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Gursinsky, Torsten. "Selenoprotein-codierende mRNAs aus Eubacterium acidaminophilum Erkennung durch den Selenocystein-spezifischen Elongationsfaktor SelB und Translation in Escherichia coli /." [S.l. : s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=967124549.
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Chang, I. S. "Carbon monoxide fermentation by Eubacterium limosum KIST612." Thesis, Swansea University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.636225.
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Carbon monoxide (CO)-utilising acetogens were enriched and the isolate KIST612 was selected for its abilities to tolerate high CO and acetate concentration. The isolate KIST612 was identified as Eubacterium limosum based on the morphological and biochemical characteristics. E. limosum KIST612 produced acetate and butyrate from CO. The optimum temperature and pH for the growth and organic acids formations were 37°C and 7.0, respectively. This bacterium was cultivated on phosphate-buffered basal medium (PBBM) with CO as the sole energy and carbon source. In a batch fermentation using a serum vial, E. limosum KIST612 grew at the initial growth rate of 0.15-0.16 h-1 with Ks for dissolved CO of 0.14 mM. When sufficient CO was supplied using a bubble column reactor, the maximum growth rate of E. limosum KIST612 was 0.23 h-1. The bacterial growth rate was reduced in the presence of acetate. A membrane reactor was employed to allow cell recycling continuous sparging CO fermentation to organic acid product removal. The reactor system used was a bubble column type reactor, and the overall volumetric CO mass transfer coefficient (kLa) of the reactor was 72 h-1. When the dry cell weight was 5.25 g/L in the reactor, the bacterial cell concentration did not increase at a CO partial pressure lower than 74 kPa, though CO was consumed with organic acid produced. At this stage, supply of CO mass transfer rate was lower than CO requirement to support maximum cell growth, but higher than that to maintain culture. Since CO was supplied higher than maintenance requirement under atmospheric CO pressure, bacterial cell concentration increased to 9.5 g/L.
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Gräntzdörffer, Andrea. "Formiat-Stoffwechsel in Eubacterium acidaminophilum molekulare und biochemische Charakterisierung der Wolfram- und Selen-haltigen Formiat-Dehydrogenasen sowie einer Eisen-Hydrogenase /." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=961933194.
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Kohlstock, Ulf-Martin. "Protein C von Eubacterium acidaminophilum Sequenzanalyse und Funktion der Thiole von GrdD für die Freisetzung von Acetylphosphat /." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963213261.
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Parther, Tina. "Die Peroxidase-Aktivität Selenocystein-haltiger Proteine des strikt anaeroben Bakteriums Eubacterium acidaminophilum." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=96943233X.
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Mariotto, Christian. "Production d'acides organiques à partir de substrats monocarbones par Eubacterium limosum." Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb37615744b.
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Mariotto, Christian. "Production d'acides organiques a partir de substrats monocarbones par eubacterium limosum." Toulouse, INSA, 1988. http://www.theses.fr/1988ISAT0023.
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Le metabolisme de la fermentation du methanol par eubacterium limosum est regule par la quantite d'equivalents reducteurs produits par la dissimilation du methanol. L'optimisation de la production d'acide butynique passe par ce flux de dissimilation. L'utilisation de la fermentation avec ajouts de substrats permet d'obtenir un titre maximal en acide butynique, et celle du couplage ultrafiltration-fermentation procure les valeurs maximales de la productivite en acide butynique
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Le, Bloas Pascale. "Etude des limitations et des régulations du métabolisme central de Eubacterium limosum." Toulouse, INSA, 1992. http://www.theses.fr/1992ISAT0042.
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Dans le cadre d'une etude physiologique sur e. Limosum, une procedure de dosage enzymatique/fluorimetrique des intermediaires glycolytiques et des coenzymes a ete elaboree: la methode est fiable et facilement adaptable a d'autres metabolites et microorganismes. Ces dosages ont ete appliques a des cultures discontinues de e. Limosum sur glucose et sur methanol/co#2. Les resultats ont conduit a des informations nouvelles sur les limitations et les regulations du metabolisme central de la bacterie: il apparait une correlation logique entre la concentration des enzymes et le sens ou la valeur des flux qu'elles doivent supporter. D'autre part, le pool de certains intermediaires glycolytiques, decrits comme effecteurs des enzymes irreversibles de la voie de emp, varie d'un substrat a l'autre dans le sens attendu d'une activation des enzymes glycolytiques sur glucose, d'une limitation sur methanol et vice versa pour les enzymes gluconeogeniques. Lors des cultures sur glucose, une limitation de la synthese de la formiate deshydrogenase peut etre a l'origine des limitations precoces de la fixation/reduction du co#2 puis se la pyruvate oxydoreductase, du flux glycolytique et de la croissance. Avec le methanol/co#2 comme substrat, une limitation precoce de l'utilisation du methanol est observee. Elle est concomitante d'un ralentissement des biosyntheses, attribuable a une disponibilite reduite en acetyl-coa et en atp. La limitation energetique, qui se repercute sur le flux gluconeogenique, se renforce et accentue la limitation de la croissance au-dela d'une concentration de butyrate reconnue comme inhibitrice chez e. Limosum
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Toyoda, Atsushi. "Studies on the Adhesion of the Major Rumen Cellulolytic Bacterium, Eubacterium cellulosolvens 5, to Cellulose." Kyoto University, 2003. http://hdl.handle.net/2433/148329.
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Haddock, John David. "Biochemistry and genetics of the pathway for the anaerobic degradation of aromatic compounds by Eubacterium oxidoreducens." Diss., Virginia Tech, 1990. http://hdl.handle.net/10919/39756.
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The biochemical pathway for the anaerobic degradation of gallate, pyrogallol and phloroglucinol by Eubacterium oxidoreducens was investigated. Phloroglucinol reductase was purified 90-fold, from the soluble fraction of cell extract, to electrophoretic homogeneity. The enzyme was an α₂ homodimer with a native Mr of 78,000, did not contain metals or cofactors and was specific for phloroglucinol and NADPH with a Km of 800 μM and 6.7 μM respectively at pH 6.8. The Km for phloroglucinol decreased with increasing pH. The enzyme catalyzed reaction was reversible and the equilibrium constant was 9.6. Dihydroresorcinol was a competitive inhibitor of the reverse reaction (Ki = 756 μM). Dihydrophloroglucinol produced in cell extract with H₂ as the reductant was identical to the compound produced by sodium borohydride reduction of phloroglucinol as shown by 1H NMR spectroscopy. The ¹³C NMR spectrum was consistent with the structural assignment of dihydrophloroglucinol. The mechanism of the proposed enzymatically catalyzed reaction is proposed to involve transfer of a hydride equivalent from NADPH to the carbonyl carbon of the phloroglucinol dianion.
