RESUMO
The upgrade of D-xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo-Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles, Thermotoga maritima, and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20â¯mM xylose to 16.1â¯mM inositol with a conversion efficiency of 96.6% at 70⯰C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70⯰C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production.
Assuntos
Suplementos Nutricionais/análise , Engenharia Metabólica/métodos , Açúcares/química , Madeira/química , Xilose/química , Trifosfato de Adenosina/metabolismo , Archaeoglobus/enzimologia , Archaeoglobus/metabolismo , Catálise , Inositol/metabolismo , Magnésio/metabolismo , Redes e Vias Metabólicas , NAD/metabolismo , Fosforilação , Thermotoga maritima/enzimologia , Thermus/enzimologia , Thermus/metabolismoRESUMO
Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0-2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (≤ 97.01%), digital DNA-DNA hybridization (≤ 32.7%), OrthoANI (≤ 87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C15:0, iso-C17:0 and anteiso-C15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by ß-galactosidase and ß-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198T (= CGMCC 1.13590T = KCTC XXX).
Assuntos
Arseniatos/metabolismo , Thermus/genética , Tiossulfatos/metabolismo , Respiração Celular , Genoma Bacteriano , Fontes Termais/microbiologia , Metabolismo dos Lipídeos , Oxirredução , Termotolerância , Thermus/isolamento & purificação , Thermus/metabolismoRESUMO
Sliding clamps on DNA consist of evolutionarily conserved enzymes that coordinate DNA replication, repair, and the cellular DNA damage response. MutS homolog (MSH) proteins initiate mismatch repair (MMR) by recognizing mispaired nucleotides and in the presence of ATP form stable sliding clamps that randomly diffuse along the DNA. The MSH sliding clamps subsequently load MutL homolog (MLH/PMS) proteins that form a second extremely stable sliding clamp, which together coordinate downstream MMR components with the excision-initiation site that may be hundreds to thousands of nucleotides distant from the mismatch. Specific or nonspecific binding of other proteins to the DNA between the mismatch and the distant excision-initiation site could conceivably obstruct the free diffusion of these MMR sliding clamps, inhibiting their ability to initiate repair. Here, we employed bulk biochemical analysis, single-molecule fluorescence imaging, and mathematical modeling to determine how sliding clamps might overcome such hindrances along the DNA. Using both bacterial and human MSH proteins, we found that increasing the number of MSH sliding clamps on a DNA decreased the association of the Escherichia coli transcriptional repressor LacI to its cognate promoter LacO. Our results suggest a simple mechanism whereby thermal diffusion of MSH sliding clamps along the DNA alters the association kinetics of other DNA-binding proteins over extended distances. These observations appear generally applicable to any stable sliding clamp that forms on DNA.
Assuntos
DNA Bacteriano/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Thermus/metabolismo , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , Modelos Teóricos , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
A Gram-stain-negative, lack of motility, catalase- and oxidase- positive bacterium (strain MT1(T)) was isolated from Buharkent hot spring in Aydin, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain was able to grow at 45-80 °C, pH 5.5-10.5 and with a NaCI tolerance up to 2.0% (w/v). Strain MT1(T) was able to utilize d-mannitol and l-arabinose, not able to utilize d-cellobiose as sole carbon source. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Thermus; strain MT1(T) detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. The predominant fatty acids of strain MT1(T) were iso-C(15:0) (43.0%) and iso-C(17:0) (27.4%). Polar lipid analysis revealed a major phospholipid, one major glycolipid, one major aminophospholipid, two minor aminolipids, one minor phospholipid, and several minor glycolipids. The major isoprenoid quinone was MK-8. The DNA G+C content of MT1(T) was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1(T) is considered to represent a novel species of the genus Thermus, for which the name Thermus anatoliensis sp. nov. is proposed. The type strain is MT1(T) (=NCCB 100425(T) =LMG 26880(T)).
