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Introducción: el hierro es un elemento vital que necesitan todos los organismos vivos para numerosos procesos celulares. En condiciones de déficit de hierro los microorganismos sobreviven secretando sideróforos. Éstos tienen capacidad para quelar Fe(III) con alta afinidad y selectividad. En su estructura poseen átomos de oxígeno cargados negativamente que actúan como donadores de electrones coordinándose fundamentalmente con el hierro u otros metales pesados. Esta capacidad les hace útiles no sólo en la agricultura como promotores del crecimiento de plantas, sino también en medicina, biorremediación y para el desarrollo de biosensores. Material y métodos: revisión bibliográfica sobre los sideróforos bacterianos en biotecnología. Se han seleccionado 48 artículos de una búsqueda en PubMed y Dialnet de los últimos 5 años. Objetivos: analizar las aplicaciones de los sideróforos en diferentes sectores, incluidos la agricultura, la biorremediación, los biosensores y la medicina. Resultados: la aplicación más extendida de los sideróforos es en el ámbito de la agricultura, para promover el crecimiento de las plantas como una alternativa ecológica a los pesticidas químicos. En medicina, los sideróforos se utilizan para administrar antibióticos (estrategia del caballo de Troya) a bacterias resistentes, elaboración de vacunas, como marcadores tempranos de infecciones invasivas e identificación de células cancerígenas. Por último, pueden ser empleados como biosensores para detectar la presencia de iones en muestras ambientales y biológicas. Conclusiones: las aplicaciones de los sideróforos se han extendido a multitud de campos haciendo posible una agricultura más sostenible, aumentando las posibilidades de diagnósticos precoces de enfermedades, facilitando el tratamiento de enfermedades graves o resistentes a antibióticos y en el desarrollo de vacunas. El interés creciente en sus aplicaciones está haciendo posible nuevas investigaciones y el descubrimiento de nuevos sideróforos

Las cianobacterias son seres vivos microscópicos ancestrales que han tenido una gran relevancia en el desarrollo de la vida desde sus orígenes hasta como la conocemos hoy en día. Estas bacterias poseen propiedades únicas en la naturaleza y han sido empleadas en el pasado por las antiguas civilizaciones. En los últimos años se ha tratado de profundizar en sus potenciales usos en diversos campos como la medicina, la cosmética, la producción de plásticos, la alimentación o la generación de energía mediante biocombustibles. En esta revisión bibliográfica se recopilan las posibles aplicaciones que pueden tener las cianobacterias en estos ámbitos, pero principalmente en el campo de la medicina y orientado hacia la búsqueda de compuestos bioactivos con relevancia en el tratamiento de patologías tan recurrentes en la actualidad como lo son las enfermedades infecciosas, el cáncer, las enfermedades inflamatorias o las neurodegenerativas. Muchos de los metabolitos tratados, especialmente los relevantes en el tratamiento del cáncer, se encuentran en fases tempranas de ensayo clínico, sin embargo, otros se encuentran en fases más avanzadas, e incluso algunos ya han comenzado a ser autorizados y comercializados, sentando los primeros precedentes de cara al futuro de las cianobacterias en la biomedicina.

Las bacterias se comunican intercelularmente a través de un proceso denominado quorum sensing. El quorum sensing comprende diversidad de sistemas que se encuentran mediados por moléculas señalizadoras llamadas autoinductores, y que se encargan de la regulación y expresión de numerosos procesos bacterianos, así como de la expresión de factores de virulencia en procesos infecciosos. Estos sistemas son dependientes de densidad celular, ya que requieren superar una concentración crítica de autoinductores en el medio para su activación. Debido a su implicación en el desarrollo de numerosos procesos patogénicos, durante los últimos 50 años se ha investigado tanto los diferentes autoinductores existentes como su funcionamiento. Además, la aparición de nuevas cepas multirresistentes durante las últimas décadas ha acelerado la búsqueda de nuevas estrategias terapéuticas. Más recientemente, han sido descubiertas estrategias naturales para la inhibición de estos procesos quorum sensing, es lo que se conoce como quorum quenching. Estas estrategias interfieren en el proceso de comunicación, ya sea bloqueando competitivamente receptores de membrana, inhibiendo las enzimas de síntesis de los autoinductores o degradando los autoinductores presentes en el medio. En este trabajo se expone información constatada acerca del quorum sensing y quorum quenching. Para ello, se hace un análisis de los autoinductores más extendidos entre las bacterias, así como de los sistemas en los que participan. También se describen los mecanismos de inhibición de quorum sensing y sus potenciales usos en el ámbito sanitario.