Mutant strains of E. oxidoreducens that showed no gallate decarboxylase or dihydrophloroglucinol hydrolase activity were isolated after mutagenesis with ethylmethane sulfonate and emichment with ampicillin. The decarboxylase deficient mutants were unable to grow on gallate while pyrogallol and phloroglucinol supported growth. The hydrolase deficient mutants were unable to grow on any aromatic substrates and converted gallate to pyrogallol and dihydrophloroglucinol. The conversion of gallate to non-aromatic intermediates by cell extract of the wild-type stain was dependent on the presence of 1,2,3,5-benzenetetrol for the conversion of pyrogallol to phloroglucinol and on formate for the reduction of phloroglucinol to dihydrophloroglucinol. Transhydroxylase activity involved in the conversion of pyrogallol to phloroglucinol was induced by growth on aromatic substrates. The formate dehydrogenase was located in the soluble fraction of cell extract, and activity was protected from oxygen inactivation by sodium azide. The Km for formate and NADP was 290 μM and 140 μM respectively at pH 7.5. The pH optimum for activity was 7.5 and maximum activity was observed at a temperature of 50°C.Ph. D.
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Loubière, Pascal. "Caractérisation physiologique de la fermentation acétogène de Eubacterium limosum : limitation du flux carbone et mécanismes d'inhibition." Toulouse, INSA, 1990. http://www.theses.fr/1990ISAT0012.
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Les comportements cinetiques de eubacterium limosum, bacterie methylotrophe acidogene, sont clairement determines sur methanol et glucose. Le role exact des co-substrats possibles du methanol (co#2 ou acetate) est elucide et les limitations des flux carbones qui imposent les vitesses de croissance sont definies. Une strategie de selection passive a permis l'obtention d'un mutant capable de se developper sur methanol/acides organiques en absence de co#2. Des conditions precises de melanges de substrats, glucose et methanol, ont conduit, d'une part a une estimation des goulets d'etranglement et des regulations metaboliques mises en jeu, d'autre part a une amelioration de la croissance de e. Limosum en termes de taux de croissance maximal, de rendement en biomasse, et de production d'acides. Enfin, un transporteur de butyrate capable d'equilibrer le gradient de butyrate et le potentiel transmembranaire est mis a jour. Il resulte de son activite, certainement inductible, un cycle futile protonophore createur d'une limitation energetique expliquant le ralentissement precoce de la croissance en fermentation discontinue, et la baisse de la concentration en biomasse associee a une augmentation de la maintenance en culture continue
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Rauh, David [Verfasser], J. R. [Akademischer Betreuer] Andreesen, G. [Akademischer Betreuer] Sawers, and C. [Akademischer Betreuer] Kisker. "Das Wolframat-Bindeprotein TupA aus Eubacterium acidaminophilum / David Rauh. Betreuer: J. R. Andreesen ; G. Sawers ; C. Kisker." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2009. http://d-nb.info/1024874036/34.
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Franklund, Clifton. "Purification and Characterization of Two Oxidoreductases Involved in Bile Acid Modification by the Intestinal Anaerobe Eubacterium sp. VPI 12708." VCU Scholars Compass, 1990. https://scholarscompass.vcu.edu/etd/5597.
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Two enzymes, a 7α-hydroxysteroid dehydrogenase (7α-HSDH) and an NADH-dependent flavin oxidoreductase (NADH:FOR), have been purified to apparent electrophoretic homogeneity from the intestinal anaerobe Eubacterium sp. VPI 12708. Using a protocol consisting of four chromatographic separations, the 7α-HSDH was purified by a factor of over 1200-fold with more than a 30% final recovery. Subunit molecular mass was estimated to be 32 Kdal by SDS-PAGE, while native molecular mass estimates from gel filtration were 124 Kdal. The purified 7α-HSDH was able to utilize a variety of bile acids containing an unhindered 7α-hydroxy moiety as substrates, existing either as free acids or glycine or taurine conjugates. The presence of an oxo moiety at position 3 or 12 profoundly altered the kinetic values for this enzyme.
The structural gene for the 7α-HSDH was cloned on a 3.8 Kb Kpnl-Pstl fragment and was sequenced using the dideoxy chain termination method. An open reading frame of 798 bp encoding a 266 amino acid protein was detected. The N terminal amino acid sequence of the purified protein was identical to the first 22 amino acids predicted from the open reading frame. Putative transcriptional promotor and terminator regions along with a tentative ribosome binding site were also located. Northern blot analysis indicated that this protein was expressed constitutively on an approximately 1 Kb monocistronic message. During sequence analysis, the 7α-HSDH was found to be highly homologous to several members of the short-chain, non-zinc alcohol/polyol dehydrogenase superfamily.
Using a five step protocol, the NADH:FOR was also purified to homogeneity. A final purification of greater than 5OO-fold with an 11 % recovery was obtained. The purified protein had a subunit molecular mass of 72 Kdal and a native mass of 210 Kdal, suggesting that it exists either as a dimer or a trimer. Northern blot, Western blot, and activity stains of native gels all indicated that the NADH:FOR is a cholate-inducible protein. N-terminal amino acid sequence determination revealed a significant homology to enoate reductase from Clostridium kluyveri. Since the enoate reductase is involved in the reduction of a variety of α/β unsaturated carboxylates, this homology may be indicative of the physiological function of the NADH:FOR in Eu. sp VPI 12708.
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Schulze, Bettina. "Untersuchungen zur Umwandlung von 5-Hydroxybenzimidazol und 5-Hydroxy-6-methylbenzimidazol in den Basenteilen von Vitamin B 12 bei Eubacterium limosum /." [S.l. : s.n.], 1997. http://www.gbv.de/dms/bs/toc/238402347.pdf.
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Camprubí, Font Carla. "Genetics and transcriptomics of adherent-invasive Escherichia coli (AIEC): new approaches to uncover molecular markers for its rapid identification." Doctoral thesis, Universitat de Girona, 2019. http://hdl.handle.net/10803/672302.
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The adherent-invasive Escherichia coli (AIEC) pathotype could play a role in the course of Crohn’s disease. This is characterized by its capacity to adhere to and to invade intestinal epithelial cells as well as to replicate and survive within macrophages. At present, identification of the AIEC pathotype relies on time-consuming techniques based on the phenotypic screening of cultured bacteria, which are not standardized. In this thesis, we focused on the study of AIEC genetics in order to look for key characteristics that could assist the development of a molecular tool for the identification of the pathotype.