Assuntos
Fontes Termais/microbiologia , Thermus/classificação , Thermus/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , DNA Ribossômico/genética , Ácidos Graxos/metabolismo , Glicolipídeos/metabolismo , Fosfolipídeos/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Thermus/genética , Thermus/metabolismo , TurquiaRESUMO
Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalização , Cristalografia por Raios X , Escherichia coli/metabolismo , Humanos , Conformação Molecular , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Peptídeos/química , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Ribossomos/química , Estereoisomerismo , Thermus/metabolismoRESUMO
To understand how macromolecular crowding affects enzyme activity, we quantified the Michaelis-Menten kinetics of mitochondrial malate dehydrogenase (MDH) in the presence of hen egg white (HEW), lysozyme, bovine serum albumin (BSA), gum arabic, poly(vinylpyrrolidone) (PVP), and dextrans of various molecular weights. Although crowding tended to decrease Km and Vmax values, the magnitude depended on the crowding agent, reaction direction, and isozyme (mitochondrial porcine heart or thermophlic TaqMDH from Thermus flavus). Crowding slowed oxaloacetate reduction more significantly than malate oxidation, which may suggest that mitochondrial enzymes have evolved to function optimally under the crowded constraints in which they are immersed. Since direct comparisons of neutral to charged crowders are underrepresented in the literature, we performed these studies and found that neutral crowding agents lowered Vmax values more than charged crowders of similar size. The exception was hen egg white, a mixture of charged proteins that caused the largest observed decreases in both Km and Vmax. Finally, the data provide insight about the mechanism by corroborating MDH subunit dependence.
Assuntos
Malato Desidrogenase/metabolismo , Mitocôndrias/enzimologia , Thermus/enzimologia , Animais , Bovinos , Galinhas , Dextranos/química , Goma Arábica/química , Cinética , Substâncias Macromoleculares/química , Malatos/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Muramidase/química , Ácido Oxaloacético/metabolismo , Oxirredução , Povidona/química , Soroalbumina Bovina/química , Suínos , Thermus/química , Thermus/metabolismoRESUMO
Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein's stepping motion at high interhead separations. On the basis of these results, we propose a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.
Assuntos
Trifosfato de Adenosina/química , Dineínas/química , Microtúbulos/química , Adenosina Trifosfatases/química , Animais , Citoplasma/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/química , Método de Monte Carlo , Movimento (Física) , Mutação , Nucleotídeos/química , Nucleotídeos/genética , Óptica e Fotônica , Conformação Proteica , Engenharia de Proteínas/métodos , Multimerização Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Ouriços-do-Mar , Estresse Mecânico , Thermus/metabolismoRESUMO
The high-resolution structure of the eukaryotic ribosome from yeast, determined at 3.0-Å resolution, permitted the unambiguous determination of the protein side chains, eukaryote-specific proteins, protein insertions, and ribosomal RNA expansion segments of the 80 proteins and â¼5,500 RNA bases that constitute the 80S ribosome. A comparison between this first atomic model of the entire 80S eukaryotic ribosome and previously determined structures of bacterial ribosomes confirmed early genetic and structural data indicating that they share an evolutionarily conserved core of ribosomal RNA and proteins. It also confirmed the conserved organization of essential functional sites, such as the peptidyl transferase center and the decoding site. New structural information about eukaryote-specific elements, such as expansion segments and new ribosomal proteins, forms the structural framework for the design and analysis of experiments that will explore the eukaryotic translational apparatus and the evolutionary forces that shaped it. New nomenclature for ribosomal proteins, based on the names of protein families, has been proposed.
Assuntos
Fungos/metabolismo , Proteínas Ribossômicas/química , Ribossomos/química , Bactérias/química , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Modelos Moleculares , Conformação Molecular , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Thermus/metabolismoRESUMO
Single-molecule trajectory analysis has suggested DNA repair proteins may carry out a one-dimensional (1D) search on naked DNA encompassing >10,000 nucleotides. Organized cellular DNA (chromatin) presents substantial barriers to such lengthy searches. Using dynamic single-molecule fluorescence resonance energy transfer, we determined that the mismatch repair (MMR) initiation protein MutS forms a transient clamp that scans duplex DNA for mismatched nucleotides by 1D diffusion for 1 s (~700 base pairs) while in continuous rotational contact with the DNA. Mismatch identification provokes ATP binding (3 s) that induces distinctly different MutS sliding clamps with unusual stability on DNA (~600 s), which may be released by adjacent single-stranded DNA (ssDNA). These observations suggest that ATP transforms short-lived MutS lesion scanning clamps into highly stable MMR signaling clamps that are capable of competing with chromatin and recruiting MMR machinery, yet are recycled by ssDNA excision tracts.