Los biosensores son instrumentos analíticos constituidos por un componente biológico y otro fisicoquímico, capaces de detectar un evento de biorreconocimiento producido por la interacción con un analito de interés, y junto a un sistema transductor adecuado, convertir dicho evento en una señal medible. Su acoplamiento con distintas biomoléculas de alta afinidad permite la detección sensible y selectiva de una gran variedad de analitos. El uso conjunto de diferentes tecnologías y estrategias de biodetección, señalización y optimización permiten el diseño, a conveniencia, de una gran variedad de biosensores con aplicaciones muy versátiles y útiles en diferentes campos. Están presentes en una variedad muy amplia de áreas de trabajo; como el diagnóstico clínico, la supervisión ambiental, el control de alimentos, el descubrimiento de fármacos, la investigación forense y biomédica. Tradicionalmente, los biosensores basados en anticuerpos, también conocidos como inmunosensores, han tenido mayor protagonismo en el ámbito clínico, debido a sus buenas propiedades de afinidad y sensibilidad. Sin embargo, la detección limitada sólo a sustancias inmunogénicas y la necesidad de desarrollar dispositivos de diagnóstico más baratos, sencillos, específicos y sensibles, junto a la posibilidad de ampliar el rango de detección para otro tipo de analitos, han dado pie a la investigación de múltiples variedades de biosensores. Entre ellos, los aptasensores. En este trabajo bibliográfico, se profundiza sobre las ventajas que proporcionan los biosensores basados en aptámeros, sus métodos de producción y clasificación; exponiendo a su vez el enorme potencial que tienen los aptámeros, por sus características únicas, como moléculas de biorreconocimiento en biosensores. A su vez, también se hace una breve comparación con el uso de los anticuerpos en biosensores

The halophilic broad salt-gi owing bacterium Nfiromofio/o#octer solezipens is a natui al producer of ectoines, compatible solutes with piomising biotechnological applications as power ful biostabilizei s in Biomedicine, kJolecular Biology, and Dermopharmacy, among other s. These solutes are synthesized and intracellularly accumulated by this bacterium, in iesponse to increasing salinity in the external medium, and thus adapting its metabolism to support this biosynthetic route. Due to the complex regulation of this metabolic adaptation, novel metabolic engineering appi oaches based on Systems Biology (omics analysis and genome-scale metabolic models) that allows the rational rewiring of cell metabolism to inci ease bioproduction have been applied for ectoines overproduction. To accomplish this purpose, in this Doctoral Thesis a piuned genome-scale metabolic model of this bacterium, obtained from a pievious one (iFP764), was iefined and used to piedict potential genetic targets by using differ ent algorithms. As a iesult, they suggested a set of genes to knockout and/or to be up/down-regulated to obtain overpioducing str ains at high and low salinities. In order to 3n s3/ico analyze the effect of these peitui Nations in the distribution of metabolic fluxes, additional computational methods were successfully applied to simulate the cellular metabolic iesponse at different salinities in mutant str ains of N. solezipens. kJoi eover, an integrative analysis of multiomics data derived from ti anscriptomic, pioteomic and metabolomics, obtained at differ ent conditions of salinities and temperatui e, was also car ried out to find metabolic cori elations that could complement these analysis. Furthermore, a number of new genetic tools have been impi oved and developed in this work to assist in the /u r/ro implementation of some of the genetic modification str ategies predicted by the metabolic model. Finally, multiple knockout mutants of U. sa/ez/peus were obtained and phenot

The halophilic broad salt-gi owing bacterium Nfiromofio/o#octer solezipens is a natui al producer of ectoines, compatible solutes with piomising biotechnological applications as power ful biostabilizei s in Biomedicine, kJolecular Biology, and Dermopharmacy, among other s. These solutes are synthesized and intracellularly accumulated by this bacterium, in iesponse to increasing salinity in the external medium, and thus adapting its metabolism to support this biosynthetic route. Due to the complex regulation of this metabolic adaptation, novel metabolic engineering appi oaches based on Systems Biology (omics analysis and genome-scale metabolic models) that allows the rational rewiring of cell metabolism to inci ease bioproduction have been applied for ectoines overproduction. To accomplish this purpose, in this Doctoral Thesis a piuned genome-scale metabolic model of this bacterium, obtained from a pievious one (iFP764), was iefined and used to piedict potential genetic targets by using differ ent algorithms. As a iesult, they suggested a set of genes to knockout and/or to be up/down-regulated to obtain overpioducing str ains at high and low salinities. In order to 3n s3/ico analyze the effect of these peitui Nations in the distribution of metabolic fluxes, additional computational methods were successfully applied to simulate the cellular metabolic iesponse at different salinities in mutant str ains of N. solezipens. kJoi eover, an integrative analysis of multiomics data derived from ti anscriptomic, pioteomic and metabolomics, obtained at differ ent conditions of salinities and temperatui e, was also car ried out to find metabolic cori elations that could complement these analysis. Furthermore, a number of new genetic tools have been impi oved and developed in this work to assist in the /u r/ro implementation of some of the genetic modification str ategies predicted by the metabolic model. Finally, multiple knockout mutants of U. sa/ez/peus were obtained and phenot