To sum up, the results of this work provide meaningful information that contributes to our understanding of AIEC genomics. In this case, two putative molecular markers resulting from a combination of genetic and/or phenotypic features have been presented and these could assist in AIEC screening. Finally, gene expression results provide new insights to better describe genes putatively involved in AIEC virulenceEl patotip adherent-invasiu d’Escherichia coli (AIEC) podria jugar un paper en el transcurs de la malaltia de Crohn. Aquest es caracteritza per tenir capacitat d’adhesió i invasió a cèl·lules de l’epiteli intestinal a més de replicar-se i sobreviure en macròfags. Actualment la única manera d’identificar aquests bacteris és analitzant aquestes característiques fenotípiques, un mètode poc estandarditzat i que requereix molt temps i dedicació. En la present tesi ens hem centrat en estudiar genèticament el patotip AIEC per tal de buscar característiques clau que puguin ajudar en el desenvolupament d’una eina molecular per a la seva identificació. En resum, els resultats d'aquest treball proporcionen informació significativa que contribueix a la comprensió de la genètica del patotip AIEC. En aquest cas, s'han presentat dos possibles marcadors moleculars resultants d'una combinació de característiques genètiques i/o fenotípiques que podrien ajudar en la detecció d’AIEC. Finalment, els resultats d'expressió gènica proporcionen noves idees per descriure millor els gens implicats en la virulència del patotip AIEC
Programa de Doctorat en Biologia Molecular, Biomedicina i Salut
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Grobost, Béatrice. "Identification de 32 souches de bacilles à gram positif asporulés anaérobies stricts : comparaison des tests conventionnels et des galeries d'identification rapide." Bordeaux 2, 1992. http://www.theses.fr/1992BOR2P066.
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Baylis, H. A. "The ribosomal RNA genes of Streptomyces coelicolor A3(2)." Thesis, University of East Anglia, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374251.
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Cummings, Stephen Paul. "Physiological adaptations to increasing salinity of a novel eubacterial halophile." Thesis, University of Sheffield, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387713.
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Richardson, Nathan J. "Eubacterial sensors for pollution monitoring and surface water intake protection." Thesis, Cranfield University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305409.
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Antoun, Ayman. "Mechanism and Regulation of Initiation of Protein Synthesis in Eubacteria." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5907.
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Boulahrouf, Abderrahmane. "La microflore responsable de la degradation des polyosides parietaux dans le gros intestin de la souris et du lapin : etude ecologique, facteurs de la colonisation, effets de la concentration en cellulose du regime, caracterisation des especes et activites in vitro." Clermont-Ferrand 2, 1988. http://www.theses.fr/1988CLF21114.
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Saleem, Batcha Raspudin [Verfasser]. "Crystallographic investigations of the stringent factor from Eubacteria / Raspudin Saleem Batcha." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2014. http://d-nb.info/1054366799/34.
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Newton, D. Trevor. "Mechanistic studies of methionyl-tRNA formyltransferase and the importance of formylation in eubacteria." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0003/MQ43192.pdf.
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Fernández, Jerí Yadira. "Identificación molecular de Eubacterias halófilas moderadas productoras de exopolisacáridos aisladas en las salinas de Atacocha – Ayacucho." Master's thesis, Universidad Nacional Mayor de San Marcos, 2007. https://hdl.handle.net/20.500.12672/10922.
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Selecciona, aisla e identifica bacterias halófilas moderadas productoras de Exopolisacáridos (EPS), de interés biotecnológico procedentes de las minas salinas de Atacocha - Ayacucho, se sembraron salmueras al 5% en el medio agua de sales suplementado con extracto de levadura al 0.5%, se seleccionaron 20 aislados productores de exopolisacáridos en función a sus características fenotípicas en el medio sólido maltosa levadura suplementado con 7.5 % de NaCl. Pruebas morfológicas, fisiológicas, bioquímicas y nutricionales fueron utilizadas en la determinación de las características fenotípicas. El Análisis de Restricción del los genes ribosómicos 16S amplificados por la Reacción en Cadena de la Polimerasa (ARDRA) se empleo para analizar la diversidad genética de los aislados productores de EPS. La secuenciación de los genes ribosómicos 16S de seis aislados productores de EPS permitió la identificación del género y especie. Todos los aislados fueron bacilos Gran negativos y crecieron en un rango óptimo de sal de 3 al 15%, a temperaturas entre 15 a 37 ºC y pH de 5 a 9, con características fenotípicas diferentes. El análisis UPGMA de los perfiles generados por ARDRA demostró que existen 18 especies diferentes. Las secuencias de los genes ribosómicos 16S analizados para los aislados ATA3, ATA4, ATA-11, ATA9, ATA17 y ATA20 mostraron que ATA4, ATA-11, ATA9 y ATA17 pertenecen al género Halomonas y ATA3 y ATA20 pertenecen al género Chromohalobacter.Tesis
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POSSOT, ODILE. "Etude du gene glna de methanococcus voltae et construction d'un vecteur d'integration permettant d'exprimer un marqueur de resistance eubacterien chez cette archaebacterie methanogene." Paris 7, 1990. http://www.theses.fr/1990PA077080.
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Un fragment d'adn de mc. Voltae portant de l'homologie au gene glna d'anabaena a ete sequence. Une phase ouverte de lecture de 1. 338 pb, a ete identifiee a glna sur la base de sa similitude avec les autres genes glna et de sa capacite de complementer un mutant d'e. Coli, elle code pour un polypeptide de 446 aminoacides et de 50. 142 daltons. Le gene glna de mc. Voltae a le plus de similitude avec les genes de bacillus subtilis et de clostridium acetobutylicum. Il est transcrit en un messager monocistronique; les regions encadrant l'orf glna n'ont aucune homologie avec d'autres genes ou sequences. Des mesures de l'activite glutamine synthetase indiquent que l'enzyme est partiellement reprime a de hautes concentrations en nh4#+. L'etude de la sensibilite de mc. Voltae a divers composes a permis de mettre en evidence six antibiotiques capables d'inhiber sa croissance. La caracterisation, in vitro, de leurs cibles biochimiques chez mc. Voltae a montre qu'elles sont identiques a celles connues chez les eubacteries: l'acide pseudomonique inhibe l'ile-arnt synthetase, la puromycine et l'acide fusidique inhibent la synthese proteique. Les effets de la coumermycine, de la novobiocine et de la ciprofloxacine, etudies notamment chez les methanogenes suggerent que les arachaebacteries possedent une adn gyrase de type eubacterien. Des vecteurs d'integration bases sur le vecteur puc18 d'e. Coli ont ete construits en introduisant, en particulier, dans le gene hisa de mc. Voltae, le gene de resistance a la puromycine (pac de streptomyces alboniger) ou le gene de resistance a l'acide fusidique (cat du transposon tn9) flanques de signaux forts de transcription de mc. Voltae. Apres transformation de cette souche, des bacteries ayant acquis la resistance a la puromycine par integration du vecteur dans le chromosome, ont pu etre selectionnees. Le gene pac d'origine bacterienne s'exprime de facon stable chez mc. Voltae
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Ortiz, Álvarez Rüdiger. "Microbial community assembly and biogeography in the Pyrenean lacustrine district." Doctoral thesis, Universitat de Girona, 2018. http://hdl.handle.net/10803/663398.