Assuntos
Proteínas de Bactérias/metabolismo , Reparo de Erro de Pareamento de DNA , DNA Bacteriano/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Thermus/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Ligação Proteica , Thermus/química , Thermus/genéticaRESUMO
Translation on the ribosome is controlled by external factors. During polypeptide lengthening, elongation factors EF-Tu and EF-G consecutively interact with the bacterial ribosome. EF-Tu binds and delivers an aminoacyl-tRNA to the ribosomal A site and EF-G helps translocate the tRNAs between their binding sites after the peptide bond is formed. These processes occur at the expense of GTP. EF-Tu:tRNA and EF-G are of similar shape, share a common binding site, and undergo large conformational changes on interaction with the ribosome. To characterize the internal motion of these two elongation factors, we used 25 ns long all-atom molecular dynamics simulations. We observed enhanced mobility of EF-G domains III, IV, and V and of tRNA in the EF-Tu:tRNA complex. EF-Tu:GDP complex acquired a configuration different from that found in the crystal structure of EF-Tu with a GTP analogue, showing conformational changes in the switch I and II regions. The calculated electrostatic properties of elongation factors showed no global similarity even though matching electrostatic surface patches were found around the domain I that contacts the ribosome, and in the GDP/GTP binding region.
Assuntos
Guanosina Trifosfato/química , Simulação de Dinâmica Molecular , Fator G para Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/química , Aminoacil-RNA de Transferência/química , Ribossomos/química , Sítios de Ligação , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Movimento (Física) , Fator G para Elongação de Peptídeos/biossíntese , Fator G para Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/biossíntese , Fator Tu de Elongação de Peptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas/fisiologia , Conformação Proteica , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo , Thermus/enzimologia , Thermus/metabolismoRESUMO
The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well.
Assuntos
Peptídeos/química , Ribossomos/química , Aminoácidos/química , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Guanosina Trifosfato/química , Cinética , Modelos Químicos , Conformação Molecular , Conformação de Ácido Nucleico , Peptidil Transferases/química , RNA Mensageiro/metabolismo , RNA de Transferência/química , Thermus/metabolismoRESUMO
Quality control mechanisms during protein synthesis are essential to fidelity and cell survival. Leucyl-tRNA synthetase (LeuRS) misactivates non-leucine amino acids including isoleucine, methionine, and norvaline. To prevent translational errors, mischarged tRNA products are translocated 30A from the canonical aminoacylation core to a hydrolytic editing-active site within a completely separate domain. Because it is transient, the tRNA translocation mechanism has been difficult to isolate. We have identified a "translocation peptide" within Escherichia coli LeuRS. Mutations in the translocation peptide cause tRNA to selectively bypass the editing-active site, resulting in mischarging that is lethal to the cell. This bypass mechanism also rescues aminoacylation of an editing site mutation that hydrolyzes correctly charged Leu-tRNA(Leu). Thus, these LeuRS mutants charge tRNA(Leu) but fail to translocate these products to the hydrolytic site, where they are cleared to guard against genetic code ambiguities.
Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Edição de RNA , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Sobrevivência Celular , Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Mutação , Peptídeos/química , Estrutura Terciária de Proteína , RNA de Transferência/metabolismo , Homologia de Sequência de Aminoácidos , Thermus/metabolismoRESUMO
DNA mismatch repair is initiated by the recognition of mismatches by MutS proteins. The mechanism by which MutS searches for and recognizes mismatches and subsequently signals repair remains poorly understood. We used single-molecule analyses of atomic force microscopy images of MutS-DNA complexes, coupled with biochemical assays, to determine the distributions of conformational states, the DNA binding affinities, and the ATPase activities of wild type and two mutants of MutS, with alanine substitutions in the conserved Phe-Xaa-Glu mismatch recognition motif. We find that on homoduplex DNA, the conserved Glu, but not the Phe, facilitates MutS-induced DNA bending, whereas at mismatches, both Phe and Glu promote the formation of an unbent conformation. The data reveal an unusual role for the Phe residue in that it promotes the unbending, not bending, of DNA at mismatch sites. In addition, formation of the specific unbent MutS-DNA conformation at mismatches appears to be required for the inhibition of ATP hydrolysis by MutS that signals initiation of repair. These results provide a structural explanation for the mechanism by which MutS searches for and recognizes mismatches and for the observed phenotypes of mutants with substitutions in the Phe-Xaa-Glu motif.