The halophilic broad salt-gi owing bacterium Nfiromofio/o#octer solezipens is a natui al producer of ectoines, compatible solutes with piomising biotechnological applications as power ful biostabilizei s in Biomedicine, kJolecular Biology, and Dermopharmacy, among other s. These solutes are synthesized and intracellularly accumulated by this bacterium, in iesponse to increasing salinity in the external medium, and thus adapting its metabolism to support this biosynthetic route. Due to the complex regulation of this metabolic adaptation, novel metabolic engineering appi oaches based on Systems Biology (omics analysis and genome-scale metabolic models) that allows the rational rewiring of cell metabolism to inci ease bioproduction have been applied for ectoines overproduction. To accomplish this purpose, in this Doctoral Thesis a piuned genome-scale metabolic model of this bacterium, obtained from a pievious one (iFP764), was iefined and used to piedict potential genetic targets by using differ ent algorithms. As a iesult, they suggested a set of genes to knockout and/or to be up/down-regulated to obtain overpioducing str ains at high and low salinities. In order to 3n s3/ico analyze the effect of these peitui Nations in the distribution of metabolic fluxes, additional computational methods were successfully applied to simulate the cellular metabolic iesponse at different salinities in mutant str ains of N. solezipens. kJoi eover, an integrative analysis of multiomics data derived from ti anscriptomic, pioteomic and metabolomics, obtained at differ ent conditions of salinities and temperatui e, was also car ried out to find metabolic cori elations that could complement these analysis. Furthermore, a number of new genetic tools have been impi oved and developed in this work to assist in the /u r/ro implementation of some of the genetic modification str ategies predicted by the metabolic model. Finally, multiple knockout mutants of U. sa/ez/peus were obtained and phenot

The halophilic broad salt-gi owing bacterium Nfiromofio/o#octer solezipens is a natui al producer of ectoines, compatible solutes with piomising biotechnological applications as power ful biostabilizei s in Biomedicine, kJolecular Biology, and Dermopharmacy, among other s. These solutes are synthesized and intracellularly accumulated by this bacterium, in iesponse to increasing salinity in the external medium, and thus adapting its metabolism to support this biosynthetic route. Due to the complex regulation of this metabolic adaptation, novel metabolic engineering appi oaches based on Systems Biology (omics analysis and genome-scale metabolic models) that allows the rational rewiring of cell metabolism to inci ease bioproduction have been applied for ectoines overproduction. To accomplish this purpose, in this Doctoral Thesis a piuned genome-scale metabolic model of this bacterium, obtained from a pievious one (iFP764), was iefined and used to piedict potential genetic targets by using differ ent algorithms. As a iesult, they suggested a set of genes to knockout and/or to be up/down-regulated to obtain overpioducing str ains at high and low salinities. In order to 3n s3/ico analyze the effect of these peitui Nations in the distribution of metabolic fluxes, additional computational methods were successfully applied to simulate the cellular metabolic iesponse at different salinities in mutant str ains of N. solezipens. kJoi eover, an integrative analysis of multiomics data derived from ti anscriptomic, pioteomic and metabolomics, obtained at differ ent conditions of salinities and temperatui e, was also car ried out to find metabolic cori elations that could complement these analysis. Furthermore, a number of new genetic tools have been impi oved and developed in this work to assist in the /u r/ro implementation of some of the genetic modification str ategies predicted by the metabolic model. Finally, multiple knockout mutants of U. sa/ez/peus were obtained and phenot