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The microbial metacommunity of the Pyrenees mountain range has never been studied at the regional level. In this PhD thesis, the main goal is to describe the taxonomic diversity and reveal the ecological processes (biotic and abiotic) behind the spatial distribution and heterogeneity of surface communities of alpine lakes. With a sampling of more than 300 lakes during summer of 2011 and high-througput sequencing of ribosomal markers (16S and 18S rRNA), we analized the microbial profiles of archaeal, bacterial and eukaryotic communities. We highlighted the discovery and description of novel and unknown lineages such as Pacearchaeota or Woesearchaeota (Archaea) or Chytridiomycota (Eukarya). We analyzed the role of environmental filtering and dispersal processes, as well as potential species interactions as drivers of local community assemblagesLa metacomunidad microbiana de Pirineos nunca ha sido estudiada a nivel de región completa. En esta tesis se busca describir la diversidad taxonómica y revelar los procesos ecológicos (bióticos y abióticos) que pueden estar tras la distribución espacial y la heterogeneidad de las comunidades superficiales de lagos alpinos. Mediante un muestreo de más de 300 lagos durante el verano de 2011 y técnicas de secuenciación masiva de genes ribosómicos (16S y 18S rRNA) se obtuvieron los perfiles microbianos de comunidades de arqueas, bacterias y eucariotas. Se puso énfasis en el descubrimiento y descripción de linajes nóveles y desconocidos como Pacearchaeota o Woesearchaeota (Archaea) o Chytridiomycota (Eukarya). Se analizan el filtro ambiental y procesos de dispersión, así como las interacciones potenciales entre especies como responsables del ensamblaje de las comunidades locales
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Wagener, Nadine [Verfasser]. "Kristallstruktur der Selen-abhängigen Nicotinat-Dehydrogenase aus Eubacterium barkeri / vorgelegt von Nadine Wagener." 2008. http://d-nb.info/1004864604/34.
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Parther, Tina [Verfasser]. "Die Peroxidase-Aktivität Selenocystein-haltiger Proteine des strikt anaeroben Bakteriums Eubacterium acidaminophilum / von Tina Parther." 2003. http://d-nb.info/96943233X/34.
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Liao, Daiqing. "The organization, expression, function and evolution of some essential genes from the hyperthermophilic eubacterium thermotoga maritima." Thesis, 1993. http://hdl.handle.net/2429/1701.
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The hyperthermophilic eubacterium Thermotoga maritima grows optimally at 80°C near marine geothermal locales. Phylogenetic analyses based on various molecular sequences indicate that T. maritima and other hyperthermophilic prokaryotes have very deep phylogenetic placements; i.e., that they have diverged early from the ancestor of living organisms. Thus, studies on the biochemistry and molecular biology of hyperthermophilic organisms such as T. maritima may shed light on the early evolution of life, as well as enhance our understanding of life at high temperature. In this study, a 5,800-base-pair DNA fragment from the chromosome of T. maritima was cloned and sequenced. This fragment encodes five tRNAs, the ribosomal protein L33, an integral membrane protein, SecE, which is probably involved in protein translocation, the transcription factor NusG, four large subunit ribosomal proteins (L11, L1, L10 and L12), and the N-terminus of the RNA polymerase 0 subunit. The transcriptional patterns of this gene cluster were analyzed using S1 nuclease protection and primer extension techniques. The tRNAgenes and the protein-encoding genes are co transcribed, except the 13 gene, which is transcribed separately. The following regulatory sequence elements were identified in this cloned fragment: five promoters (Pi and P2 in front of the first and second methionine tRNAs, respectively, PLio in the L1-L10 intergenic space, PL12 at the end of the L10 gene, and PR in the L12-(3 intergenic region), a transcription attenuator upstream of the L10 gene, a transcription terminator located between theL12 and the (3 subunit gene of the RNA polymerase, and an autogenous translational regulation site (the Ll binding site) located upstream of the L11 gene. The transcription factor NusG encoded in this cluster exhibits 43% amino acid sequence identity when aligned to its E. coli counterpart; the alignment is interrupted by a 171-amino-acid-long insertion into the T. maritima protein. The T. maritima NusG was over expressed in E. coli, and the recombinant NusG protein was purified. The NusG protein binds to DNA cooperatively, but nonspecifically. Two types of NusG-DNA complexes have been observed. The first type forms instantly and can be stained with ethidium bromide ("loose" complex); the second type forms more slowly, and is probably converted from the loose structure(s). The second type is probably more compact, as it can not be stained with ethidium bromide ("tight" complex). This protein binds to both ds- and ssDNA, but preferentially to dsDNA in a mixture of both DNA molecules. About 40 and 60NusG monomers per kilobases (pairs) of ds- and ssDNA, respectively, are required to form cooperative NusG-DNA complexes. When a relatively large amount ofNusG was added to an in vitro transcription assay, it appears to selectively suppress aberrant transcription initiation and termination, and at the same time, the production of specific transcripts is, at most, only marginally reduced. Available sequences that correspond to the E. coli ribosomal proteins L11, L1,L10 and L12 from eubacteria, archaebacteria and eukaryotes have been aligned, and the alignments were subjected to quantitative phylogenetic analysis. Eubacteria andeukaryotes each form a well-defined, coherent and non-overlapping group. Archaebacteria also form a coherent phylogenetic group by themselves, but the relationships between the major groups of archaebacteria and out groups (eubacteriaand eukaryotes) can not unambiguously be established. On the other hand, T.maritima does not appear as the deepest branch within the eubacterial kingdom; however, this placement is less definitive.
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Poehlein, Anja [Verfasser]. "Das Selenoprotein PrpU als Vermittler zwischen oxidativem und reduktivem Glycin-Metabolismus von Eubacterium acidaminophilum / von Anja Poehlein." 2008. http://d-nb.info/996019529/34.
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Gursinsky, Torsten [Verfasser]. "Selenoprotein-codierende mRNAs aus Eubacterium acidaminophilum : Erkennung durch den Selenocystein-spezifischen Elongationsfaktor SelB und Translation in Escherichia coli / von Torsten Gursinsky." 2002. http://d-nb.info/967124549/34.
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Kohlstock, Ulf-Martin [Verfasser]. "Protein C von Eubacterium acidaminophilum : Sequenzanalyse und Funktion der Thiole von GrdD für die Freisetzung von Acetylphosphat / von Ulf-Martin Kohlstock." 2001. http://d-nb.info/963213261/34.
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Hapel, Ashraf al [Verfasser]. "Nicotinatfermentation in Eubacterium barkeri : Klonierung des kompletten Nicotinat-Locus und Charakterisierung der 2-(Hydroxymethyl)glutarat-Dehydrogenase und Enamidase / vorgelegt von Ashraf al Hapel." 2005. http://d-nb.info/977126781/34.
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Gräntzdörffer, Andrea [Verfasser]. "Formiat-Stoffwechsel in Eubacterium acidaminophilum : molekulare und biochemische Charakterisierung der Wolfram- und Selen-haltigen Formiat-Dehydrogenasen sowie einer Eisen-Hydrogenase / von Andrea Gräntzdörffer." 2000. http://d-nb.info/961933194/34.
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Auger, Jérémie. "Impact des antibiotiques céfprozil et céfoxitine sur le microbiote Eggerthella lenta, lié au métabolisme du cardiotonique digoxine." Thèse, 2018. http://hdl.handle.net/1866/23628.
