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Trabajo presentado en el VII Congreso Nacional de Microbiología Infustrial y Biotecnología Microbiana, celebrado en Cádiz (España) del 6 al 8 de junio de 2018, Todas las bacterias secuenciadas del género Streptomyces poseen en su genoma información genética para producir más de 20 compuestos con actividad antibiótica de los que únicamente una pequeña parte es producida en las condiciones de cultivo empleadas en los laboratorios. El activar estas rutas silenciadas ha atraído a gran número de investigadores y son variadas las estrategias abordadas para intentar su activación sin que hasta la fecha se haya encontrado una estrategia general para activarlas. Nuestro grupo está estudiando la regulación que ejercen varios sistemas de dos componentes (SDC) así como proteínas de unión a DNA sobre la producción de antibióticos empleando como organismo modelo Streptomyces coelicolor. Una de estas proteínas es la codificada por el gen SCO4441 que es una proteína de 295 aminoácidos con un dominio HTH-Xre y que se ha propuesto que forma parte de un sistema toxina-antitoxina junto con el gen codificado por SCO4442 que codifica una proteína de 63 aa con un dominio DUF397. En este sistema la primera proteína actuaría como antitoxina y la segunda sería la toxina (Shao, 2011). La sobreexpresión individual o conjunta de estos genes bajo el control de un promotor fuerte demostró la sobreexpresión de la toxina no tenía un efecto tóxico en las condiciones de cultivo ensayadas. Sin embargo, la sobreexpresión de la antitoxina o de ambos genes en conjunto conllevaba un elevado incremento en la producción de los antibióticos coloreados producidos por S. coelicolor. Actualmente hemos extendido este estudio a otras especies en las que hemos sobreexpresado estos genes y analizado su efecto en la producción de antibióticos.

Louise Erdrich''s The Round House (2012) is not only an original detective novel but a moving postcolonial narrative which denounces the individual and collective trauma that sexism, gender violence and racism cause to Native American communities in the USA. The novel''s interest also lies in how Erdrich problematizes the stereotypes traditionally attached to Indians as well as the victim/victimizer dichotomy by including a protagonist who is simultaneously a victim and a perpetrator traumatized by his own acts. The purpose of this paper is precisely to explore the figure of the protagonist from the perspective of perpetrator trauma-a neglected approach by critics-through a non-Eurocentric viewpoint in line with the current tendency of the decolonization of trauma studies. By so doing, I will demonstrate that under the novel''s numerous layers lies Erdrich''s core denunciation: the complex and unfair long-process situation of (neo)colonialism that Native Americans still endure in the 21st century.

Resumen del trabajo presentado al 7th Congress of European Microbiologists, celebrado en Valencia (España) del 9 al 13 de julio de 2017., [Backgrounds]: Streptomyces is a promising bacterial expression system that has been used to produce high levels of several proteins. In previous work, we described a new strategy for the stabilization of expression plasmids in Streptomyces based on the use of a toxin-antitoxin system (yefM/yoeBsl) as selection marker. We demonstrated the effectiveness of this system for the production of high levels of proteins without the use of antibiotics during the production step. However, the host used and the expression vectors still have the antibiotics resistance genes needed for its construction. [Objectives]: The objective of our work is the improvement of this system by the elimination of the antibiotic resistance genes still present in the host and in the expression plasmid. [Methods]: We have integrated the toxin gene (yoeBsl) in the genome with a new plasmid, pTES-tox, that has the target sites for the Cre recombinase (loxP) flanking the toxin gene and the phage attachment site (attP). Thus, after the expression of Cre recombinase we have deleted the plasmid backbone leaving only the toxin gene in the genome. In the same way, we have introduced the target sites for the Dre recombinase (rox) flanking the neomycin gene in the expression plasmid, the subsequent expression of the recombinase allowed us to delete it. [Conclusions]: We describe a system completely free of antibiotic resistance genes that is useful for the production of high yields of proteins in Streptomyces. This system could be a powerful tool for using Streptomyces as host to produce proteins at industrial and pharmaceutical level.

Resumen del trabajo presentado al 13th Horizons in Molecular Biology Congress, celebrado en Göttingen (Alemania) del 12 al 15 de septiembre de 2016., The two-component systems (TCS) are important global pathways of regulation in bacteria. Our group study the TCSs involved in antibiotic regulation in Streptomyces. In previous work we have shown the great potential of Streptomyces coelicolor strains altered in TCSs to express different clusters from other organisms that present low antibiotic production. In this work we have generated new strains of S. coelicolor in which the TCS AbrB have been removed. The AbrB is a negative regulator of antibiotic production and morphological differentiation. Moreover, we have generated a second elimination of AbrA TCS from the mutant AbrB, getting a double mutant strain. The AbrA is also a negative regulator previously studied in our group. Both mutants were obtained by the REDIRECT procedure. The comparative analysis of mutants with the wild type strain was carried out in different solid and liquid mediums. The phenotype of both mutants was highly medium-dependent. We have found, however, that the double elimination of two negative TCSs regulators (AbrA and AbrB) is beneficial to increase the production of endogenous antibiotics in some culture medium such as LB or YEPD. However, in many of the conditions tested, the production was similar to that obtained with the single mutants. We believe that in the absence of AbrA and AbrB systems, others TCSs could be playing an important role in the regulation of the observed responses in these strains. The usefulness of these new strains of S. coelicolor in the heterologous expression of antibiotic routes has been initially checked with the Tetracenomycin C pathway, an aromatic polyketide produced by S. glaucescens. We have observed a high increase in Tetracenomycin production in the mutants strains compared with the wild type.

