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Determination of bacterial vesicles interactions in the most abundant marine cyanobacteria and its potential applications

Periodic Reporting for period 1 - VESYNECH (Determination of bacterial vesicles interactions in the most abundant marine cyanobacteria and its potential applications)

Berichtszeitraum: 2019-04-15 bis 2021-04-14

In recent years, there has been an increasing recognition of the importance of marine microbes, coupled with the discovery of a vast microbial diversity in the oceans. Remarkable examples of this importance are the cyanobacteria, which are responsible for more than 50% of the oxygen production on Earth. Marine picocyanobacteria (i.e. Synechococcus) dominate most phytoplankton assemblages in oceans interacting over different spatial and temporal scales. Interactions mediated by uptake of proteins, lipids or metabolites, quorum signaling, viruses or by the secretion of membrane vesicles, have a large ecological impact on community structure. VESYNECH is an ambitious project with the aim of gaining a comprehensive understanding of the role of marine microbial vesicles of Synechococcus and its implications in the microbial diversity in the ocean. The abundant and ecological importance of these marine microorganisms as contributors of global primary production in the ocean reinforces the relevance of this project. To fully understand how these vesicles are implicated in these interactions, I will characterize the microbial vesicles of different strains of the cyanobacterium Synechococcus. Furthermore, I will determine the potential functions of vesicles from marine cyanobacteria and its role in the horizontal gene transfer. Finally, I will explore the content of these vesicles to unveil possible bioactive potential of genes and enzymes.

Specific objectives of the project:
Objective 1: To characterize the variety and composition of vesicles present in cultures of Synechococcus in response to stress.
Objective 2: To identify the possible role of the Synechococcus vesicles in HGT.
Objective 3: To explore possible changes in the transcriptome of the marine Synechococcus sp. PCC 7002 under the presence of vesicles of Synechococcus sp. WH 8102.
During these two years of the project, I have been studying and characterizing vesicles of the most abundant phototrophic cyanobacteria Synechococcus to gain insight into the ecology and function of these membrane vesicles.
It is known that starvation can induce extensive stress response pathways in bacterial cells, however we have demonstrated in this project that outer membrane vesicles production increase during phosphorus starvation condition, even when the culture looks stable. However, when the nutrient starvation is nitrogen, vesicles tend to decrease with a low protein concentration inside, what it might make sense since nitrogen is considered the primary nutrient limiting in the development of cyanobacteria.
Moreover, in this project we have demonstrated that light shock stimulates vesiculation more than 5 times and with more than double of protein. We found that under light shock vesicles released proteins involved in stress tolerance as superoxide dismutase, chaperones, pigments but also ferredoxins and GS-GOGAT proteins which are involved in the electrons transfer from the photosystem. Moreover, in control samples we identified photosystem I and II proteins which were not identified under the treatment what would demonstrate damage to PSII and PSI than the wild type. The protein CtpA, encoding the carboxyl-terminal processing protease involved in the maturation of the PSII core subunit D1 also increased during the treatment.
Due to the damaging nature of Reactive Oxygen Species (ROS), very efficient scavenging (or antioxidant) mechanisms exist in chloroplasts to prevent oxidative damage: 1O2 can be quenched by pigments and the superoxide dismutase. The excess of electrons in the photosynthetic electron chain could explain the increase of proteins involved in the transfers of electrons as ferrodoxins and GS-GOGAT. Light stress is deleterious for the photosynthesis, and that the full recovery of damaged PSII from light shock might involve the neo-synthesis of a large number of PSII subunits and not just the reassembly of pre-existing subunits after D1 replacement.
Another stress-specific response related to photosynthesis is the significant induction of proteins coding for all ATPase subunits in response to high light stress. This suggests that, although cells seemingly actively respond to the increase in irradiance by reducing the number of photosystems and their light harvesting capacity (as demonstrated by the downregulation of most genes involved in biosynthesis of PSI as well as PSII subunits), they are able to use the extra photons provided by the increase in light intensity to enhance energy production.
The reason why we found all these proteins inside vesicle might be related with the abundance of the proteins existing in the membrane, before vesiculation. Moreover, all these results accumulate evidence that membrane vesicles endow bacteria with an extraordinary capacity to survive in stressful conditions and are considered to be a potent bacterial innate immune element.

During the project I promoted a new collaboration between Prof. Steve Biller and Penny Chisholm with the Prof. García-Fernández, leaders of two groups that have not collaborated before. For that, I spent a short stay in Penny´s lab at the Massachusetts Institute of Technology (Cambridge, USA) to exchange information in this subject. During this stay, Prof. Biller taught me to isolate, concentrate and characterize marine vesicles. Moreover, we started a few experiments working with Synechococcus WH7803 and WH8102 under different kind of stress. These experiments helped me to set up protocols in my lab and bring us the axenic cultures I needed to start the project at the University of Córdoba. Steve also taught me to quantify vesicles with the nanoparticles tracking analysis (Nanosight).

Achieving a greater understanding of the ocean and, particularly, of the organisms driving key biogeochemical process such as primary production, is crucial towards developing efficient environmental management policies by the public administration. For that reason, in this project I created a dissemination blog called “Capitana Vesy” (http://www.uco.es/investigacion/proyectos/capitanavesy/index.php/es/) in English and Spanish languages, thanks the educational platform “Scientific Culture and Innovation Unit” at the University of Córdoba, who help me with the creation of a public image for a exhibition ship and the creation a blog where I share the multidisciplinary talks, conferences in schools and presentations to the general public.
I have gained an outstanding training in characterization of marine vesicles at Penn Chisholm Lab, and I was able to transfer this knowledge and experience at the University of Córdoba. The fellowship gave me the opportunity to contact with people from the field, widen my network of contacts and building collaborations for the next phase of my career. Moreover, I had the opportunity to develop my own line of research and applied and got a new project as a PI. It also has allowed me to stimulate my creativity and acquire new complementary perspectives in my research.
This project had an important impact in my career because not only gave me the opportunity to open a new line of research but also it helped me to gain my actual position as Interim Substitute Professor at the University of Córdoba. In this project we got an interesting and new results about the knowledge of vesicles that expect to produce three articles in top-ranked journals.
Membrane vesicles of the marine cyanobacteria Synechococcus