Chromohalobacter salexigens es un microorganismo extremófilo modelo de máximo interés y aplicabilidad en Biotecnología Microbiana, ya que sintetiza en respuesta a estrés osmótico y/o por temperatura, los solutos compatibles ectoína e hidroxiectoína, compuestos con propiedades estabilizadoras por lo que son de gran interés en Dermofarmacia, Biología Molecular, Agricultura o Biomedicina. La síntesis de ectoínas y su regulación es un proceso en el que intervienen complejos mecanismos transcripcionales y postranscripcionales, además de la implicación de reguladores globales y específicos. Por otro lado, se ha puesto de manifiesto la importancia de la regulación de la homeostasis del hierro en la adaptación a la salinidad, y por tanto en la síntesis de estos solutos. Así, el regulador global Fur es el encargado de mantener un control preciso de los niveles de hierro intracelulares existiendo, en C. salexigens, dos parálogos pertenecientes a la superfamilia Fur, Fur1 y Fur2. Además, para poder sintetizar la cantidad necesaria de estos solutos en condiciones de estrés, en C. salexigens se produce una adaptación metabólica en función de la salinidad y temperatura, ya que la síntesis de ectoínas depende de intermediarios procedentes de rutas del metabolismo central. Así, para poder dilucidar posibles modificaciones genéticas que permitan la obtención de cepas mejoradas en la producción de estos compuestos, se hace necesario un estudio más global e integrado tanto del metabolismo, como de los reguladores implicados en dicho proceso, así como de otros factores ambientales que puedan intervenir. En los últimos años se ha desarrollado un modelo metabólico de C. salexigens a escala genómica, que permitirá la predicción de estrategias de Ingeniería Metabólica para optimizar la producción de ectoínas en este microorganismo. Para la mejora de este modelo se está desarrollando un modelo de regulación que integre tanto las redes de señalización como las redes transcripcionales respons

La Biotecnología abarca una gran variedad de técnicas basadas en el uso de organismos vivos o sus componentes subcelulares. Presenta un amplio espectro de aplicaciones y ofrece nuevas vías para el desarrollo de tratamientos o terapias preventivas para un conjunto diverso de enfermedades como son el cáncer, las enfermedades infecciosas o las enfermedades neurodegenerativas. Dos de las técnicas biotecnológicas más utilizadas y en las que se centra esta revisión son la terapia génica (uso de vectores virales) y la inmunoterapia (uso de anticuerpos monoclonales). Los avances y usos de estas técnicas se explican en su aplicación a las enfermedades neurodegenerativas. Estas se caracterizan por la pérdida progresiva de la función neuronal y la muerte selectiva de poblaciones de neuronas vulnerables. La mayoría son trastornos multigénicos, con distinta penetración de los genes alterados, y multifactoriales, influencia de factores genéticos, ambientales y envejecimiento, donde los eventos críticos son el plegamiento incorrecto, la agregación y la acumulación de proteínas en el cerebro. Su evaluación se basa en la diferenciación de los depósitos de proteínas anormales y la valoración de las distintas etapas de cada enfermedad. En esta revisión se han escogido tres patologías: la enfermedad de Alzheimer, caracterizado por ovillos neurofibrilares y placas neuríticas, la enfermedad de Parkinson, caracterizado por cuerpos de Lewys, y la enfermedad de Corea de Huntington, caracterizada por agregados de huntingtina. Estas técnicas demuestran los beneficios terapéuticos del uso de la biotecnología en estas patologías. No obstante, actualmente estas enfermedades siguen siendo incurables y tampoco existe ningún marcador de enfermedad presintomática, por lo que no es posible identificar el inicio de la pérdida de neuronas cerebrales. Se siguen necesitando investigaciones que muestren ensayos clínicos satisfactorios para encontrar nuevas vías en el tratamiento de las enfermedades neurod

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C. salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for

Chromohalobacter salexigens es un microorganismo extremófilo modelo de máximo interés y aplicabilidad en Biotecnología Microbiana, ya que sintetiza en respuesta a estrés osmótico y/o por temperatura, los solutos compatibles ectoína e hidroxiectoína, compuestos con propiedades estabilizadoras por lo que son de gran interés en Dermofarmacia, Biología Molecular, Agricultura o Biomedicina. La síntesis de ectoínas y su regulación es un proceso en el que intervienen complejos mecanismos transcripcionales y postranscripcionales, además de la implicación de reguladores globales y específicos. Por otro lado, se ha puesto de manifiesto la importancia de la regulación de la homeostasis del hierro en la adaptación a la salinidad, y por tanto en la síntesis de estos solutos. Así, el regulador global Fur es el encargado de mantener un control preciso de los niveles de hierro intracelulares existiendo, en C. salexigens, dos parálogos pertenecientes a la superfamilia Fur, Fur1 y Fur2. Además, para poder sintetizar la cantidad necesaria de estos solutos en condiciones de estrés, en C. salexigens se produce una adaptación metabólica en función de la salinidad y temperatura, ya que la síntesis de ectoínas depende de intermediarios procedentes de rutas del metabolismo central. Así, para poder dilucidar posibles modificaciones genéticas que permitan la obtención de cepas mejoradas en la producción de estos compuestos, se hace necesario un estudio más global e integrado tanto del metabolismo, como de los reguladores implicados en dicho proceso, así como de otros factores ambientales que puedan intervenir. En los últimos años se ha desarrollado un modelo metabólico de C. salexigens a escala genómica, que permitirá la predicción de estrategias de Ingeniería Metabólica para optimizar la producción de ectoínas en este microorganismo. Para la mejora de este modelo se está desarrollando un modelo de regulación que integre tanto las redes de señalización como las redes transcripcionales respons