Resumen del trabajo presentado al VI Congreso de Microbiología Industrial y Biotecnología Microbiana, celebrado en León (España) del 12 al 14 de septiembre de 2016., Las bacterias del género Streptomyces poseen un elevado potencial industrial debido a su capacidad para producir moléculas con interés en clínica, en veterinaria, o con interés fitosanitario. La secuenciación del genoma de un amplio número de especies de este género ha permitido corroborar que este potencial es aún mas amplio que el observado en el laboratorio ya que todas ellas tienen capacidad para producir más de 20 compuestos de los que únicamente una pequeña parte es producida en las condiciones de cultivo empleadas. El activar estas rutas silenciadas ha atraído a gran numero de investigadores y son variadas las estrategias abordadas para intentar su activación sin que hasta la fecha se haya encontrado una estrategia general para activarlas. Nuestro grupo esta estudiando la regulación que ejercen varios sistemas de dos componentes (SDC) sobre la producción de antibióticos empleando como organismo modelo Streptomyces coelicolor. A lo largo de este estudio hemos identificado dos SDCs que regulan negativamente la producción de antibióticos y la diferenciación y un sistema que actúa como un regulador positivo en ambos procesos. Este último sistema es un SDC especial al contener dos histidina kinasas (HK) y un único regulador de respuesta (RR) al que, las dos HKs del sistema, parecen controlar de modo diferente. La generación de cepas delecionadas en los SDC negativos y su empleo como cepas hospedadoras de rutas heterólogas ha permitido comprobar que esta es una buena estrategia para incrementar la producción heteróloga de compuestos ya conocidos. Por otro lado, hemos demostrado el gran potencial que supone la sobreexpresión del RR del SDC positivo en otros organismos para estimular la producción de sus metabolitos secundarios endógenos. Actualmente hemos extendido este estudio a otras proteínas con capacidad de unión a DNA observando que la producción de antibióticos puede verse modificada drásticamente por la sobreexpresión de alguna de estas proteínas.

Additional file 1. Supplementary tables (S1–S6) containing enzyme quantification by activity or by ImageJ.

Resumen del trabajo presentado a la 4th International Conference on Analytical proteomics, celebrada en Caparica (Portugal) del 7 al 9 de septiembre de 2015., [Purpose]: Chilling injury (CI) constitute a set of physiological disorders causing detrimental effects on quality of fruits when stored after harvest at low temperatures to retard postharvest ripening and to extend their shelf-life. We have applied proteomics analysis to identify potential protein markers of CI and cold stress, and to detect biological processes affected by CI or involved in the plant organ response to deal with the cold stress. [Experimental description]: Fruits (bell peppers and tomatoes) were subjected to postharvest storage and posterior reconditioning at room temperature (20ºC). Storage was performed at a low temperature causing CI (1 and 2ºC respectively) for one batch of fruits and at a higher non-chilling temperature (10 and 12ºC respectively) for another batch used as control. Physiological parameters were determined, such as electrolyte leakage, ethylene production by GC-FID, respiration rate by GC-TCD, lipid peroxidation by MDA production, and sugars, organic acids and ascorbic acid by HPLC-RID/UV. Transmission electron microscopy studies were performed in bell pepper fruit sections. After extraction proteins were separated by two-dimensional differential in-gel electrophoresis (2D-DIGE) and proteins spots were identified after trypsin digestion with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The search for peptides was fulfilled in batch mode using GPS Explorer v3.5 software with a licensed version of MASCOT, using viridiplantae and SGN databases. [Results and Conclusions]: A comparative proteomics analysis between fruits stored at chilling and non-chilling temperatures with posterior reconditioning was carried out. In tomato fruits storage at low temperature was ended just before visible CI symptoms appeared, but in bell pepper fruits this storage was extended until development of these symptoms. The proteomic study has allowed for the identification of processes occurring in fruit biology that are critically affected by chilling injury such as the activation of stress defense mechanisms starred by stressresponsive proteins (LEAs, HSPs and PRs), and alterations in carbohydrate/energy metabolism, particularly in enzymes from glycolysis, TCA and Calvin cycle, but also involved in stress signaling such as GAPDH, OGDH, INV and aldose-1-epimerase, this last one a potential new protein marker in cold stress. In tomato a remarkable down-regulation of a chloroplastic ATP synthase occurred, which may lead to depletion of intracellular ATP. In bell pepper fruits where CI progress was more advanced the proteomics analysis revealed alterations in redox homeostasis, protein abundance in the ascorbate-glutathione cycle is altered and CAT down-regulated., This work was supported by Fundación Séneca (1897/JLI/13).

