Estudio comparativo de rusticianinas en microorganismos acidófilos/halotolerantes
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2015
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Resumen
La biolixiviación representa una vía económica atractiva para recuperar cobre especialmente
de minerales de baja ley. Chile es sin duda una potencia mundial en el desarrollo de la
tecnología de biolixiviación. Sin embargo, un gran desafio para la industria de la
biolixiviación es la dificultad de obtener el agua dulce requerida para este proceso. Esto
también es un problema en otros países, como Australia, donde la biolixiviación es llevada a
cabo en zonas áridas. Usar agua salina, particularmente agua derivada del océano, para
reemplazar el agua dulce podría mitigar el problema. Sin embargo, muchos microorganismos
usados en los procesos de biolixiviación convencional no crecen en condiciones salinas.
Esta tesis explica cómo la rusticianina, una proteína clave de transferencia de electrones, es
capaz de funcionar en bacterias tolerantes a sal (halotolerantes) como Acidihalobacter
prosperus DSM 5130 y Acidiferrobacter murciensis SPIII/3. Una comparación
bioinformática de las proteínas rusticianina de estas dos bacterias halófilicas con proteínas
rusticianinas homólogas de microorganismos biolixiviantes no tolerante a sal como
Acidithiobacillus ferrooxidans fue realizado con el fin de evaluar si habían cambios
aminoacídicos y/o modificaciones estructurales que pudiesen ayudar a explicar la tolerancia
de sal de rusticianinas en A. prosperus y A. murciensis.
La investigación se realizó usando varios programas bioinformáticas disponibles en internet.
RAST fue utilizado para anotar los genomas de A. prosperus y A. murciensis, permitiendo la
predicción de genes codificantes de rusticianina y proteínas asociadas con el complejo
rusticianina. Las proteínas predichas de estos genes fueron curadas a mano usando un número
de programas para la predicción de propiedades de proteínas incluyendo BLAST, PFAM, TMMPRED y PSORT. MAFFT y MEGA fueron utilizados para construir árboles
filogenéticos. Swiss-Model fue utilizado para crear modelos estructurales de las diferentes
rusticianinas, las cuales fueron visualizadas con Swiss-PDBViewer.
Se realizó una búsqueda, en bases de datos, de genomas de microorganismos que contengan
genes codificadores de proteínas potencialmente oxidadoras de hierro, incluyendo la
rusticianina. La búsqueda reveló dos organismos halotolerante similares a A. ferrooxidan A. prosperus y A. murciencis.
En contraste con A. ferrooxidan que contiene solo una copia de rusticianina, A. prosperus y
A. murciencis contienen dos y tres copias respectivamente. Una comparación del modelo de
rusticianina, correspondiente a A. sugiere que las cinco rusticianinas de los
halofilicos conservan el sitio de cobre involucrado en la transferencia de electrones, pero
cada una de estas rusticianina muestra cambios aminoacídicos alrededor del sitio de unión a
cobre, principalmente tienen una tendencia a aumentar los residuos cargados positivamente.
Adicionalmente, un aumento de residuos cargados fue detectado en la superficie de estas
proteínas. El modelo presentado sugiere cómo éstas modificaciones podrían estabilizar la
msticianina en presencia de concentraciones altas de sal. El modelo presenta predicciones
que se podrían comprobar en futuras investigaciones experimentales y allana el camino para
una mayor investigación en la microbiología de biolixiviación en condiciones salinas.