Chromohalobacter salexigens es un microorganismo extremófilo modelo de máximo interés y aplicabilidad en Biotecnología Microbiana, ya que sintetiza en respuesta a estrés osmótico y/o por temperatura, los solutos compatibles ectoína e hidroxiectoína, compuestos con propiedades estabilizadoras por lo que son de gran interés en Dermofarmacia, Biología Molecular, Agricultura o Biomedicina. La síntesis de ectoínas y su regulación es un proceso en el que intervienen complejos mecanismos transcripcionales y postranscripcionales, además de la implicación de reguladores globales y específicos. Por otro lado, se ha puesto de manifiesto la importancia de la regulación de la homeostasis del hierro en la adaptación a la salinidad, y por tanto en la síntesis de estos solutos. Así, el regulador global Fur es el encargado de mantener un control preciso de los niveles de hierro intracelulares existiendo, en C. salexigens, dos parálogos pertenecientes a la superfamilia Fur, Fur1 y Fur2. Además, para poder sintetizar la cantidad necesaria de estos solutos en condiciones de estrés, en C. salexigens se produce una adaptación metabólica en función de la salinidad y temperatura, ya que la síntesis de ectoínas depende de intermediarios procedentes de rutas del metabolismo central. Así, para poder dilucidar posibles modificaciones genéticas que permitan la obtención de cepas mejoradas en la producción de estos compuestos, se hace necesario un estudio más global e integrado tanto del metabolismo, como de los reguladores implicados en dicho proceso, así como de otros factores ambientales que puedan intervenir. En los últimos años se ha desarrollado un modelo metabólico de C. salexigens a escala genómica, que permitirá la predicción de estrategias de Ingeniería Metabólica para optimizar la producción de ectoínas en este microorganismo. Para la mejora de este modelo se está desarrollando un modelo de regulación que integre tanto las redes de señalización como las redes transcripcionales respons

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C. salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for

Chromohalobacter salexigens es un microorganismo extremófilo modelo de máximo interés y aplicabilidad en Biotecnología Microbiana, ya que sintetiza en respuesta a estrés osmótico y/o por temperatura, los solutos compatibles ectoína e hidroxiectoína, compuestos con propiedades estabilizadoras por lo que son de gran interés en Dermofarmacia, Biología Molecular, Agricultura o Biomedicina. La síntesis de ectoínas y su regulación es un proceso en el que intervienen complejos mecanismos transcripcionales y postranscripcionales, además de la implicación de reguladores globales y específicos. Por otro lado, se ha puesto de manifiesto la importancia de la regulación de la homeostasis del hierro en la adaptación a la salinidad, y por tanto en la síntesis de estos solutos. Así, el regulador global Fur es el encargado de mantener un control preciso de los niveles de hierro intracelulares existiendo, en C. salexigens, dos parálogos pertenecientes a la superfamilia Fur, Fur1 y Fur2. Además, para poder sintetizar la cantidad necesaria de estos solutos en condiciones de estrés, en C. salexigens se produce una adaptación metabólica en función de la salinidad y temperatura, ya que la síntesis de ectoínas depende de intermediarios procedentes de rutas del metabolismo central. Así, para poder dilucidar posibles modificaciones genéticas que permitan la obtención de cepas mejoradas en la producción de estos compuestos, se hace necesario un estudio más global e integrado tanto del metabolismo, como de los reguladores implicados en dicho proceso, así como de otros factores ambientales que puedan intervenir. En los últimos años se ha desarrollado un modelo metabólico de C. salexigens a escala genómica, que permitirá la predicción de estrategias de Ingeniería Metabólica para optimizar la producción de ectoínas en este microorganismo. Para la mejora de este modelo se está desarrollando un modelo de regulación que integre tanto las redes de señalización como las redes transcripcionales respons

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C. salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for