Assuntos
Pareamento Incorreto de Bases , Reparo do DNA , Proteína MutS de Ligação de DNA com Erro de Pareamento/fisiologia , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Sequência de Bases , DNA/química , Hidrólise , Microscopia de Força Atômica , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Fenótipo , Conformação Proteica , Thermus/metabolismoRESUMO
Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.
Assuntos
Técnicas de Tipagem Bacteriana , Fontes Termais/microbiologia , Thermus/classificação , Microbiologia da Água , Adaptação Fisiológica , Proteínas de Bactérias/análise , Biodiversidade , DNA Bacteriano/análise , Bases de Dados Genéticas , Eletroforese em Gel de Poliacrilamida , Enzimas/análise , Evolução Molecular , Genótipo , Concentração de Íons de Hidrogênio , Islândia , Oxirredução , Fenótipo , Filogenia , RNA Ribossômico/genética , Ribotipagem , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Cloreto de Sódio/metabolismo , Temperatura , Thermus/enzimologia , Thermus/genética , Thermus/crescimento & desenvolvimento , Thermus/isolamento & purificação , Thermus/metabolismo , Thermus thermophilus/classificação , Tiossulfatos/metabolismoRESUMO
The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure of Ffh alone and in the complex with 4.5S RNA by measuring distances between different positions within Ffh and between Ffh and 4.5S RNA by fluorescence resonance energy transfer (FRET). According to the FRET distances, NG and M domains in free Ffh are in close contact, as in the A/A arrangement in the crystal structure of Ffh from Thermus aquaticus, in agreement with the formation of a crosslink between cysteine residues at two critical positions in the G and M domains. Upon Ffh binding to 4.5S RNA or a 61 nucleotide fragment comprising internal loops A-C, the G and M domains move apart to assume a more open conformation, as indicated by changes of FRET distances. The movement is smaller when Ffh binds to a 49 nucleotide fragment of 4.5S RNA comprising only internal loops A and B, i.e. lacking the binding site of the NG domain. The FRET results suggest that in the SRP complex 4.5S RNA is present in a bent, rather than extended, conformation. The domain rearrangement of Ffh that takes place upon formation of the SRP is probably important for subsequent steps of membrane targeting, including interactions with the translating ribosome and the SRP receptor.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Partícula de Reconhecimento de Sinal/química , Anisotropia , Sequência de Bases , Compostos Bicíclicos com Pontes/química , Membrana Celular/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , RNA/química , Thermus/metabolismo , Fatores de TempoRESUMO
Microbiota from multiple kingdoms (e.g., Eubacteria, Fungi, Protista) thrive at temperature optima ranging from 0-20 degrees C (psychrophiles) to 40-85 degrees C (thermophiles). In this study, we have monitored changes in adenylate levels and growth rate as a function of temperature in disparate thermally adapted organisms. Our data indicate that growth rate and adenylate levels increase with temperature in mesophilic and thermophilic species, but rapid losses of adenosine 5'-triphosphate (ATP) occur upon cold or heat shock. By contrast, psychrophilic species decrease adenylate levels but increase growth rate as temperatures rise within their viable range. Moreover, psychrophilic ATP levels fell rapidly upon heat shock, but dramatic gains in ATP (approximately 20-50%) were observed upon cold shock, even at sub-zero temperatures. These results suggest that energy metabolism in thermophiles resembles that in mesophiles, but that elevated adenylate nucleotides in psychrophiles may constitute a compensatory strategy for maintaining biochemical processes at low temperature.