Full textAbstract:
La digoxine est un cardiotonique largement employé pour contrôler les symptômes de l'insuffisance cardiaque et de la fibrillation auriculaire. Il est connu depuis les années 1980 que le métabolite principal de la digoxine, la dihydrodigoxine, est produit exclusivement par le microbiome intestinal (métabolisme de premier passage) et plus précisément la bactérie Eggerthella lenta. Aux États-Unis, c'est 14% des participants à une étude qui excrétaient 40% et plus de la dose sous la forme de ce métabolite rapidement éliminable et ayant perdu son affinité pour sa cible. De plus, chaque année, la digoxine est le médicament qui engendre le plus d'hospitalisations pour effets secondaires toxiques. Les effets secondaires très problématiques de la digoxine sont souvent déclenchés par l'ajout d'antibiotiques (surtout les macrolides) à la prescription de digoxine. La théorie explorée ici explique les évènements de toxicité chez les patients métabolisateurs. Ces derniers ont une dose quotidienne de maintien de digoxine plus élevée pour compenser l'action de la bactérie et, lorsque ces patients reçoivent un antibiotique pour une infection non reliée à leur condition cardiaque, l'arrêt du métabolisme par le microbiome engendre une augmentation de la biodisponibilité de la digoxine. Si la concentration plasmatique du médicament augmente trop, les effets secondaires peuvent aller jusqu'à causer la mort. Dans le présent projet, nous avons vérifié la sensibilité de E. lenta à deux antibiotiques de la famille des céphalosporines de seconde génération, in vivo et in vitro. Pour les 18 volontaires qui ont été exposés à 2x500mg de céfprozil durant une semaine, il y a une tendance à la baisse de l'abondance de la bactérie d'intérêt (par 58,3% par rapport au niveau initial), mais pas de significativité au niveau des tests statistiques. Pour les échantillons complets de microbiome fécal, mis en culture avec et sans antibiotiques, il y a une différence statistiquement significative avec une valeur-p de 0,0457, alors que la croissance de E. lenta a été impactée négativement par l'ajout de céfprozil au milieu de culture. Les résultats valident une prémisse importante pour la démonstration du rôle du microbiome dans la pharmacocinétique de la digoxine et la gestion clinique du médicament cardiotonique.Digoxin is a widely used cardiotonic drug in the management of heart failure and atrial fibrillation. It has been known since the early 1980's that the main metabolite of digoxin, dihydrodigoxin, is synthesized by the gut microbiome during first pass metabolism and is exclusively produced by the bacteria Eggerthella lenta. In a clinical study done in the U.S.A., there were 14% of high metabolizers, for whom over 40% of the oral digoxin dose is transformed to the inactive metabolite and rapidly eliminated. Digoxin toxicity is the leading cause of hospitalization from medication's secondary effects. The toxicity events are often associated with the addition of an antibiotic (mostly from the macrolides class) to the patient's drugs regiments. The theory explored in this project could help explain the toxicity events in metabolizers. These patients have a higher daily digoxin maintenance dose to counteract the effects of the microbiome and are then prescribed antibiotics for an infection unrelated to their heart condition. The antibiotic alters E. lenta negatively, which cannot metabolize digoxin anymore and therefore augments the bioavailability of the cardiotonic. If the plasmatic concentration reaches dangerous levels (over 2ng/ml of plasma), the patients face adverse effects that include death. In the present project, we evaluated the susceptibility of E. lenta to two second generation cephalosporins, in vivo and in vitro. With the 18 healthy volunteers that were exposed to 2x500mg of cefprozil daily for 7 days, we observed a diminution of the abundance of the bacteria of interest by 58,3% from the initial levels. This change did not however produce statistically significant tests results. For the complete fecal microbiome that were cultivated in vitro, with or without cefprozil, the difference between the two conditions resulted in a statistically significant p-value of 0.0457, confirming the sensitivity of E. lenta to this cephalosporin. These results validate an important premise for the demonstration of the importance of the gut microbiome in the pharmacokinetics of digoxin and the clinical management of the drug to avoid toxicity events in clinical practice.
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Yen, Te-Ming, and 顏德民. "Evolution divergence of archaeal Rib7 and eubacterial RibG reductases: substrate specificity and cofactor preference." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/yy75t6.
Full textAbstract:
博士國立陽明大學
生化暨分子生物研究所
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Archaeal/fungal Rib7 and eubacterial RibG possess a reductase domain for ribosyl reduction in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for an amino and a carbonyl group of the pyrimidine ring, respectively. Structure-based mutational analysis displayed that no detectable activity was observed for the Bacillus subtilis RibG K151A, K151D, and K151E mutants, and the Methanosarcina mazei Rib7 D33N, D33K, and E156Q variants, while a decreased activity by 2-3 orders of magnitude compared with the wild-type for the M. mazei Rib7 N9A, S29A, D33A, and D57N variants. Our results suggest that the eubacterial conserved Lys151 in B. subtilis RibG, while the archaeal conserved Asp33 together with Arg36 in M. mazei Rib7, ensure the specific substrate recognition. Unexpectedly, an endogenous NADPH cofactor is observed in M. mazei Rib7, in which the 2’-phosphate group interacts with Ser88, and Arg91. Replacement of Ser88 with glutamate or Arg91 with alanine eliminates the endogenous NADPH binding. Activity assay in the presence of 1 mM DAROPP and 0.25 mM NAD(P)H, revealed that wild type and the S88E mutant have a clear cofactor preference, they were better at recognizing NADPH/NADH and NADH/NADPH by ~3 fold, respectively. In addition, the lower melting temperature (Tm) of ~10℃ for the S88E and R91A mutants suggests that nature had evolved a tightly bound NADPH to greatly enhance the structural stability of archaeal Rib7. On the other hand, RibGs from Thermotoga maritime, Escherichia coli, and Acinetobacter baumannii exist as a dimer through a similar reductase interface. However, B. subtilis RibG forms as a tetramer through the reductase and its unique deaminase interface. To investigate the functional role of the tetramer in B. subtilis RibG, seven mutants were generated through replacement of the deaminase interfacial residues with glutamate. Sedimentation velocity analyses revealed that several mutants existed as dimer or partial dimer. The activity assay showed that the dimeric and dimeric/tetrameric mutants have similar reductase activity to wild type but with a lack of detectable deaminase activity. Moreover, the stability assay displayed the lower Tm of 3-7℃ and lower resistance toward guanidinium chloride denaturation for all the variants. These data imply that the tetramer of B. subtilis RibG may maintain the active conformation of the deaminase domain and enhance the structural stability.
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Anuradha, S. "Mechanism Of Ribosome Recycling In Eubacteria, And The Impact Of rRNA Methylations On Ribosome Recycling And Fidelity Of Initiation In Esherichia coli." Thesis, 2009. http://hdl.handle.net/2005/629.