Trabajo presentado al congreso "III Jornadas Bienales de jóvenes investigadores en proteómica" celebrado en Santiago de Compostela del 9 al 10 de febrero de 2012., Tomate (Solanum lycopersicum) y pimiento (Capsicum annuum), son dos especies hortofrutícolas pertenecientes a la familia Solanaceae susceptibles a sufrir daños por frío cuando se almacenan a bajas temperaturas, lo que afecta seriamente a la calidad de los frutos, siendo causa de importantes pérdidas económicas en el sector agro-alimentario. Una de las principales respuestas celulares al estrés por bajas temperaturas consiste en el mantenimiento de la homeostasis osmótica por la acumulación de osmolitos compatibles, para lo que tiene lugar la reprogramación de metabolismos energéticos como la glicólisis, el ciclo de Calvin y el ciclo de Krebs. Se ha abordado este estudio desde una perspectiva proteómica, comparando el proteoma de tomate y de pimiento sometidos a daños por frío para investigar las diferentes respuestas del metabolismo energético a las bajas temperaturas en frutos de estas dos especies. El análisis proteómico se ha realizado mediante electroforesis 2D-DIGE seguida de la identificación de las proteínas diferencialmente expresadas por MALDI-TOF-MS. Previamente a comparar los perfiles proteómicos de ambas especies, dentro de cada una de ellas se ha realizado una comparativa del perfil de frutos conservados a temperatura que inducen daños por frío y de frutos conservados a temperatura que no inducen dichos daños, con el fin de identificar las proteínas diferencialmente expresadas debido a la fisiopatía. Se han observado alteraciones en la fermentación y en el ciclo de Calvin en pimiento y cambios en el metabolismo de carbohidratos compuestos sacarosa), metabolismo de monosacáridos (glucosa) y cadena de transporte de electrones en tomate. En ambos frutos se han detectado modificaciones en la expresión de enzimas de la glicolisis y del ciclo de Krebs por efecto de la conservación refrigerada, hecho que pone de manifiesto la importancia que estos dos metabolismos parecen tener en la respuesta a los daños por frío., Este trabajo de investigación ha sido financiado por el Ministerio de Ciencia e Innovación (MICINN) (proyecto PIE2009-40I080), y por la Agencia de Ciencia y Tecnología de la Región de Murcia (Fundación SENECA) (proyecto 04553/GERM/06). PSB y ISE están contratadas en el marco del programa JAE-DOC del CSIC.

This work has been supported by grants from Ministerio de Ciencia e Innovación (EUI2008-03631 to R.I.S.; BFU2009-07565/BMC, 70% funded by FEDER, and the programme CONSOLIDER-INGENIO 2010, CSD2009-00006, both to J.M.-D.), Spain. This material is based on work supported in part by the National Science Foundation under Grant 0742976 to L.J.S. Thanks are due to Dr. M. Bibb for S. coelicolor M510, M511 and M512 strains and to M.J. Jiménez Rufo for her excellent technical work.

Trabajo presentado a la conferencia "Molecular Basis of Plant Stress" celebrado en Bulgaria del 21 al 23 de septiembre de 2011.-- FP7 REGPOT Project "BioSupport"., Tomato (Solanum lycopersicum L.) is the second most cultivated horticultural crop in the world in terms of harvested area and production, after the potato (FAOSTAT 2011, data from 2009). A main problem regarding its postharvest life is its sensitivity to low temperature stress during refrigerated storage, which constitutes a main obstacle for its long-term commercialization. This sensitivity is reflected in the physiopathy of chilling injury (CI), which negatively affects the final fruit quality. The main symptoms of CI-affected fruits are skin depressions, tissue decomposition and impaired ripening, which results in deficient flavour and aroma. Physiological and biochemical events involved in CI progress have been extensively described, but the precise molecular mechanisms that ultimately regulate the plant response to cold stress remain unclear. In order to investigate this response at molecular level in tomato fruit a proteomics strategy has undertaken. The proteome analysis provides a direct insight on the changes undergone by proteins, which are the major functional determinants of the cell machinery, in a certain biological situation such as low temperature stress. The protein expression profile of chilled tomato fruits has been compared with fruits stored at nonchilling temperature (control). The protein analysis has been performed by two-dimensional differential-in-gel-electrophoresis (2D-DIGE), and mass spectrometry of protein spots was applied to identify proteins differentially expressed. Comparative analysis revealed significant changes in abundance of 31 identified proteins between the proteomes of chilled and control fruits. Major modifications in the expression profile are related to those proteins specifically involved in stress (chaperonins and heat-shock proteins); cell wall biochemistry (hydrolytic enzymes), and carbohydrate metabolism (enzymes involved in glycolisis, tricarboxylic acids cycle, photosynthesis and sucrose biosynthesis)., This work was supported by the Spanish Ministry of Science and Innovation (MICINN) through grant and PIE2009-40I080, and by the Council of Science and Technology from the Spanish Region of Murcia (Fundación SENECA) through grant no. 04553/GERM/06.