Whispering waves: unveiling the secrets of marine vesicles, from nature to the laboratory

• Autores: Nadefa Adda Nekrouf
• Directores de la Tesis: Manuel Martínez García (dir. tes.)
• Lectura: En la Universitat d'Alacant / Universidad de Alicante ( España ) en 2024
• Idioma: inglés
• Número de páginas: 218
• Tribunal Calificador de la Tesis: José Manuel García Fernández (presid.), Esther Rubio Portillo (secret.), Marta Sebastián (voc.)
• Programa de doctorado: Programa de Doctorado en Biología Experimental y Aplicada por la Universidad de Alicante
• Materias:
  • Ciencias de la vida
• Enlaces
  • Tesis en acceso abierto en: RUA
• Resumen

  • Extracellular Vesicles (EVs) are prevalent across a spectrum of environments, spanning from terrestrial to marine ecosystems, where they are presumed to serve multifaceted functions. EVs are acknowledged for containing a myriad of biomolecules, among which DNA is notable. Despite their acknowledged importance, there is a notable dearth of comprehensive studies delving into the seasonal dynamics of EVs within natural marine habitats. Furthermore, the genetic information enclosed within these EVs remains largely untapped, representing a significant avenue for further exploration. The primary focus of this doctoral thesis lies in the characterization of EVs derived from environmental samples obtained from marine ecosystems, specifically the Mediterranean Sea and the Atlantic Ocean. Additionally, the study extends to the analysis of EVs originating from copiotrophic strains isolated within laboratory settings. This investigation aims to unveil the intricate structure and metagenomic content of EVs present in varied marine environments, thus elucidating their pivotal role in ecological processes. This thesis is structured into three chapters as outlined below: 1. Seasonal characterization of EVs from the Mediterranean Sea 2. Vibrio spp. vesicles microcosm experiment 3. Exploring prokaryotic DNA in the biological nanoparticle fraction of the deep Atlantic Ocean The summary of the chapters is as follows: 1. Seasonal characterization of EVs from the Mediterranean Sea Here, we carried out a field seasonal (1 year) sampling at the Mediterranean Sea (Cape Huertas, Alicante, Spain). Each month, surface seawater was collected, and microbial abundance and diversity, EV abundance and dynamics, and DNA content were monitored. For studying EV fractions, seawater samples were pumped through a 0.2 μm filter and concentrated using a tangential flow filter equipped with a 30 kDa membrane. The concentrate was then filtered again through a 0.2 μm syringe filter to ensure no cells remained, followed by pelleting via ultracentrifugation at 100,000 g for 6 hours at 4 °C. EVs were subsequently purified using an Optiprep (iodixanol) density gradient, ranging from 20% to 45%. The pellet from each fraction was resuspended in 0.5 mL of HEPES buffer with 0.85% NaCl at pH 7.4. EV abundance was assessed using Nanoparticle Tracking Analysis (NTA) with a NanoSight LM10HS instrument equipped with fluorescence and dispersion modes. Furthermore, to explore the ultrastructure of membrane EVs isolated from Mediterranean coastal waters, EVs were visualized using transmission electron microscopy (TEM) and Field Emission Scanning Electron Microscopy (FESEM). DNA from both prokaryotes and EV fractions of 25% and 20% was sequenced and analyzed. The Nanosight analysis revealed the highest abundance of EVs in Optiprep fractions 20% and 25%, corresponding to densities between 1.127 and 1.150 g/mL. Our NTA data obtained annually from the dispersion mode showed a wide range of EV concentrations, varying from 3.25x104 to 1.05x106 EVs/mL in seawater, fluctuating alongside microbial abundance. EVs were more abundant in the summer and spring seasons while lower in the winter. On average, the rate of EV production during the seasonal sampling ranged from 1 to 4 EVs per cell for almost all analyzed samples. In our taxonomy analysis covering both 25% and 20% fractions, we identified essential species significantly influencing EV production. The notable occurrence of the “Unclassified” category underscores the presence of sequences lacking clear taxonomic designations. These findings illuminate the considerable taxonomic diversity among bacteria involved in EV production, spanning oligotrophic species like Pelagibacter to copiotrophic ones like Alteromonas. In addition, photosynthetic eukaryotes, such as Micromonas, were putatively dominant in the vesicle production. Genes putatively belonging to viruses were