Assuntos
Monofosfato de Adenosina/metabolismo , Aclimatação/fisiologia , Trifosfato de Adenosina/química , Metabolismo Energético , Escherichia coli/metabolismo , Temperatura Alta , Modelos Biológicos , Pseudomonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Choque , Especificidade da Espécie , Sporothrix/metabolismo , Temperatura , Thermus/metabolismo , Fatores de TempoRESUMO
Thermophilic bacterium strain C2, which has the ability to transform crude oils, was isolated from the reservoir of the Shengli oil field in East China. The Gram-negative, rod-shaped, nonmotile cells were grown at a high temperature, up to 83 degrees C, in the neutral to alkaline pH range. Depending on the culture conditions, the organism occurred as single rods or as filamentous aggregates. Strain C2 was grown chemoorganotrophically and produced metabolites, such as volatile fatty acids, 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl)ester, dibutyl phthalate, and di-n-octyl phthalate. It could metabolize different organic substrates (acetate, D-glucose, fructose, glycerol, maltose, pyruvate, starch, sucrose, xylose, hexadecane). The G+C content (68 mol%) and the 16S rRNA sequence of strain C2 indicated that the isolate belonged to the genus Thermus. The strain affected different crude oils and changed their physical and chemical properties. The biochemical interactions between crude oils and strain C2 follow distinct trends characterized by a group of chemical markers (saturates, aromatics, resins, asphaltenes). Those trends show an increase in saturates and a decrease in aromatics, resins, and asphaltenes. The bioconversion of crude oils leads to an enrichment in lighter hydrocarbons and an overall redistribution of these hydrocarbons.
Assuntos
Petróleo/metabolismo , Microbiologia do Solo , Thermus/isolamento & purificação , Thermus/metabolismo , Alcanos/análise , Técnicas de Tipagem Bacteriana , Composição de Bases , Biodegradação Ambiental , China , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/química , DNA Ribossômico/isolamento & purificação , Dibutilftalato/análise , Metabolismo Energético/fisiologia , Ácidos Graxos Voláteis/análise , Genes de RNAr , Hidrocarbonetos/metabolismo , Concentração de Íons de Hidrogênio , Ácidos Ftálicos/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Temperatura , Thermus/citologia , Thermus/crescimento & desenvolvimentoRESUMO
Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S(O) as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA-DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.
Assuntos
Ferro/metabolismo , Thermus/classificação , Thermus/metabolismo , Composição de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Ácidos Graxos/análise , Biologia Molecular , Oxirredução , Fenótipo , Filogenia , Microbiologia do Solo , África do Sul , Thermus/genética , Thermus/isolamento & purificaçãoRESUMO
A gene (thaI) corresponding to L-arabinose isomerase from Thermus strain IM6501 was cloned by PCR. It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da. The deduced amino acid sequence had 96.8% identity with the L-arabinose isomerase of Geobacillus stearothermophilus. Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin. The purified ThaI was thermostable with maximal activity at 60 degrees C at pH 8 for 30 min of reaction. Zn2+ and Ni2+ inactivated the catalytic activity of ThaI, 5 mM Mn2+ enhanced the bioconversion yield by 90%. The bioconversion yield of 54% from D-galactose to D-tagatose was obtained by recombinant ThaI at 60 degrees C over 3 d.
Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Hexoses/síntese química , Thermus/enzimologia , Thermus/genética , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/isolamento & purificação , Bacillaceae/enzimologia , Bacillaceae/genética , Clonagem Molecular , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose/química , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Concentração de Íons de Hidrogênio , Controle de Qualidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Especificidade da Espécie , Temperatura , Thermus/classificação , Thermus/metabolismoRESUMO
Predicted highly expressed (PHX) and putative alien genes determined by codon usages are characterized in the genome of Deinococcus radiodurans (strain R1). Deinococcus radiodurans (DEIRA) can survive very high doses of ionizing radiation that are lethal to virtually all other organisms. It has been argued that DEIRA is endowed with enhanced repair systems that provide protection and stability. However, predicted expression levels of DNA repair proteins with the exception of RecA tend to be low and do not distinguish DEIRA from other prokaryotes. In this paper, the capability of DEIRA to resist extreme doses of ionizing and UV radiation is attributed to an unusually high number of PHX chaperone/degradation, protease, and detoxification genes. Explicitly, compared with all current complete prokaryotic genomes, DEIRA contains the greatest number of PHX detoxification and protease proteins. Other sources of environmental protection against severe conditions of UV radiation, desiccation, and thermal effects for DEIRA are the several S-layer (surface structure) PHX proteins. The top PHX gene of DEIRA is the multifunctional tricarboxylic acid (TCA) gene aconitase, which, apart from its role in respiration, also alerts the cell to oxidative damage.