RESUMEN
Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC-MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.
Asunto(s)
Proteínas Arqueales/clasificación , Proteoma/clasificación , Pyrococcus furiosus/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Datos de Secuencia Molecular , Señales de Clasificación de Proteína , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Pyrococcus furiosus/química , Pyrococcus furiosus/genética , Vías SecretorasRESUMEN
On behalf of the German chapter of the International Association for the Study of Pain (IASP) recommendations for German pain treatment services have been developed for the first time. The criteria were based on the IASP recommendations but adapted to the specific German situation. According to the structure and process criteria four different levels of pain treatment services can be distinguished. The aim of the recommendations is to serve as a guide for future development and implementation of pain therapy and quality assurance.
Asunto(s)
Dolor Crónico/terapia , Clínicas de Dolor/clasificación , Atención Ambulatoria/organización & administración , Dolor de Espalda/terapia , Dolor Crónico/clasificación , Alemania , Trastornos de Cefalalgia/terapia , Implementación de Plan de Salud/organización & administración , Necesidades y Demandas de Servicios de Salud/organización & administración , Investigación sobre Servicios de Salud/organización & administración , Humanos , Clínicas de Dolor/organización & administración , Garantía de la Calidad de Atención de Salud/organización & administración , Sociedades MédicasRESUMEN
A specific 16S rRNA-targeted oligonucleotide probe (PIR1223) for the genus Pirellula and a species-specific probe (RB454) for Pirellula sp. strain SH1 have been designed and optimized. Together with the already existing order-specific probe PLA886, the two newly designed probes were used to detect and identify planctomycetes, pirellulae, and close relatives of Pirellula sp. strain SH1 in different habitats. With the help of these probes for detection and identification, bacteria of the genus Pirellula were detected and cultivated from tissue of the Mediterranean sponge Aplysina aerophoba and from the water column of the Kiel Fjord. An unexpected result was the close phylogenetic relationship of the isolate from the sponge and the brackish water habitat Kiel Fjord as revealed by DNA/DNA hybridization.
Asunto(s)
Bacterias/genética , ADN/genética , Sondas de Oligonucleótidos , Poríferos/microbiología , Microbiología del Agua , Animales , Bacterias/ultraestructura , Composición de Base , Secuencia de Bases , Alemania , Mar Mediterráneo , Microscopía Electrónica , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Alineación de SecuenciaRESUMEN
A novel thermophilic, hydrogen-oxidizing bacterium, VM1T, has been isolated from a marine hydrothermal area of Vulcano Island, Italy. Cells of the strain were gram-negative rods, 2-4 microm long and 1-1.5 microm wide with four to seven monopolarly inserted flagella. Cells grew chemolithoautotrophically under an atmosphere of H2/CO2 (80:20) in the presence of low concentrations of O2 (optimum 1-2%). Carbohydrates and peptide substrates were not utilized, neither for energy generation nor as a source of cellular carbon. Growth of VM1T occurred between 45 and 80 degrees C with an optimum at 65 degrees C. Growth was observed between pH 5 and 7. NaCl stimulated growth in the range 0.5-6% with an optimum at 2-3%. Hydrogen could not be replaced by elemental sulfur or thiosulfate as electron donors. Nitrate and sulfate were not used as electron acceptors. The major respiratory lipoquinone was a new menathioquinone. Analysis of the fatty acids of VM1T revealed straight-chain saturated C18:0 and the unsaturated C18:1omega9c and C20:1omega9c as major components. The G+C content of the total DNA was 43 mol%. Phylogenetic analysis placed strain VM1T near the members of the genera Hydrogenobacter, Thermocrinis and Aquifex on a separate deep-branching phylogenetic lineage. Therefore, it is proposed that strain VM1T (= DSM 12046T = JCM 10974T) represents a novel species within a new genus, for which the name Hydrogenothermus marinus gen. nov., sp. nov., is proposed. In addition, it is shown that Calderobacterium hydrogenophilum should be transferred to the genus Hydrogenobacter; the name Hydrogenobacter hydrogenophilus comb. nov. (DSM 2913T = JCM 8158T) is proposed for this organism. Furthermore, on the basis of 16S rRNA sequence analysis, Hydrogenobacter acidophilus is only distantly related to Hydrogenobacter species. Owing to this finding and its growth at low pH, the name Hydrogenobaculum acidophilum gen. nov., comb. nov., is proposed for Hydrogenobacter acidophilus. The type strain is JCM 8795T (= DSM 11251T).