Full textAbstract:
The studies reported in this thesis address, firstly, aspects of ribosome recycling in eubacteria, and secondly, a preliminary characterization of an EFG-like locus from Mycobacterium smegmatis. A hitherto unsuspected role of the ribosome recycling factor in governing the fidelity of initiation has been discovered during the course of this work. A summary of the relevant literature is presented in chapter 1. Section I of the ‘General Introduction’ provides a brief review of the current understanding of protein biosynthesis, with a special emphasis on ribosome recycling and the fidelity of translation initiation. Section II provides a brief introduction to mycobacterial translation, and known deviations from the E. coli prototype are highlighted. This is followed by three chapters containing experimental work, as summarized below.
(i) Role of elongation factor G in governing specificity of ribosome recycling
In eubacteria and the eukaryotic organelles, the post-termination ribosome complexes are recycled by the combined action of ribosome recycling factor (RRF) and elongation factor G (EFG). Earlier studies both from our laboratory and other laboratories have revealed the existence of specific interactions between RRF and EFG that are crucial for ribosome recycling, using ribosomes from E. coli and factors from both E. coli and heterologous sources such as Mycobacterium tuberculosis, Thermus thermophilus etc. In this study, to further understand the mechanism of ribosome recycling, we employed polysomes from both E. coli and M. smegmatis and monitored ribosome recycling in in vitro assays using RRF and EFG from both these sources; in addition, in vivo assays were performed in E. coli using either temperature-sensitive strains or strains carrying a deletion in frr (encoding RRF) or fusA (encoding EFG) genes. It was found that, in E. coli, RRF from Mycobacterium tuberculosis and M. smegmatis function with MtuEFG or MsmEFG but not with EcoEFG. In vitro assays revealed that the mycobacterial EFGs facilitate recycling of both the mycobacterial and E. coli polysomes not only with mycobacterial RRFs but also with EcoRRF. In contrast, although EcoEFG binds to mycobacterial polysomes, carries out GTP hydrolysis and is reported to sustain translocation on mycobacterial ribosomes, its activity in recycling mycobacterial polysomes was undetectable with EcoRRF, as well as with the mycobacterial RRFs. Such an observation allowed us to infer that EFG establishes specific interactions with the ribosome that are crucial for ribosome recycling but not for translocation, suggesting that translocation and ribosome recycling are distinct functions of EFG. In addition, a number of EFG chimeras generated by swapping corresponding domains between Msm- and Eco-EFGs were analyzed for their ability to sustain translocation and/or ribosome recycling in E. coli and M. smegmatis, using a combination of in vivo (for E. coli) and in vitro (for both E. coli and M. smegmatis) approaches. Our observations reveal that a dual set of specific interactions of EFG with RRF and ribosome is essential for ribosome recycling. While the RRF-EFG specific interactions are predominantly localized to the domains IV and V of EFG, the EFG-ribosome specific interactions that are crucial for ribosome recycling are not localized to a specific region of EFG but are found throughout the molecule. Our novel observations also emphasize the importance of using ribosomes from heterologous sources to understand the mechanism of this crucial process.
(ii) Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli
Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions; however, very little is known about the role of these rRNA modifications in ribosome function. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of the intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. The isolation of a folD122 mutant strain of E. coli with a deficiency in rRNA methylations, as well as the availability of E. coli strains deficient for various individual methyltransferases that modify specific rRNA residues, provided us with a genetic tool to assay the role of rRNA methylations in ribosome recycling. We observed that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, a compromise in the RRF activity was found to afford increased initiation with a mutant tRNAfMet wherein the three consecutive G-C base pairs (29GGG31:39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNAMet (29UCA31:39ψGA41). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. In addition, it was also found that IF3 and rRNA methylations, both of which are known to affect fidelity of initiation, exert their effects through distinct mechanisms, despite the proximity of a cluster of methylated rRNA residues to the IF3 binding site on the 30S subunit.
(iii) Characterization of the role of EFG2, an EFG-like locus in Mycobacterium smegmatis
Several bacteria, including various species of mycobacteria (with the exception of Mycobacterium leprae) contain a second EFG-like locus, denoted as fusA2, which shows considerable homology to fusA (encoding EFG). A comparison of the sequences of EFG and EFG2 from various bacteria reveals that EFG2 contains a GTPase domain and domains with significant homology to EFG domains IV and V, suggesting that it may function as an elongation factor. With the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like protein factors has not been studied so far. Hence, it was of interest to characterize EFG2. In the current study, EFG2 from M. smegmatis was characterized both by in vitro biochemical assays as well as by in vivo experiments targeted to investigate the biological significance of EFG2 in mycobacteria. It was found that, unlike EFG, MsmEFG2 could not sustain either translocation or ribosome recycling in E. coli. Despite the fact that the purified MsmEFG2 could bind guanine nucleotides, it lacked the ribosome-dependent GTPase activity characteristic of EFG and other translation GTPases, suggesting that it was unlikely to function as an elongation factor. However, EFG2 was found to be expressed in stationary phase cultures of M. smegmatis. To understand the biological significance of EFG2, fusA2 was disrupted in M. smegmatis. The viability of the M. smegmatis mc2155 fusA2::kan derivative indicates that MsmfusA2 is a non-essential gene. While disruption of the fusA2 gene (encoding EFG2) in M. smegmatis does not appear to affect its growth and survival in log phase or stationary phase or under hypoxic conditions, preliminary experiments indicate that disruption of fusA2 confers a fitness disadvantage to M. smegmatis when competed against M. smegmatis mc2155 (with wild type fusA2 locus).
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Gaur, Rahul. "Metabolism Of Queuosine, A Modified Nucleoside, In Escherichia Coli And Caenorhabditis Elegans And Dual Function Of Bovine Mitochondrial Initiation Factor 2 As Initiation Factors 1 And 2 In Escherichia Coli." Thesis, 2007. http://hdl.handle.net/2005/543.
Full textAbstract:
The studies reported in this thesis address firstly, the biology of a modified nucleoside, Queuosine (Q) and secondly, the properties of mitochondrial translation initiation factor 2. A summary of the relevant literature on both these topics is presented in Chapter 1. Section I of this ‘General Introduction’ summarizes the literature on biosynthesis and physiological importance of Queuosine. Section II is a brief review of the current understanding of translation initiation in Eubacteria. Information about the mitochondrial translation initiation apparatus also features as a subsection. The next chapter (Chapter 2), describes the ‘Materials and Methods’ used throughout the experimental work presented in the thesis. It is followed by three chapters containing experimental work as described below:-
i) Biosynthesis of Queuosine (Q) in Escherichia coli
Q is a hypermodification of guanosine found at the wobble position of tRNAs with GUN anticodons. Q is thought to be produced via a complex multistep pathway, the details of which are not known. It was found in our laboratory that a naturally occurring strain of E. coli B105 lacked Q modification in the tRNAs. As the known enzymes of Q biosynthesis were functional in this strain, it presented us with the opportunity to uncover novel component(s) of Q biosynthetic pathway. In the present work, a genetic screen was developed to map the defect in E. coli B105 to a previously uncharacterised gene, ybaX, predicted to code for a 231 amino acid long protein with a pI of 5.6. Further genetic analyses showed that YbaX functions at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. The gene ybaX has been renamed as queC. Using a combination of bioinformatics based prediction and gene knockouts, we have also been able to place two more genes, queD and queE at the initial step in Q biosynthesis, suggesting that the initial reaction of Q biosynthesis might be more complex and mechanistically different than what has been proposed earlier.
