Examinando por Autor "Jedlicki, Eugenia"

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    Bioinformatic prediction and experimental verification of Fur-regulated genes in the extreme acidophile Acidithiobacillus ferrooxidans
    (2007-04) Quatrini, Raquel; Lefimil, Claudia; Veloso, Felipe A.; Pedroso, Inti; Holmes, David S.; Jedlicki, Eugenia
    The γ-proteobacterium Acidithiobacillus ferrooxidans lives in extremely acidic conditions (pH 2) and, unlike most organisms, is confronted with an abundant supply of soluble iron. It is also unusual in that it oxidizes iron as an energy source. Consequently, it faces the challenging dual problems of (i) maintaining intracellular iron homeostasis when confronted with extremely high environmental loads of iron and (ii) of regulating the use of iron both as an energy source and as a metabolic micronutrient. A combined bioinformatic and experimental approach was undertaken to identify Fur regulatory sites in the genome of A. ferrooxidans and to gain insight into the constitution of its Fur regulon. Fur regulatory targets associated with a variety of cellular functions including metal trafficking (e.g. feoPABC, tdr, tonBexbBD, copB, cdf), utilization (e.g. fdx, nif, transcriptional regulation (e.g. phoB, irr, iscR) and redox balance (grx), trx, gst) were identified. Selected predicted Fur regulatory sites were confirmed by FURTA, EMSA and in vitro transcription analyses. This study provides the first model for a Fur-binding site consensus sequence in an acidophilic iron-oxidizing microorganism and lays the foundation for future studies aimed at deepening our understanding of the regulatory networks that control iron uptake, homeostasis and oxidation in extreme acidophiles. © 2007 The Author(s).
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    Differential expression of two bc1 complexes in the strict acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans suggests a model for their respective roles in iron or sulfur oxidation
    (2007-01) Bruscella, Patrice; Appia-Ayme, Corinne; Levicán, Gloria; Ratouchniak, Jeanine; Jedlicki, Eugenia; Holmes, David S.; Bonnefoy, Violaine
    Three strains of the strict acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans, including the type strain ATCC 23270, contain a petllABC gene cluster that encodes the three proteins, cytochrome c1, cytochrome b and a Rieske protein, that constitute a bc1, electron-transfer complex. RT-PCR and Northern blotting show that the petllABC cluster is co-transcribed with cycA, encoding a cytochrome c belonging to the c4 family, sdrA, encoding a putative short-chain dehydrogenase, and hip, encoding a high potential iron-sulfur protein, suggesting that the six genes constitute an operon, termed the petll operon. Previous results indicated that A. ferrooxidans contains a second pet operon, termed the petl operon, which contains a gene cluster that is similarly organized except that it lacks hip. Real-time PCR and Northern blot experiments demonstrate that petl is transcribed mainly in cells grown in medium containing iron, whereas petll is transcribed in cells grown in media containing sulfur or iron. Primer extension experiments revealed possible transcription initiation sites for the petl and petll operons. A model is presented in which petl is proposed to encode the bc1, complex, functioning in the uphill flow of electrons from iron to NAD(P), whereas petll is suggested to be involved in electron transfer from sulfur (or formate) to oxygen (or ferric iron). A. ferrooxidans is the only organism, to date, to exhibit two functional bc1 complexes. © 2007 SGM.
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    Expression and activity of the Calvin-Benson-Bassham cycle transcriptional regulator CbbR from Acidithiobacillus ferrooxidans in Ralstonia eutropha
    (Oxford University Press, 2015-08) Esparza, Mario; Jedlicki, Eugenia; Dopson, Mark; Holmes, David S.
    Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbRAF) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbRAF function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbRAF was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbRAF was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbRAF activity. © FEMS 2015.