Asunto(s)
Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/crecimiento & desarrollo , Hidrógeno/metabolismo , Agua de Mar/microbiología , Composición de Base , Carbono/metabolismo , ADN Ribosómico/análisis , Ácidos Grasos/análisis , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/aislamiento & purificación , Lípidos/análisis , Datos de Secuencia Molecular , Oxidación-Reducción , Filogenia , Quinonas/metabolismo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , TemperaturaRESUMEN
Transcription of archaeal non-stress genes involves the basal factors TBP and TFB, homologs of the eucaryal TATA-binding protein and transcription factor IIB, respectively. No comparable information exists for the archaeal molecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE. These do not occur in some archaeal species, but are present in others possibly due to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with eukaryotic-like transcription machinery. Among the Archaea with the genes, those from the mesophilic methanogen Methanosarcina mazeii are the only ones whose basal (constitutive) and stress-induced transcription patterns have been determined. To continue this work, tbp and tfb were cloned from M. mazeii, sequenced, and the encoded recombinant proteins characterized in solution, separately and in complex with each other and with DNA. M. mazeii TBP ranks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N-terminal third. M. mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs. Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M. mazeii molecules are closer to the homologs from Archaeoglobus fulgidus than to any other. Antigenically, M. mazeii TBP and TFB are close to archaeal homologs within each factor family, but the two families are unrelated. The purified recombinant factors were functionally active in a cell-free in vitro transcription system, and were interchangeable with the homologs from Methanococcus thermolithotrophicus. The M. mazeii factors have a similar secondary structure by circular dichroism (CD). The CD spectra changed upon binding to the promoters of the stress genes grpE, dnaK, and dnaJ, with the changes being distinctive for each promoter; in contrast, no effect was produced by the promoter of a non-stress-gene. Factor(s)-DNA modeling predicted that modifications of H bonds are caused by TBP binding, and that these modifications are distinctive for each promoter. It also showed which amino acid residues would contact an extended TATA box with a B recognition element, and evolutionary conservation of the TBP-TFB-DNA complex orientation between two archaeal organisms with widely different optimal temperature for growth (37 and 100 degrees C).
Asunto(s)
Proteínas Arqueales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Genes Arqueales/genética , Methanosarcina , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Sistema Libre de Células , Dicroismo Circular , ADN de Archaea/química , ADN de Archaea/genética , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/inmunología , Células Eucariotas/química , Evolución Molecular , Enlace de Hidrógeno , Metales/metabolismo , Methanosarcina/química , Methanosarcina/genética , Methanosarcina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/inmunología , Conformación de Ácido Nucleico , Filogenia , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Factores de Transcripción/inmunología , Transcripción Genética/genéticaRESUMEN
A novel thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus hirschii Yel5aT (= DSM 11420T = JCM 10831T) has been isolated from the Angel Terrace Spring, Yellowstone National Park. The isolate was rod-shaped (1.0-1.5 x 0.8 microm) with a polarly inserted flagellum. Cells grew chemolithoautotrophically under an atmosphere of H2 and CO2 (80:20) in the presence of low concentrations of O2 (optimum 2.5%). Organotrophic growth occurred on complex organic substrates such as yeast extract and peptone and on organic acids. Carbohydrates and amino acids were not utilized. The strain grew between 50 and 67 degrees C; optimal growth occurred at a temperature of 63 degrees C. The pH optimum was 6.5. NaCl inhibited growth at concentrations higher than 1.5%. The major respiratory lipoquinone was ubiquinone-8. Analysis of fatty acids of Yel5aT revealed a straight-chain saturated C16:0 as the major component followed by cyclo C17:0 and cyclo C19:0. The G+C content of total DNA was 61 mol%. Phylogenetic analysis placed the strain in the beta-proteobacteria. The 16S rDNA sequence of strain Yel5aT was related to that of Hydrogenophilus thermoluteolus. To our knowledge, Hydrogenophilus hirschii is the most thermophilic micro-organism found within the proteobacteria that grows in the temperature range 50-68 degrees C.
