Análisis multi-ómico de la respuesta a estrés osmótico y térmico en la bacteria extremófila crhomohalobacter salexigens
- Autores: Manuel Salvador de Lara
- Directores de la Tesis: Montserrat Argandoña Bertrán (dir. tes.), Carmen Vargas Macías (dir. tes.), Joaquín José Nieto Gutiérrez (dir. tes.)
- Lectura: En la Universidad de Sevilla ( España ) en 2014
- Idioma: español
- Tribunal Calificador de la Tesis: Francisco Rodríguez Valera (presid.), Maria Encarnación Mellado Durán (secret.), Manuel Cánovas Díaz (voc.), Eduardo Díaz Fernández (voc.), Laszlo N. Csonka (voc.)
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- Resumen
Chromohalobacter salexigens is a halophilic bacterium which naturally produces ectoine and hydroxyectoine, two compatible solutes that are accumulated in response to osmotic and heat stress, respectively. Ectoines have great biotechnological potential in dermopharmacy, cosmetics, molecular biology, and biomedicine. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism and high anaplerotic activity is needed to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Consequently, its metabolism has to be adapted to support this biosynthetic route. We have evidence for the existence of a limited catabolism of glucose at low salinity due to a low demand for ectoines synthesis, leading to a higher overflow metabolism.
Because of the complexity of the metabolic pathways for ectoines synthesis and their regulation, and their connection with central metabolism, a global and integrative approach such as Systems Biology is required, in order to develop new metabolic engineering strategies for the optimization of the industrial production of ectoines by C. salexigens. In this work, we performed a multi-omic (transcriptomic, proteomic and metabolomic) analysis of the response of C. salexigens to osmotic and heat stress, conditions that trigger ectoine and hydroxyectoine synthesis, respectively. Cells were cultured under conditions of maximal production of ectoine (2.5 M NaCl at 37 °C) or hydroxyectoine (2.5 M NaCl at 45 °C), as well as at low salinity (0.6 M NaCl at 37 °C) for differential analysis. Transcriptomics was performed by RNA-seq in a SOLID platform, proteomics by differential 2D-DIGE, and metabolomics by HPLC-MS and GC-MS. In a first approach, we used the Cytoscape software to associate data from differential transcriptomics to the C. salexigens protein interaction network (from the STRING database).
This analysis showed functional groups that were differentially expressed in each assayed condition. Subsequently, the global information related to metabolism was implemented and analyzed through the Pathway Projector web tool. The most relevant results related to stress response mechanisms, including metabolism (in particular glucose assimilation via Entner-Doudoroff and pentose phosphate pathways, ectoines synthesis, and energy production, among others), are described. Our findings highlighted specific metabolic adaptations to environmental conditions in this compatible solute-producing halophilic bacterium. These results will be overlaid in a high-quality genome-based metabolic model, and will serve to facilitate the rational design of new metabolic engineering strains. In addition, we describe the characterization of a C. salexigens mutant affected in the general stress factor RpoS, especially concerning its involvement in the control of ectoine synthesis and/or metabolic adaptations to osmotic and heat stress conditions. This is a first step towards the construction of a transcriptional regulatory model, which will link the C. salexigens genes involved in central and ectoines metabolism with the external conditions through a network of osmosensors and transcriptional factors.
