the field strength was varied, all three of these strains resulted in a one-to-one correspondence between the appearance of current oscillations and successful ET. Oscillations accompanied ET of both C. thermocellum strains only at field strengths of > or =12 kV/cm, and ET was only observed above the same threshold. Similarly, for T. saccharolyticum, oscillations were only observed at field strengths of > or =10 kV/cm, and ET was only observed above the same threshold. When a passive electrical filter consisting of an inductor and resistor in parallel was added to the system to prevent the development of oscillations, ET efficiencies were reduced dramatically for all three strains at all field strengths tested. The maximum tested field strength, 25 kV/cm, resulted in the maximum measured transformation efficiency for all three strains. At this field strength, the efficiency of ET in the absence of oscillations was decreased compared to that observed in the presence of oscillations by 500-fold for C. thermocellum ATCC 27405, 2,500-fold for C. thermocellum DSM 1313, and 280-fold for T. saccharolyticum. Controls using the same apparatus with Escherichia coli cells or a resistor with a value representative of the direct current resistance of typical cell samples did not develop oscillations, and ET efficiencies obtained with E. coli were the same with or without the electrical filter included in the pulse generator circuit. The results are interpreted to indicate that spontaneously arising oscillations have a large beneficial effect on transformation efficiency in the system employed here and that the development of oscillations in this system is affected by the cell species present. (+info)
Reclassification of Thermoanaerobium acetigenum as Caldicellulosiruptor acetigenus comb. nov. and emendation of the genus description.
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Although the type species of the genus Thermoanaerobium, Thermoanaerobium brockii, was transferred to Thermoanaerobacter, Thermoanaerobium acetigenum was not transferred. Therefore, Thermoanaerobium acetigenum should be reclassified. Based on 16S rRNA gene sequence analysis and re-examination of physiological properties of the type strain, X6B(T) (=DSM 7040(T) = ATCC BAA-1149(T)), we propose that Thermoanaerobium acetigenum should be reclassified as Caldicellulosiruptor acetigenus comb. nov. Strain X6B(T) contains two separate 16S rRNA genes bracketing another species in the phylogenetic 16S rRNA gene-based tree. (+info)
Thermoanaerobacterium aciditolerans sp. nov., a moderate thermoacidophile from a Kamchatka hot spring.
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An anaerobic, moderately thermoacidophilic bacterium, strain 761-119T, was isolated from an acidic hot spring in the Orange Field of the Uzon Caldera (Kamchatka, far-eastern Russia). Cells were spore-forming, Gram-positive rods, possessing one polar flagellum. Growth of strain 761-119T was observed between 37 and 68 degrees C and in the pH(20 degrees C) range 3.2-7.1. No growth was observed within 5 days of incubation at or below 35 degrees C and at or above 70 degrees C, as well as at or below pH(20 degrees C) 2.8 and at or above pH(20 degrees C) 7.5. The optimal temperature and pH(20 degrees C) for growth were 55 degrees C and pH(20 degrees C) 5.7, respectively. A wide range of carbohydrates and polysaccharides were fermented, as well as peptides and proteinaceous substrates. The main products of glucose fermentation were acetate, ethanol, lactate, H2 and CO2. The DNA G+C content was 34 (+/-0.5) mol%. 16S rRNA gene sequence analysis indicated that strain 761-119T belonged to the genus Thermoanaerobacterium. The level of 16S rRNA gene sequence similarity with other Thermoanaerobacterium species was 86.5-97.8 %, with the only moderately acidophilic member of this genus, Thermoanaerobacterium aotearoense, being one of its closest relatives. DNA-DNA hybridization with T. aotearoense showed 33 % relatedness. Thus, morphological (one polar flagellum) and physiological characteristics (lower pH limit of growth at pH(20 degrees C) 3.2 compared with T. aotearoense) and 16S rRNA gene sequence analyses revealed that strain 761-119T represents a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium aciditolerans sp. nov. is proposed, with the type strain 761-119T (=DSM 16487T=VKM B-2363T). (+info)
In situ analysis of sulfur species in sulfur globules produced from thiosulfate by Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes.
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The Firmicutes Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes convert thiosulfate, forming sulfur globules inside and outside cells. X-ray absorption near-edge structure analysis revealed that the sulfur consisted mainly of sulfur chains with organic end groups similar to sulfur formed in purple sulfur bacteria, suggesting the possibility that the process of sulfur globule formation by bacteria is an ancient feature. (+info)
A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides.
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CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6-fold by ATP. Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100-10000-fold higher than for other CBS-domain-containing proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+-translocating PPase with a comparable PP(i)-hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP]+[ADP]) ratios respectively. (+info)