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    Extending the models for iron and sulfur oxidation in the extreme Acidophile Acidithiobacillus ferrooxidans
    (BioMed Central Ltd., 2009-08-24) Quatrini, Raquel; Appia-Ayme, Corinne; Denis, Yann; Jedlicki, Eugenia; Holmes, David S.; Bonnefoy, Violaine
    Background: Acidithiobacillus ferrooxidans gains energy from the oxidation of ferrous iron and various reduced inorganic sulfur compounds at very acidic pH. Although an initial model for the electron pathways involved in iron oxidation has been developed, much less is known about the sulfur oxidation in this microorganism. In addition, what has been reported for both iron and sulfur oxidation has been derived from different A. ferrooxidans strains, some of which have not been phylogenetically characterized and some have been shown to be mixed cultures. It is necessary to provide models of iron and sulfur oxidation pathways within one strain of A. ferrooxidans in order to comprehend the full metabolic potential of the pangenome of the genus. Results: Bioinformatic-based metabolic reconstruction supported by microarray transcript profiling and quantitative RT-PCR analysis predicts the involvement of a number of novel genes involved in iron and sulfur oxidation in A. ferrooxidans ATCC23270. These include for iron oxidation: cup (copper oxidase-like), ctaABT (heme biogenesis and insertion), nuoI and nuoK (NADH complex subunits), sdrA1 (a NADH complex accessory protein) and atpB and atpE (ATP synthetase F0 subunits). The following new genes are predicted to be involved in reduced inorganic sulfur compounds oxidation: a gene cluster (rhd, tusA, dsrE, hdrC, hdrB, hdrA, orf2, hdrC, hdrB) encoding three sulfurtransferases and a heterodisulfide reductase complex, sat potentially encoding an ATP sulfurylase and sdrA2 (an accessory NADH complex subunit). Two different regulatory components are predicted to be involved in the regulation of alternate electron transfer pathways: 1) a gene cluster (ctaRUS) that contains a predicted iron responsive regulator of the Rrf2 family that is hypothesized to regulate cytochrome aa3 oxidase biogenesis and 2) a two component sensor-regulator of the RegB-RegA family that may respond to the redox state of the quinone pool. Conclusion: Bioinformatic analysis coupled with gene transcript profiling extends our understanding of the iron and reduced inorganic sulfur compounds oxidation pathways in A. ferrooxidans and suggests mechanisms for their regulation. The models provide unified and coherent descriptions of these processes within the type strain, eliminating previous ambiguity caused by models built from analyses of multiple and divergent strains of this microorganism. © 2009 Quatrini et al; licensee BioMed Central Ltd.
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    Genes and pathways for CO2fixation in the obligate, chemolithoautotrophic acidophile, Acidithiobacillus ferrooxidans, Carbon fixation in A. ferrooxidans
    (BMC, 2010) Esparza, Mario; Crdenas, Juan Pablo; Bowien, Botho; Jedlicki, Eugenia; Holmes, David S.
    Background. Acidithiobacillus ferrooxidans is chemolithoautotrophic -proteobacterium that thrives at extremely low pH (pH 1-2). Although a substantial amount of information is available regarding CO2uptake and fixation in a variety of facultative autotrophs, less is known about the processes in obligate autotrophs, especially those living in extremely acidic conditions, prompting the present study. Results. Four gene clusters (termed cbb1-4) in the A. ferrooxidans genome are predicted to encode enzymes and structural proteins involved in carbon assimilation via the Calvin-Benson- Bassham (CBB) cycle including form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39) and the CO2- concentrating carboxysomes. RT-PCR experiments demonstrated that each gene cluster is a single transcriptional unit and thus is an operon. Operon cbb1 is divergently transcribed from a gene, cbbR, encoding the LysR-type transcriptional regulator CbbR that has been shown in many organisms to regulate the expression of RubisCO genes. Sigma70-like -10 and -35 promoter boxes and potential CbbR-binding sites (T-N11-A/TNA-N7TNA) were predicted in the upstream regions of the four operons. Electrophoretic mobility shift assays (EMSAs) confirmed that purified CbbR is able to bind to the upstream regions of the cbb1, cbb2 and cbb3 operons, demonstrating that the predicted CbbR-binding sites are functional in vitro. However, CbbR failed to bind the upstream region of the cbb4 operon that contains cbbP, encoding phosphoribulokinase (EC 2.7.1.19). Thus, other factors not present in the assay may be required for binding or the region lacks a functional CbbR-binding site. The cbb3 operon contains genes predicted to encode anthranilate synthase components I and II, catalyzing the formation of anthranilate and pyruvate from chorismate. This suggests a novel regulatory connection between CO 2fixation and tryptophan biosynthesis. The presence of a form II RubisCO could promote the ability of A. ferrooxidans to fix CO2at different concentrations of CO2. Conclusions. A. ferrooxidans has features of cbb gene organization for CO2-assimilating functions that are characteristic of obligate chemolithoautotrophs and distinguish this group from facultative autotrophs. The most conspicuous difference is a separate operon for the cbbP gene. It is hypothesized that this organization may provide greater flexibility in the regulation of expression of genes involved in inorganic carbon assimilation. © 2010 Esparza et al; licensee BioMed Central Ltd.
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    Regulación de los niveles intracelulares del represor transcripcional Fur de Acidithiobacillus ferrooxidans
    (Universidad Andrés Bello, 2011) Lefimil Puente, Claudia Andrea; Holmes, David; Jedlicki, Eugenia
    El hierro es un nutriente esencial para todos los organismos, pero en exceso conduce a daño macromolecular vía estrés oxidativo (reacción de Fenton). El regulador transcripcional Fur (Ferric Uptake Regulator) ha sido mostrado de ser el responsable en coordinar los procesos fisiológicos involucrados en la captación, incorporación y almacenamiento (homeostasis) de hierro en todas las bacterias Gram negativas estudiadas a la fecha. At. ferrooxidans, una bacteria Gram negativa, es un interesante e inusual modelo para estudiar la homeostasis de hierro debido a que vive a pH 2 donde la biodisponibilidad de hierro es muy alta, y porque además necesita balancear el uso de hierro como un micronutriente versus su uso como fuente de energía. En muchos organismos estudiados, Fur ha sido encontrado en elevados niveles intracelulares pero muy poco es conocido sobre los mecanismos moleculares involucrados en la regulación de su expresión. En esta tesis, se observó que los niveles de Fur en At. ferrooxidans varían dependiendo de la fase de crecimiento en que se encuentre la bacteria, incrementándose en fase logarítmica tardía. Sin embargo, estos cambios no fueron debidos a variaciones en los niveles de mRNA de Fur AF, que permanecen constantes. También se determinó que ambos, el mRNA y la proteína FurAF, varían en respuesta a cambios en el sustrato energético tilizado en el medio de cultivo, sugiriendo la posibilidad de que estas variaciones se deban a regulación a nivel transcripcional y post-transcripcional. Predicciones bioinformáticas de sitios de unión a factores de transcripción en la región promotora de furAF, o cercana a ésta, junto a los ensayos in vivo de expresión de genes en el sistema heterólogo de E. coli son consistentes con la idea de que furAF puede ser transcrito por la RNA polimerasa asociada a diferentes factores sigma, incluyendo sigma70, sigma32 y sigma54. Predicciones bioinformáticas también sugieren que la transcripciónde furAF podría ser regulada por el activador CRP (CAP) indicando un potencial nexo entre el metabolismo del carbono y la homeostasis de hierro. El análisis de estabilidad del mRNA de furAF mostraron que éste posee una elevada vida media de 0,5-4 horas (comparada con 1-20 minutos para un típico mRNA), y que su vida media varía dependiendo del medio de cultivo en que At. ferrooxidans es crecido. Además, se detectó un RNA, denominado frr, que es transcrito de manera antisentido a fur. Análisis de su transcripción, incluyendo su sitio promotor y terminador, acoplado con una predicción de su estructura secundaria sugieren un modelo en que Frr puede unir el mRNA de FurAF promoviendo el acceso del ribosoma e incrementando los niveles de FurAF· Este es un nuevo mecanismo para la regulación post-transcripcional de Fur.