ii) Caenorhabditis elegans as a Model System to Study Queuosine Metabolism in Metazoa
Animals are thought to obtain Q (or its analogs) as a micronutrient from dietary sources such as gut microflora, and the corresponding base is then inserted in the substrate tRNAs by tRNA guanine transglycosylase (TGT). In animal cells, changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumor growth but the precise function of Q in animal tRNAs remains unknown. A major obstacle in the study of Q metabolism in higher organisms has been the requirement of a chemically defined medium to cause Q depletion in animals. Having discovered that E. coli B105 has a block in the initial step of Q biosynthesis, we reasoned that this strain could be used as a Q- diet for organisms like C. elegans, which naturally feed on bacteria. An analysis of C. elegans tRNA revealed that as in the other higher animals, tRNAs in the worm C. elegans, are modified by Q and its sugar derivatives. When the worms were fed on Q deficient E. coli B105, Q modification was absent from the worm tRNAs suggesting that C. elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C. elegans as a model organism, the speed and simplicity of conferring a Q deficient phenotype on it, make it an ideal system to investigate the function of Q modification in tRNA. By microinjecting tgt-1-gfp constructs into C. elegans, we could also demonstrate that a major form of TGT is localised to the nucleus, suggesting that insertion of Q into the tRNAs could be occurring in the nucleus.
iii) Dual Function of Bovine Mitochondrial Initiation Factor 2 as Initiation Factors 1 and 2 in Escherichia coli
Translation initiation factors 1 and 2 (IF1 and IF2) are known as ‘universal translation initiation factors’ due to the presence of their homologs in all living organisms. Homologs of these factors are also present in the chloroplast, however, a unique situation exists in the mitochondria where IF2 homolog (IF2mt) is known to occur but an IF1 like factor is not found. We have engineered a system of E. coli knockouts to allow the study of IF2mt in a prokaryotic milieu. We found that the bovine IF2mt complements an E. coli strain wherein the gene for IF2 is knocked out, providing the first proof of a mitochondrial translation initiation factor working in a eubacterial system. This conservation of function is especially interesting in light of the recent reports revealing significant differences between the mitochondrial and eubacterial ribosomes. Further, we found that the IF2mt can also support a double knockout of IF1 and IF2 genes in E. coli, suggesting that IF2mt possesses both IF1 and IF2 like activities in E. coli. This finding offers an explanation for the lack of an IF1 like factor in mitochondria. Molecular modeling of bovine IF2mt indicated that a conserved insertion found in all mitochondrial IF2s, may form a protruding α-helix that could stabilize IF2mt on ribosomes. This insertion could in principle function as IF1 and we have explored the role of this conserved insertion both in vivo and in vitro, by generating mutants of IF2mt and EcoIF2, to lose or gain the conserved insertion respectively.
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Ayyub, Shreya Ahana. "The Role of Initiation Factor 3 : Insights from E. Coli, Mitochondria and Mycoplasma." Thesis, 2016. http://etd.iisc.ernet.in/handle/2005/2977.
Full textAbstract:
The process of translation initiation is the most highly regulated step of protein synthesis. In bacteria, three initiation factors (IF1, IF2 and IF3) play crucial roles during initiation. IF3 acts as an anti-association factor for the two ribosomal subunits. Eubacterial IF3 also permits initiator tRNA (i-tRNA) selection at the P site of the ribosome. Two features of i-tRNA, i. e. the characteristic 3GC base pairs in the anticodon stem and the cognate interaction of the anticodon sequence with the initiation codon of the mRNA contribute to IF3 based selection and/or proofreading. However, the exact mechanism of this discrimination and the contribution of the individual domains towards this process of selection/ proofreading are unclear. Further, there are exceptional instances in the natural world where either the codon-anticodon interaction or the anticodon stem composition deviates from the norm. For instance, in mammalian mitochondria, non-AUG codons such as AUU and AUA are present in the genome although they are notoriously poor initiation codons. In addition, some species of Mycoplasma have i-tRNAs with variations in the typically conserved 3GC base pairs of the anticodon stem. In this study, we have investigated the mechanism of proofreading activity of IF3 of E. coli, mitochondrial and mycoplasmal origins.
Part I: Proofreading function of IF3 in E. coli
IF3 is composed of N and C terminal domains joined by a flexible linker region. By means of complete and partial IF3 knockouts, we show that the C-terminal domain (CTD) is essential for the survival of E. coli while the N-terminal (NTD) is required for cellular fitness. Using reporter assays, we have established the role of the NTD in proofreading, while polysome profile analyses reaffirm that the CTD alone can bind to the 30S and carry out ribosome anti-association. Therefore, we show that the CTD is the ribosome binding and anti-association domain, while the NTD is the major proofreading domain. Unpublished cryoEM structures from Prof. Ramakrishnan’s lab indicate that the NTD of IF3 pushes the i-tRNA at its elbow and helps in P site accommodation of the i-tRNA. We propose that when the codon-anticodon interaction is non-cognate or if the 3GC base pairs of the anticodon stem are not intact, then the dynamic action of the NTD destabilises the tRNA at the P site and leads to its rejection.
Part II: Proofreading function of mitochondrial IF3 (IF3mt)
Of the 13 protein-coding genes in mammalian mitochondria, 3 utilise the non-canonical AUA codon and one utilises the non-canonical start codon AUU. Since IF3mt does not possess many of the generally conserved residues implicated in proofreading, we decided to characterise the proofreading function of IF3mt and its role in initiation with non-canonical start codons. Structurally, IF3mt is similar to EcoIF3 with its N and C terminal domains joined by a linker region. However, IF3mt additionally possesses N- and C-terminal extensions which are generally disordered in structure. In vivo studies of mitochondrial translation factors have been mired by the lack of methodologies to manipulate mitochondria. We have developed an E. coli strain to study the proofreading functions of mitochondrial IF3 (IF3mt) with the help of reporter genes. Consistent with its function in mitochondria, IF3mt allowed promiscuous initiation from non-AUG codons. However, IF3mt avoided initiation with i-tRNAs lacking evolutionarily conserved 3GC pairs in anticodon stems. Interestingly, expression of IF3mt N-terminal domain or IF3mt devoid of its typical N-, and C-terminal extensions significantly improved its proofreading activity. Our immunoblot assays from polysome profile fractions indicate that the IF3mt derivative lacking extensions is capable of superior 30S ribosome binding. The two derivatives of IF3mt missing the Next (IF3mtΔNext) or both the Next and Cext (IF3mtΔNextCext) display an affinity for the 50S ribosome. We propose that the extensions of IF3mt may have evolved to reduce the affinity of IF3mt to the ribosome and thereby permit initiation with non-canonical start codons like AUU and AUA. Our studies suggest that E. coli provides an excellent heterologous model to study distinctive features of mitochondrial factors.