Chromohalobacter salexigens es un microorganismo extremófilo modelo de máximo interés y aplicabilidad en Biotecnología Microbiana, ya que sintetiza en respuesta a estrés osmótico y/o por temperatura, los solutos compatibles ectoína e hidroxiectoína, compuestos con propiedades estabilizadoras por lo que son de gran interés en Dermofarmacia, Biología Molecular, Agricultura o Biomedicina. La síntesis de ectoínas y su regulación es un proceso en el que intervienen complejos mecanismos transcripcionales y postranscripcionales, además de la implicación de reguladores globales y específicos. Por otro lado, se ha puesto de manifiesto la importancia de la regulación de la homeostasis del hierro en la adaptación a la salinidad, y por tanto en la síntesis de estos solutos. Así, el regulador global Fur es el encargado de mantener un control preciso de los niveles de hierro intracelulares existiendo, en C. salexigens, dos parálogos pertenecientes a la superfamilia Fur, Fur1 y Fur2. Además, para poder sintetizar la cantidad necesaria de estos solutos en condiciones de estrés, en C. salexigens se produce una adaptación metabólica en función de la salinidad y temperatura, ya que la síntesis de ectoínas depende de intermediarios procedentes de rutas del metabolismo central. Así, para poder dilucidar posibles modificaciones genéticas que permitan la obtención de cepas mejoradas en la producción de estos compuestos, se hace necesario un estudio más global e integrado tanto del metabolismo, como de los reguladores implicados en dicho proceso, así como de otros factores ambientales que puedan intervenir. En los últimos años se ha desarrollado un modelo metabólico de C. salexigens a escala genómica, que permitirá la predicción de estrategias de Ingeniería Metabólica para optimizar la producción de ectoínas en este microorganismo. Para la mejora de este modelo se está desarrollando un modelo de regulación que integre tanto las redes de señalización como las redes transcripcionales respons

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C. salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for

Chromohalobacter salexigens es un microorganismo extremófilo modelo de máximo interés y aplicabilidad en Biotecnología Microbiana, ya que sintetiza en respuesta a estrés osmótico y/o por temperatura, los solutos compatibles ectoína e hidroxiectoína, compuestos con propiedades estabilizadoras por lo que son de gran interés en Dermofarmacia, Biología Molecular, Agricultura o Biomedicina. La síntesis de ectoínas y su regulación es un proceso en el que intervienen complejos mecanismos transcripcionales y postranscripcionales, además de la implicación de reguladores globales y específicos. Por otro lado, se ha puesto de manifiesto la importancia de la regulación de la homeostasis del hierro en la adaptación a la salinidad, y por tanto en la síntesis de estos solutos. Así, el regulador global Fur es el encargado de mantener un control preciso de los niveles de hierro intracelulares existiendo, en C. salexigens, dos parálogos pertenecientes a la superfamilia Fur, Fur1 y Fur2. Además, para poder sintetizar la cantidad necesaria de estos solutos en condiciones de estrés, en C. salexigens se produce una adaptación metabólica en función de la salinidad y temperatura, ya que la síntesis de ectoínas depende de intermediarios procedentes de rutas del metabolismo central. Así, para poder dilucidar posibles modificaciones genéticas que permitan la obtención de cepas mejoradas en la producción de estos compuestos, se hace necesario un estudio más global e integrado tanto del metabolismo, como de los reguladores implicados en dicho proceso, así como de otros factores ambientales que puedan intervenir. En los últimos años se ha desarrollado un modelo metabólico de C. salexigens a escala genómica, que permitirá la predicción de estrategias de Ingeniería Metabólica para optimizar la producción de ectoínas en este microorganismo. Para la mejora de este modelo se está desarrollando un modelo de regulación que integre tanto las redes de señalización como las redes transcripcionales respons

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C. salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for

Los biopolímeros están ganando interés no solo en la industria alimentaria, textil, agrícola, cosmética o como alternativa a los plásticos, sino también en el ámbito farmacéutico y biomédico. Estos polímeros pueden ser obtenidos a partir de fuentes animales, vegetales o microorganismos. Esta revisión se centrará en los biopolímeros que son obtenidos por fermentación microbiana ya que, a diferencia de las otras alternativas de producción, nos aporta ventajas como mayor rentabilidad del proceso, modificación de los microorganismos para incrementar la producción, uso de inductores específicos y mejora de las rutas de su biosíntesis. Los polihidroxialcanoatos y los exopolisacáridos microbianos son biopolímeros con mayor interés farmacéutico y biomédico. Los primeros se acumulan como gránulos insolubles en el interior de diversos microorganismos, los segundos, sin embargo, son producidos y secretados al exterior de la célula. Ambos biopolímeros microbianos se caracterizan por poseer propiedades especiales que otros polímeros sintéticos no tienen, como biocompatibilidad, biodegradabilidad y no toxicidad hacia las células y tejidos humanos o animales. Además, posibilitan la modificación de muchas de sus propiedades como la rigidez, elasticidad, cristalinidad y degradabilidad, de forma sencilla dando lugar a una mejor adaptación del biopolímero a su uso en diversas aplicaciones. Entre sus numerosas aplicaciones farmacéuticas destaca la modificación de la liberación del fármaco, trasporte dirigido del fármaco hacia su diana terapéutica, atravesar barreras fisiológicas y la protección de agentes terapéuticos inestables en condiciones fisiológicas. Así mismo, tienen un papel fundamental en aplicaciones biomédicas para la formación de productos sanitarios, como vendas y apósitos, andamios en ingeniería de tejido que son utilizados en la reparación de tejidos óseos, cartílagos, vasculares, piel, medula ósea y conductos nerviosos. También han demostrado ser buenos adyuvantes de v