Asunto(s)
Betaproteobacteria/clasificación , Calor , Hidrógeno/metabolismo , Microbiología del Agua , Composición de Base , Betaproteobacteria/aislamiento & purificación , Betaproteobacteria/ultraestructura , ADN Ribosómico/genética , Ácidos Grasos/análisis , Fenómenos Geológicos , Geología , Datos de Secuencia Molecular , Oxidación-Reducción , Quinonas/análisis , ARN Ribosómico 16S/genéticaRESUMEN
A spore-forming, halophilic bacterium was isolated from surface sediment located on the beach of Palaeochori Bay near to a shallow water hydrothermal vent area, Milos, Greece. The bacterium, designated SH 714T, consisted of motile, strictly aerobic rods which contained an Orn-D-Glu type murein and a G+C content of 35 mol%. Thin sections showed a cell wall typical for Gram-positive bacteria; the peptidoglycan layer, however, was very thin. The Gram-reaction of the organism was negative. Comparative 16S rRNA gene sequencing demonstrated that the isolate represents a new line of descent within the spore-forming rods branching at the periphery of the rRNA group 1 Bacillus (Bacillus sensu stricto). The nearest phylogenetic neighbours of the unknown bacterium were Bacillus haloalkaliphilus, Marinococcus albus and Halobacillus species. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as Filobacillus milensis gen. nov., sp. nov. The type strain is SH 714T (= DSM 13259T = ATCC 700960T).
Asunto(s)
Bacillus/clasificación , Peptidoglicano/química , Microbiología del Agua , Bacillus/química , Bacillus/genética , Bacillus/ultraestructura , Bacterias Aerobias , ADN Ribosómico , Sedimentos Geológicos , Ácido Glutámico/análisis , Calor , Datos de Secuencia Molecular , Ornitina/análisis , ARN Ribosómico 16S , Agua de Mar , Terminología como AsuntoRESUMEN
Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus, in contrast to polymerase II, does not require hydrolysis of the beta-gamma bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex spanned at least the DNA region from -11 to -1 at a tRNA(Val) promoter. The Methanococcus TBP-TFB promoter complex protected the DNA region from -40 to -14 on the noncoding DNA strand and the DNA segment from -36 to -17 on the coding DNA strand from DNase I digestion. This DNase I footprint was extended only to the downstream end by the addition of the RNA polymerase to position +17 on the noncoding strand and to position +13 on the coding DNA strand.
Asunto(s)
Proteínas Arqueales/metabolismo , ADN de Archaea/metabolismo , Methanococcus/metabolismo , Transcripción Genética , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Sistema Libre de Células , Huella de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/metabolismo , Desoxirribonucleasas/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólisis , Methanococcus/genética , Datos de Secuencia Molecular , Permanganato de Potasio , Regiones Promotoras Genéticas , TATA Box/genética , TATA Box/fisiología , Proteína de Unión a TATA-Box , Temperatura , Moldes Genéticos , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.
Asunto(s)
ADN de Archaea/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica Arqueal , Regiones Promotoras Genéticas , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Arqueales , Secuencia de Bases , Sitios de Unión , ADN de Archaea/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Escherichia coli , Proteína Reguladora de Respuesta a la Leucina , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na2S) was replaced by sodium sulfite (Na2SO3), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H2S was simply added to the headspace of serum bottles by a syringe. The use of H2S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably.
Asunto(s)
Archaea/crecimiento & desarrollo , Archaea/metabolismo , Sustancias Reductoras/metabolismo , Sulfitos/metabolismo , Anaerobiosis , División Celular , Medios de Cultivo/química , Sulfuro de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Methanococcus/crecimiento & desarrollo , Methanococcus/metabolismo , TemperaturaRESUMEN
RNA polymerase from the hyperthermophile archaeon Pyrococcus furiosus (Pfu) forms specific and transcriptionally active complexes with its conjugate transcription factors TBP (the archaeal TATA binding protein homolog) and TFB (the archaeal homolog of eukaryotic RNA polymerase II and III transcription factors TFIIB and Brf) at the Pfu glutamate dehydrogenase promoter. A photochemical crosslinking method was used to map the vicinity of the catalytic subunits of Pfu RNA polymerase to DNA locations distributed along the polymerase-promoter interface. The largest component of this archaeal polymerase is split into two subunits, A' and A", whose relatively sharp boundary of DNA crosslinking (probed on the transcribed strand) is centered five to six base pairs downstream of the transcriptional start site. A strong argument based on this information, on the well-defined homology between the core bacterial, archaeal and eukaryotic RNA polymerase subunits, and on the recently determined structure of a bacterial RNA polymerase specifies the directionality of DNA in the archaeal transcription complex and its trajectory downstream of the transcriptional start site.