Part III: Fidelity of translation initiation in mycoplasma
One of the many singular features of mycoplasma is the presence of many anticodon stem variants of the i-tRNA across different species. In general, i-tRNAs are characterized by the presence of the typical feature of the conserved 3 consecutive GC base pairs (GC/GC/GC) in the anticodon stem. However, many mycoplasmal species have i-tRNAs with AU/GC/GC, GC/GC/GU or AU/GC/GU sequences. Interestingly, the mycoplasmal species which harbour the AU/GC/GU i-tRNA are also human pathogens. Therefore, we decided to investigate whether these organisms possess any unique features to accommodate the i-tRNA variants, by investigating the usage of Shine Dalgarno sequences and by carrying out multiple sequence alignments of genes encoding initiation factors, ribosomal proteins S9 and S13 and 16S rRNA. Since IF3 plays a crucial role in i-tRNA selection, we carried out computational analysis of mycoplasmal IF3 sequences, which revealed many interesting features. Most striking amongst them was the variation of the highly conserved R at position 131 in some species. Interestingly, these were the very mycoplasmal species which possessed the anticodon stem variant AU/GC/GU, suggesting a strong correlation between these two features. It is known that the R131P mutation of EcoIF3 is characterised by an enormous loss of proofreading activity. It seemed unusual that such compromised proofreading would be tolerated in the cell, so we decided to investigate other components of the translational machinery as well. The C-terminal SKR tail of the ribosomal protein S9, which contacts the P-site tRNA, is highly conserved across bacteria. Analysis of the C-terminal sequences of S9 proteins in various mycoplasmal species revealed a surprising variation- the presence of a TKR tail in strains with the AU/GC/GU tRNA. In this study we have investigated the co-occurrence of S9 and IF3 variations in i-tRNA selection in E. coli. We see that the R131P polymorphism of IF3 leads to a tremendous loss of proofreading, but this loss is significantly tempered by the presence of the S9 TKR variation. Our bioinformatics studies revealed that the mycoplasmal species which are sustained on AU/GC/GU i-tRNAs also tend to use a higher percentage of non-AUG codons. By means of our reporter assays in E. coli, we have shown once again that the R131P polymorphism of IF3 leads to a tremendous increase in initiation with the non-canonical start codon AUA, but this increase is significantly tempered by the presence of the S9 TKR variation.
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Selvaraj, M. "Structural Studies on Mycobacterium Tuberculosis Peptidyl-tRNA Hydrolase and Ribosome Recycling Factor, Two Proteins Involved in Translation." Thesis, 2013. http://etd.iisc.ernet.in/handle/2005/2816.
Full textAbstract:
Protein synthesis is a process by which organisms manufacture their proteins that perform various cellular activities either alone or in combination with other similar or different molecules. In eubacteria, protein synthesis proceeds at a rate of around 15 amino acids per second. The ribosomes, charged tRNAs and mRNAs can be considered as the core components of protein synthesis system which, in addition, involves a panel of non-ribosomal proteins that regulate the speed, specificity and accuracy of the process. Peptidyl-tRNA hydrolase (Pth) and ribosome recycling factor (RRF) are two such non-ribosomal proteins involved in protein synthesis. These two proteins are essential for eubacterial survival and the work reported in this thesis involves structural characterization of these two proteins from the bacterial pathogen, Mycobacterium tuberculosis.
The protein structures were solved using established techniques of protein crystallography. Hanging drop vapour diffusion method and crystallization under oil using microbatch plates were the methods employed for protein crystallization. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator in all the cases. The data were processed using DENZO and MOSFLM. The structures were solved by molecular replacement method using the program PHASER. Structure refinements were carried out using programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, CHIMERA, and PYMOL were used for structure validation and analysis of the refined structures.
Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA that has dropped off from ribosome before reaching the stop codon, in order to avoid the toxicity resulting from peptidyl-tRNA accumulation and to free the tRNA to make it available for further rounds in protein synthesis. To begin with, the structure of the enzyme from M. tuberculosis (MtPth) was determined in three crystal forms. This structure and the structure of the same enzyme from Escherichia coli (EcPth) in its crystal differ substantially on account of the binding of the C-terminus of the E.coli enzyme to the peptide binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in open conformation when the enzyme is in the free state as in the crystals of MtPth. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the EcPth crystal mimics the peptide-bound conformation of the enzyme. The peptide stretch involving a loop and a helix, referred to earlier, now closes on the bound peptide. Concurrently, a gate connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of the two residues. Thus, the crystal structure of MtPth when compared with that of EcPth, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule.
A discrepancy between the X-ray results and NMR results, which subsequently became available, led to X-ray studies on new crystal forms of the enzyme. The results of these studies and those of the enzyme from different sources that became available, confirmed the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding site and tRNA binding site. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movement is not, however, observed in the NMR structure of MtPth.
The discrepancy between the X-ray and NMR structures of MtPth in relation to the functionally important plasticity of the molecule, referred to earlier, also led to molecular dynamics simulations. The X-ray and the NMR studies along with the simulations indicated an inverse correlation between crowding and molecular volume. A detailed comparison of proteins for which X-ray and the NMR structures are available appears to confirm this correlation. In consonance with the reported results of the investigation in cellular components and aqueous solutions, the comparison indicates that the crowding results in compaction of the molecule as well as change in its shape, which could specifically involve regions of the molecule important for function. Crowding could thus influence the action of proteins through modulation of the functionally important plasticity of the molecule.
After termination of protein synthesis at the stop codon, the ribosome remains as a post-termination complex (PoTC), consisting of the 30S and the 50S subunits, mRNA and a deacylated tRNA. This complex has to be disassembled so that the ribosome is available for the next round of translation initiation. Ribosome recycling factor (RRF) binds to ribosome and in concert with elongation factor G (EF.G), performs the recycling of ribosome that results in disassembly of PoTC. The structure of this L-shaped protein with two domains connected by a hinge, from Mycobacterium tuberculosis (MtRRF) was solved previously in our laboratory. The relative movement of domains lies at the heart of RRF function. Three salt bridges were hypothesized to reduce the flexibility of MtRRF when compared to the protein from E.coli (EcRRF), which has only one such salt bridge. Out of these three bridges, two are between domain 1 and domain 2, whereas the third is between the hinge region and the C-terminus of the molecule. These salt bridges were disrupted with appropriate mutations and the structure and activity of the mutants and their ability to complement EcRRF were explored. An inactive C-terminal deletion mutant of MtRRF was also studied. Major, but different, structural changes were observed in the C-terminal deletion mutant and the mutant involving the hinge region. Unlike the wild type protein and the other mutants, the hinge mutant complements EcRRF. This appears to result from the increased mobility of the domains in the mutant, as evidenced by the results of librational analysis.
In addition to the work on PTH and RRF, the author was involved during the period of studentship in carrying out X-ray studies of crystalline complexes involving amino acids and carboxylic acids, which is described in the Appendix of the thesis. The complexes studied are that of tartaric acid with arginine and lysine.