Bioleaching represents an economically attractive way to recover copper especially from low grade ores. Chile is arguably the world's leader in developing bioleaching technology. However, a major challenge for the Chilean bioleaching industry is the difficulty of obtaining sufficient fresh water required for the process. This is also a problem in other countries, such as Australia, where bioleaching is being carried out in arid conditions. Using saline water, in particular water derived from the sea, to replace fresh water would alleviate the problem. However, many microorganisms used in conventional bioleaching process are stressed or even fail to grow in saline conditions. This thesis addresses the issue of how rusticyanin, a key electron transfer protein, is able to function in the salt tolerant (halotolerant) bacteria Acidihalobacter prosperus DSM 5130 and Acidiferrobacter murciensis SPIII/3. A bioinformatics comparison of the rusticyanin proteins from these two halophilic bacteria with the homologous rusticyanin from the non-salt tolerant bioleaching microorganism Acidithiobacillus ferrooxidans was carried out in order to evaluate if there were amino acid changes and/or structural modifications that might help explain the salt tolerance of rusticyanin in A. prosperus and A. murciensis. This investigation was carried out using several bioinformatics programs available on the web. RAST was used to annotate the genomes A. prosperus and A. murciensis, allowing the prediction of genes for rusticyanin and proteins associated with the rusticyanin complex. The predicted proteins from these genes were hand curated using a number of programs for prediction of protein properties including BLAST, PFAM, TMMPRED and PSORT. MAFFT and MEGA were used to construct phylogenetic trees. Swiss-Model was used to create structural models of the different rusticyanins which were subsequently visualized using Swiss-PDBViewer. A search of genome databases for organisms that contain genes that potentially encode iron oxidizing complexes including rusticyanin, with similarity to that found in A. revealed two candidates, A. and A. that were also halotolerant. In contrast to A. that contains only one copy of rusticyanin, A. and A. contain two and three copies respectively. A comparison of the model rusticyanin from A. suggests that all five rusticyanins from the halophiles conserved the critical copper site involved in electron transfer, but each displayed amino acid changes around this site, principally having a tendency to increase positively charged residues. In addition, increased charged residues were detected on the surface of these proteins. A model is presented that suggests how these modifications might stabilize rusticyanin in the presence of high salt. The model makes testable predictions for future experimental investigations and paves the way for further research into the microbiology of bioleaching in saline conditions.
Bioleaching represents an economically attractive way to recover copper especially from low grade ores. Chile is arguably the world's leader in developing bioleaching technology. However, a major challenge for the Chilean bioleaching industry is the difficulty of obtaining sufficient fresh water required for the process. This is also a problem in other countries, such as Australia, where bioleaching is being carried out in arid conditions. Using saline water, in particular water derived from the sea, to replace fresh water would alleviate the problem. However, many microorganisms used in conventional bioleaching process are stressed or even fail to grow in saline conditions. This thesis addresses the issue of how rusticyanin, a key electron transfer protein, is able to function in the salt tolerant (halotolerant) bacteria Acidihalobacter prosperus DSM 5130 and Acidiferrobacter murciensis SPIII/3. A bioinformatics comparison of the rusticyanin proteins from these two halophilic bacteria with the homologous rusticyanin from the non-salt tolerant bioleaching microorganism Acidithiobacillus ferrooxidans was carried out in order to evaluate if there were amino acid changes and/or structural modifications that might help explain the salt tolerance of rusticyanin in A. prosperus and A. murciensis. This investigation was carried out using several bioinformatics programs available on the web. RAST was used to annotate the genomes A. prosperus and A. murciensis, allowing the prediction of genes for rusticyanin and proteins associated with the rusticyanin complex. The predicted proteins from these genes were hand curated using a number of programs for prediction of protein properties including BLAST, PFAM, TMMPRED and PSORT. MAFFT and MEGA were used to construct phylogenetic trees. Swiss-Model was used to create structural models of the different rusticyanins which were subsequently visualized using Swiss-PDBViewer. A search of genome databases for organisms that contain genes that potentially encode iron oxidizing complexes including rusticyanin, with similarity to that found in A. revealed two candidates, A. and A. that were also halotolerant. In contrast to A. that contains only one copy of rusticyanin, A. and A. contain two and three copies respectively. A comparison of the model rusticyanin from A. suggests that all five rusticyanins from the halophiles conserved the critical copper site involved in electron transfer, but each displayed amino acid changes around this site, principally having a tendency to increase positively charged residues. In addition, increased charged residues were detected on the surface of these proteins. A model is presented that suggests how these modifications might stabilize rusticyanin in the presence of high salt. The model makes testable predictions for future experimental investigations and paves the way for further research into the microbiology of bioleaching in saline conditions.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Esta tesis se realizó en el Laboratorio Centro de Bioinformática y Biología Genómica de Fundación Ciencia y Vida. Fue financiada por el proyecto FONDECYT N° 1130683 titulado "Comparative genomic, metagenomic and bioinformatic approaches to generating fundamental and applied knowledge for bioleaching and other acidic econiches".
Esta tesis se realizó en el Laboratorio Centro de Bioinformática y Biología Genómica de Fundación Ciencia y Vida. Fue financiada por el proyecto FONDECYT N° 1130683 titulado "Comparative genomic, metagenomic and bioinformatic approaches to generating fundamental and applied knowledge for bioleaching and other acidic econiches".
Palabras clave
Lixiviación Bacteriana, Rusticianina