Los biopolímeros están ganando interés no solo en la industria alimentaria, textil, agrícola, cosmética o como alternativa a los plásticos, sino también en el ámbito farmacéutico y biomédico. Estos polímeros pueden ser obtenidos a partir de fuentes animales, vegetales o microorganismos. Esta revisión se centrará en los biopolímeros que son obtenidos por fermentación microbiana ya que, a diferencia de las otras alternativas de producción, nos aporta ventajas como mayor rentabilidad del proceso, modificación de los microorganismos para incrementar la producción, uso de inductores específicos y mejora de las rutas de su biosíntesis. Los polihidroxialcanoatos y los exopolisacáridos microbianos son biopolímeros con mayor interés farmacéutico y biomédico. Los primeros se acumulan como gránulos insolubles en el interior de diversos microorganismos, los segundos, sin embargo, son producidos y secretados al exterior de la célula. Ambos biopolímeros microbianos se caracterizan por poseer propiedades especiales que otros polímeros sintéticos no tienen, como biocompatibilidad, biodegradabilidad y no toxicidad hacia las células y tejidos humanos o animales. Además, posibilitan la modificación de muchas de sus propiedades como la rigidez, elasticidad, cristalinidad y degradabilidad, de forma sencilla dando lugar a una mejor adaptación del biopolímero a su uso en diversas aplicaciones. Entre sus numerosas aplicaciones farmacéuticas destaca la modificación de la liberación del fármaco, trasporte dirigido del fármaco hacia su diana terapéutica, atravesar barreras fisiológicas y la protección de agentes terapéuticos inestables en condiciones fisiológicas. Así mismo, tienen un papel fundamental en aplicaciones biomédicas para la formación de productos sanitarios, como vendas y apósitos, andamios en ingeniería de tejido que son utilizados en la reparación de tejidos óseos, cartílagos, vasculares, piel, medula ósea y conductos nerviosos. También han demostrado ser buenos adyuvantes de v

Chromohalobacter salexigens is a halophilic bacterium which naturally produces ectoine and hydroxyectoine, two biotechnologically important compatible solutes that are accumulated in response to osmotic and heat stress, respectively. The exploitation of C. salexigens in biotechnology as a producer of ectoines requires an integral understanding of the regulatory pathways controlling ectoines metabolism. The availability of its genomic sequence, physiological, biochemical, genetic, and high performance transcriptomics data, previously obtained from our Research Group, together with regulatory studies (this work), will allow us to obtain a more global knowledge of the metabolism of the ectoines in C. salexigens. The aim of this PhD was two-fold. On the one hand, to gain insight on the regulatory circuits controlling the metabolism of ectoines in C. salexigens. On the other, to progress in the biomedical use of ectoine for the prevention and treatment of neurodegenerative diseases. The findings presented in this work provide clues for a better understanding of the complex regulation of ectoine metabolism and osmoadaptation in this moderately halophilic bacterium, and its contribution to adaptation to extreme environments. Our results will be overlayed on the existing high-quality genome-based metabolic model and global analyzes, in order to facilitate the rational design of new strains for Systems metabolic engineering. Additionally, we have investigated the in vitro and in vivo application of ectoine as neuroprotective- and inflammation-preventive- treatment in an Aβ murine model of Alzheimer's disease. Thus, we have got more insight in its potential applications in biomedicine, especially in neurodegenerative disorders as Alzheimer’s disease (AD). EupR is the first-described response regulator of a two-component system involved in bacterial osmoregulation (Rodríguez-Moya et al., 2010). Through in silico studies, EupK was selected as its putative cognate histidine kina

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as

Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not c

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperatur

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperatur

Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not c

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperatur

Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not c

Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not c

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperatur

Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not c

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperatur

Background The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Results Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect − mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L−1) and volumetric ectoine yields (up to 4.21 g L−1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic

Background The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Results Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect − mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L−1) and volumetric ectoine yields (up to 4.21 g L−1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic

Background The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Results Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect − mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L−1) and volumetric ectoine yields (up to 4.21 g L−1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic

Background The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Results Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect − mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L−1) and volumetric ectoine yields (up to 4.21 g L−1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic

Background The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Results Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect − mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L−1) and volumetric ectoine yields (up to 4.21 g L−1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic

La neuropatía óptica hereditaria de Leber es una enfermedad rara ligada al ADN mitocondrial, de manera que es transmitida verticalmente de madres a hijos. Usualmente, son las mujeres quienes portan la enfermedad, siendo los hombres quienes la padecen, normalmente estableciéndose en la segunda o tercera década de vida. Se trata de una mutación a nivel de la cadena respiratoria mitocondrial. Dependiendo de la mutación que se produzca, se verá afectada una u otra subunidad del complejo I. Independientemente del tipo de mutación, el resultado es el mismo: se ve afectado el flujo de electrones a través de la cadena respiratoria, ya que los electrones no pueden fluir desde el complejo I hasta los complejos sucesivos, de manera que se disminuye la síntesis de ATP y aumenta la concentración de radicales libres, lo cual provoca una peroxidación lipídica a nivel de las células ganglionares de la retina. El resultado, una pérdida de la visión central bilateral, inicialmente a nivel de un solo ojo, pero que puede evolucionar hasta afectar a ambos ojos. Se han estudiado distintas estrategias terapéuticas a lo largo de los años: desde fármacos análogos de la CoQ10 que corrijan ese defecto en la cadena respiratoria, hasta tratamientos sintomáticos mediante modificación de la dieta, pero ninguno de ellos ha resultado eficaz. En la última década se han venido estudiando distintas estrategias terapéuticas basándose en la terapia génica, terapia celular y modificación de la línea germinal: conociendo exactamente el 4 gen que se muta en esta enfermedad, se pueden diseñar vectores virales o no virales, o trasplantar células madre, que porten un gen silvestre que corrija dicha mutación e impedir que la enfermedad llegue a término o ralentizar su evolución

La neuropatía óptica hereditaria de Leber es una enfermedad rara ligada al ADN mitocondrial, de manera que es transmitida verticalmente de madres a hijos. Usualmente, son las mujeres quienes portan la enfermedad, siendo los hombres quienes la padecen, normalmente estableciéndose en la segunda o tercera década de vida. Se trata de una mutación a nivel de la cadena respiratoria mitocondrial. Dependiendo de la mutación que se produzca, se verá afectada una u otra subunidad del complejo I. Independientemente del tipo de mutación, el resultado es el mismo: se ve afectado el flujo de electrones a través de la cadena respiratoria, ya que los electrones no pueden fluir desde el complejo I hasta los complejos sucesivos, de manera que se disminuye la síntesis de ATP y aumenta la concentración de radicales libres, lo cual provoca una peroxidación lipídica a nivel de las células ganglionares de la retina. El resultado, una pérdida de la visión central bilateral, inicialmente a nivel de un solo ojo, pero que puede evolucionar hasta afectar a ambos ojos. Se han estudiado distintas estrategias terapéuticas a lo largo de los años: desde fármacos análogos de la CoQ10 que corrijan ese defecto en la cadena respiratoria, hasta tratamientos sintomáticos mediante modificación de la dieta, pero ninguno de ellos ha resultado eficaz. En la última década se han venido estudiando distintas estrategias terapéuticas basándose en la terapia génica, terapia celular y modificación de la línea germinal: conociendo exactamente el 4 gen que se muta en esta enfermedad, se pueden diseñar vectores virales o no virales, o trasplantar células madre, que porten un gen silvestre que corrija dicha mutación e impedir que la enfermedad llegue a término o ralentizar su evolución

Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544) or cy 2 (bll2388), and genes involved in nitric oxide detoxification (blr2806-09) and copper homeostasis ( copCAB ), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK -like gene (bll3466), and bll4130, which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC . P1 matched with the previously mapped 5 9 end of norC mRNA which we demonstrate in this work to be under FixK 2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c -staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS

Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544) or cy 2 (bll2388), and genes involved in nitric oxide detoxification (blr2806-09) and copper homeostasis ( copCAB ), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK -like gene (bll3466), and bll4130, which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC . P1 matched with the previously mapped 5 9 end of norC mRNA which we demonstrate in this work to be under FixK 2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c -staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS

Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544) or cy 2 (bll2388), and genes involved in nitric oxide detoxification (blr2806-09) and copper homeostasis ( copCAB ), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK -like gene (bll3466), and bll4130, which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC . P1 matched with the previously mapped 5 9 end of norC mRNA which we demonstrate in this work to be under FixK 2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c -staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS

Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544) or cy 2 (bll2388), and genes involved in nitric oxide detoxification (blr2806-09) and copper homeostasis ( copCAB ), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK -like gene (bll3466), and bll4130, which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC . P1 matched with the previously mapped 5 9 end of norC mRNA which we demonstrate in this work to be under FixK 2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c -staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS