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Los vectores plasmídicos son uno de los sistemas más eficientes para la modificación genética en bacterias, pero existe una falta de estandarización de estas herramientas. La librería SEVA-DB es una iniciativa que promueve la utilización de plásmidos sintéticos basados en elementos modulables para facilitar el diseño de los dispositivos genéticos. Sin embargo, no existen publicaciones que estudien de forma sistemática su viabilidad en cianobacterias. En el presente trabajo se establecieron cuáles de estos vectores son más eficientes, mediante diferentes métodos de transferencia del ADN, en el organismo modelo Synechocystis sp. PCC 6803. De entre los siete plásmidos probados, sólo los basados en el origen de replicación RSF1010 presentaron colonias y en estas, la electroporación fue el método más eficiente. Adicionalmente, se diseñó un nuevo vector sintético adaptado al estándar SEVA y este presentó una ventaja conjugativa respecto a los disponibles en la librería. Estos nuevos plásmidos fueron viables tanto en el organismo modelo estudiado, como en la cianobacteria filamentos Anabaena sp. PCC 7120. Este trabajo desarrolla y expande la utilización de los plásmidos SEVA para establecer metodologías optimizadas con el fin de facilitar la biología sintética en cianobacterias.

La autofagia es un proceso catabólico ampliamente conservado en eucariotas por el cual se degradan componentes celulares incluyendo proteínas, ribosomas u orgánulos completos para mantener la homeostasis celular. La síntesis de ácidos grasos se localiza en el estroma del cloroplasto de plantas y algas. Los ácidos grasos sintetizados de novo por la enzima ácido graso sintasa (FAS) se utilizan como precursores de los lípidos esenciales para la estructura del cloroplasto y su función. El objetivo principal de esta tesis es investigar la conexión entre el proceso de autofagia y la inhibición de la síntesis de ácidos grasos en el cloroplasto de la microalga modelo Chlamydomonas reinhardtii. Los resultados de esta tesis demuestran que la inhibición de la enzima FAS mediante la droga cerulenina, un inhibidor específico de esta enzima, activa el proceso de autofagia. El tratamiento con esta droga disminuye la abundancia del lípido monogalactosil diacilglicerol (MGDG), el lípido mayoritario de las membranas plastídicas. Además, este tratamiento disminuye la eficiencia fotosintética y aumenta los niveles de especies reactivas del oxígeno (ROS). Igualmente, mediante microscopía electrónica de transmisión se observó que las membranas tilacoidales presentan un alto grado de empaquetamiento y un mayor número de membranas por apilamiento en células tratadas con cerulenina. Por otro lado, el análisis transcriptómico realizado en estas células mostró la inducción de genes de respuesta a estrés en el cloroplasto y la acumulación de transcritos relacionados con la detoxificación de ROS. Así, nuestros resultados indican que existe una relación entre el metabolismo de lípidos, la respuesta a estrés en el cloroplasto y el proceso de autofagia en Chlamydomonas reinhardtii. Para profundizar en el estudio de la inhibición de la enzima FAS y su conexión con la autofagia en este organismo modelo, se generó un mutante deficiente de autofagia mediante el sistema de edición genético CRISPR/Cas9.

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

Fatty acids are synthesized in the stroma of plant and algal chloroplasts by the fatty acid synthase complex. Newly synthesized fatty acids are then used to generate plastidial lipids that are essential for chloroplast structure and function. Here, we show that inhibition of fatty acid synthesis in the model alga Chlamydomonas reinhardtii activates autophagy, a highly conserved catabolic process by which cells degrade intracellular material under adverse conditions to maintain cell homeostasis. Treatment of Chlamydomonas cells with cerulenin, a specific fatty acid synthase inhibitor, stimulated lipidation of the autophagosome protein ATG8 and enhanced autophagic flux. We found that inhibition of fatty acid synthesis decreased monogalactosyldiacylglycerol abundance, increased lutein content, down-regulated photosynthesis, and increased the production of reactive oxygen species. Electron microscopy revealed a high degree of thylakoid membrane stacking in cerulenin-treated cells. Moreover, global transcriptomic analysis of these cells showed an up-regulation of genes encoding chloroplast proteins involved in protein folding and oxidative stress and the induction of major catabolic processes, including autophagy and proteasome pathways. Thus, our results uncovered a link between lipid metabolism, chloroplast integrity, and autophagy through a mechanism that involves the activation of a chloroplast quality control system.

Cyanobacteria form a diverse group of oxygenic photosynthetic prokaryotes considered to be the antecessor of plant chloroplast. They contain four different thioredoxins isoforms, three of them corresponding to m, x and y type present in plant chloroplast, while the fourth one (named TrxC) is exclusively found in cyanobacteria. TrxC has a modified active site (WCGLC) instead of the canonical (WCGPC) present in most thioredoxins. We have purified it and assayed its activity but surprisingly TrxC lacked all the classical activities, such as insulin precipitation or activation of the fructose-1,6-bisphosphatase. Mutants lacking trxC or over-expressing it were generated in the model cyanobacterium Synechocystis sp. PCC 6803 and their phenotypes have been analyzed. The ∆trxC mutant grew at similar rates to WT in all conditions tested although it showed an increased carotenoid content especially under low carbon conditions. Overexpression strains showed reduced growth under the same conditions and accumulated lower amounts of carotenoids. They also showed lower oxygen evolution rates at high light but higher Fv’/Fm’ and Non-photochemical-quenching (NPQ) in dark adapted cells, suggesting a more oxidized plastoquinone pool. All these data suggest that TrxC might have a role in regulating photosynthetic adaptation to low carbon and/or high light conditions.

Autophagy is an intracellular catabolic system that delivers cytoplasmic constituents and organelles in the vacuole. This degradative process is mediated by a group of proteins coded by autophagy-related (ATG) genes that are widely conserved from yeasts to plants and mammals. Homologs of ATG genes have been also identified in algal genomes including the unicellular model green alga Chlamydomonas reinhardtii. The development of specific tools to monitor autophagy in Chlamydomonas has expanded our current knowledge about the regulation and function of this process in algae. Recent findings indicated that autophagy is regulated by redox signals and the TOR network in Chlamydomonas and revealed that this process may play in important role in the control of lipid metabolism and ribosomal protein turnover in this alga. Here, we will describe the different techniques and approaches that have been reported to study autophagy and autophagic flux in Chlamydomonas

Autophagy is an intracellular catabolic system that delivers cytoplasmic constituents and organelles in the vacuole. This degradative process is mediated by a group of proteins coded by autophagy-related (ATG) genes that are widely conserved from yeasts to plants and mammals. Homologs of ATG genes have been also identified in algal genomes including the unicellular model green alga Chlamydomonas reinhardtii. The development of specific tools to monitor autophagy in Chlamydomonas has expanded our current knowledge about the regulation and function of this process in algae. Recent findings indicated that autophagy is regulated by redox signals and the TOR network in Chlamydomonas and revealed that this process may play in important role in the control of lipid metabolism and ribosomal protein turnover in this alga. Here, we will describe the different techniques and approaches that have been reported to study autophagy and autophagic flux in Chlamydomonas.