Asunto(s)
ADN de Archaea/química , ADN de Archaea/metabolismo , Conformación de Ácido Nucleico , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genética , Factor de Transcripción TFIIB , Transcripción Genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Reactivos de Enlaces Cruzados , ADN de Archaea/genética , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Arqueales/genética , Glutamato Deshidrogenasa/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteína de Unión a TATA-Box , Moldes Genéticos , Factores de Transcripción/metabolismo , Rayos UltravioletaRESUMEN
Two novel, hyperthermophilic, anaerobic, heterotrophic archaea were isolated from shallow hydrothermal vents off Palaeochori Bay, Milos, Greece. Strain P5T (BK17S6-3-b2T) is an irregular coccus, with a single polar flagellum, growing optimally at 90 degrees C, pH 6 and 2% NaCl. The DNA G+C content was 45 mol%. Due to its morphology, phylogenetic analyses based on 16S rRNA gene sequencing, DNA-DNA hybridization experiments, physiological properties and nutritional features, this strain represents a new species within the genus Thermococcus for which the name Thermococcus aegaeicus is proposed. The type strain is P5T (= DSM 12767T = JCM 10828T). Strain p8T (BK20S6-10-b1T) is a coccus that forms aggregates. It grew optimally at 85 degrees C, pH 6 and 3% NaCl. The DNA G+C content was 38 mol%. Physiological properties and sequence analysis of the 165 rRNA gene, as well as DNA-DNA hybridization experiments, indicate that this strain is a new species belonging to the genus Staphylothermus for which the name Staphylothermus hellenicus is proposed. The type strain is P8T (= DSM 12710T = JCM 10830T).
Asunto(s)
Desulfurococcaceae/clasificación , Agua de Mar/microbiología , Temperatura , Thermococcus/clasificación , Composición de Base , ADN Ribosómico/análisis , Desulfurococcaceae/genética , Desulfurococcaceae/crecimiento & desarrollo , Desulfurococcaceae/aislamiento & purificación , Grecia , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Thermococcus/genética , Thermococcus/crecimiento & desarrollo , Thermococcus/aislamiento & purificaciónRESUMEN
Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100 degrees. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100 degrees. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg(2+) ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95 degrees was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degrees and 90 degrees. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter.
Asunto(s)
ADN-Topoisomerasas de Tipo I , ADN de Archaea/genética , Pyrococcus furiosus/genética , Transcripción Genética , Proteínas Arqueales/metabolismo , Sistema Libre de Células , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Archaea/química , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica Arqueal , Semivida , Calor , Conformación de Ácido Nucleico , Desnaturalización Proteica , Pyrococcus furiosus/fisiología , ARN de Archaea/biosíntesis , ARN de Archaea/química , ARN de Archaea/genética , ARN Mensajero/biosíntesis , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum DeltaH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the M. thermoautotrophicum RNAP and TFB has been demonstrated.
Asunto(s)
Proteínas Arqueales , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica Arqueal , Methanobacterium/genética , Factor de Transcripción TFIIB , Transcripción Genética , Secuencia de Aminoácidos , Centrifugación por Gradiente de Densidad , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Hidrógeno/metabolismo , Metano/metabolismo , Methanobacterium/enzimología , Methanobacterium/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pruebas de Precipitina , Proteína de Unión a TATA-Box , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The genetic organization, expression, and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus were analyzed. This locus includes the celB gene, which codes for an intracellular beta-glucosidase, and a divergently orientated gene cluster, adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular beta-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth of P. furiosus on either the beta-1,4-linked glucose dimer cellobiose or the beta-1,3-linked glucose polymer laminarin, the activities of both beta-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.
Asunto(s)
Alcohol Deshidrogenasa/genética , Celulasa/genética , Regulación de la Expresión Génica Arqueal , Familia de Multigenes , Operón , Pyrococcus furiosus/genética , Transcripción Genética , beta-Glucosidasa/genética , Alcohol Deshidrogenasa/metabolismo , Secuencia de Bases , Celulasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos , Intrones , Cinética , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Pyrococcus furiosus/enzimología , ARN Mensajero/genética , Mapeo Restrictivo , TATA Box , beta-Glucosidasa/metabolismoRESUMEN
The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.
Asunto(s)
Ferredoxinas/metabolismo , Gliceraldehído 3-Fosfato/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Pyrococcus furiosus/genética , Secuencia de Aminoácidos , Clonación Molecular , Genes Arqueales , Gluconeogénesis/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glucólisis/fisiología , Datos de Secuencia Molecular , Pyrococcus furiosus/enzimología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The DNA binding and compacting activities of the recombinant (r) archaeal histones rHMfA and rHMfB from Methanothermus fervidus, and rHPyA1 from Pyrococcus species GB-3a, synthesized in Escherichia coli, have been shown to be completely resistant to incubation for 4h at 95 degrees C in the presence of 1M KCl. Continued incubation of rHMfA and rHMfB at 95 degrees C resulted in a gradual loss of these activities, and rHMfA and rHMfB lost activity more rapidly at 95 degrees C when the salt environment was reduced to 200 mM K Cl. rHPyA1, in contrast, retained full activity even after a 60-h incubation at 95 degrees C in 1 M KCl, and reducing the salt concentration did not affect the heat resistance of rHPyA1. rHPya1-DNA complexes remained intact at 100 degrees C, and rHPyA1 bound to the template DNA in in vitro transcription reaction mixtures assembled using Pyrococcus furiosus components at 90 degrees C. Transcription in vitro from the P. furiosus gdh promoter was reduced by rHPyA1 binding, in a manner that was dependent on the histone-to-DNA ratio and on the topology of the DNA template. Transcription from circular templates was more sensitive to rHPyA1 binding than transcription from a linear template, consistent with rHPyA1 binding introducing physical barriers to transcription and causing changes in the topology of circular templates that also reduced transcription.
Asunto(s)
Archaea/metabolismo , ADN de Archaea/metabolismo , Histonas/química , Histonas/genética , Transcripción Genética , Secuencia de Aminoácidos , Archaea/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
The gene coding for ornithine carbamoyltransferase (OTCase, argF) in the hyperthermophilic archaea Pyrococcus furiosus was cloned by complementation of an OTCase mutant of Escherichia coli. The cloned P. furiosus argF gene also complemented a similar mutant of Saccharomyces cerevisiae. Sequencing revealed an open reading frame of 314 amino acids homologous to known OTCases and preceded by a TATA box showing only limited similarity with the Euryarchaeota consensus sequence. This is in accordance with the comparatively low in vitro promoter activity observed in a cell-free purified transcription system. Transcription initiates in vivo as well as in vitro at a guanine, 22 nucleotides downstream of the TATA box. Upstream from argF is a putative gene for diphthine synthetase, a eukaryotic enzyme assumed to occur also in archaea but not in bacteria.
Asunto(s)
Archaea/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Ornitina Carbamoiltransferasa/genética , Secuencia de Aminoácidos , Archaea/enzimología , Secuencia de Bases , Clonación Molecular , ADN Recombinante , Datos de Secuencia Molecular , Ornitina Carbamoiltransferasa/metabolismo , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
A new hyperthermophilic, strictly anaerobic crenarchaeote, Stetteria hydrogenophila DSM11227 representing a new genus within the family of Desulfurococcaceae, was isolated from the sediment of a marine hydrothermal system at Paleohori Bay in Milos, Greece. Cells are gram-negative irregular and disc-shaped cocci, 0.5-1.5 microm in diameter, which are flagellate and can form cytoplasmatic protrusions up to 2 microm in length. The strain grew optimally at 95 degrees C at pH 6.0 and at a NaCl concentration of 3%. The organism grew mixotrophically on peptide substrates. It required elemental sulfur as an external electron acceptor, and in addition, its growth was completely dependent on the presence of molecular hydrogen. Sulfur could be replaced by thiosulfate. H2S, CO2, acetate, and ethanol were identified as products of metabolism. The G + C content of DNA was 65 mol%. Analysis of its phylogenetic position by sequence analysis of 16S rRNA placed this organism in the family of Desulfurococcaceae. The dependence of this organism on both hydrogen and sulfur during growth on peptide substrates distinguishes Stetteria from all previously described species of Crenarchaeota.