identified in the sequenced dataset from both the 25% and 20% EV fractions albeit the relative proportion of viral signal was overall low. Notably, a consistent pattern of lower viral signals persisted in the 20% fraction compared to the 25% fraction across all seasonal samplings. This phenomenon is likely attributed to the higher molecular weight of viruses, causing them to gravitate towards higher densities. The presence of viral contigs within EV metagenomes implies the potential encapsulation of specific viruses within these EVs although other scenarios discussed in the thesis could not be ruled out. 2. Vibrio spp. vesicles microcosm experiment In this section, EVs were isolated from four pure copiotrophic bacterial strains which were isolated in the laboratory from the same sampling location studied in Chapter 1, including Vibrio kanaloae strain 1C, Pseudoalteromonas spp., and Arcobacter roscoffensis, with a particular emphasis on the V. kanaloae strain 1C. This Vibrio strain was selected as the model organism in this thesis due to its notable production of EVs compared to the other strains. The main objective of this chapter was to explore the vesiculation responses of Vibrio cultures cultivated under different culture conditions, such as standard laboratory conditions (e.g., Erlenmeyer flask), dilution of standard culture conditions to 1/5, and culture of V. kanaloae in dialysis tubing in aquarium containing natural seawater to mimic environmental conditions. EVs in the 20% fraction from laboratory dilution 1/5 cultures and those from cultures utilizing dialysis tubing and cellulose membranes were quantified using NTA. A higher total concentration of EVs was observed in the laboratory dilution 1/5 cultures (i.e., Erlenmeyer flasks). The ratio of EVs per cell varied among the conditions, with standard culture conditions showing a higher production of EVs at an average rate of 1 vesicle per 50 cells. Conversely, in the alternative conditions, including lab dilution 1/5 and dialysis tubing membrane, vesicle production was lower, averaging less than 1 vesicle per 100 cells. Furthermore, we conducted sequencing of both the Vibrio kanaloae strain 1C genome using Pacbio long-read sequencing and the DNA enclosed within EVs (Illumina sequencing) derived from the 20% fraction isolated from cultures utilizing dialysis tubing membranes placed in a seawater aquarium. Analysis of EVs reads mapped against the V. kanaloae strain 1C genome revealed a nearly complete representation of the strain’s genetic material within the EVs. However, a specific genomic segment spanning 129 Kb, identified as putative plasmid-phage, was notably absent in the Es. 3. Exploring prokaryotic DNA in the biological nanoparticle fraction of the deep Atlantic Ocean Covering nearly 65% of the Earth’s surface, the deep sea is an extensive and primarily oligotrophic ecosystem. The plankton communities dwelling in the deep sea rely almost entirely on the organic carbon that descends from the nutrient-rich surface waters, playing a crucial role in the Earth’s biogeochemical cycles (Mizuno et al., 2016). In this third chapter, we conducted a detailed exploration of the Atlantic Ocean’s depths, collecting 10 water samples from various depths, reaching beyond 4,000 meters. This endeavor aimed to investigate the prevalence and genetic makeup of potential EVs within the biological nanoparticle fractions spanning from the surface to the deep-sea environments. To quantify the presence of these putative EVs across all collected samples, we employed NTA in fluorescence mode. The analysis revealed a noticeable decrease in EV abundance as the depth increased, a trend that persisted down to depths exceeding 4,000 meters. This chapter focuses on understanding the distribution and genetic characteristics of EVs in the Atlantic Ocean, highlighting how these components vary with ocean depth. The taxonomic analysis of deep and surface Atlantic Ocean samples, specifically from depths of 4,296 meters (sample 24P_4296m) and 75 meters (sample 602_75m), provided a detailed insight into the microbial diversity and potential EV production sources within these marine environments. The analysis of the 4,296-meter sample revealed a diverse and complex microbial ecosystem characterized by a significant proportion of unclassified organisms and the identification of key classes such as Gammaproteobacteria, Dehalococcoidia, Flavobacteriia, and Verrucomicrobiae involved putatively in EV production. Conversely, the 75-meter sample demonstrated a dominance of Prochlorococcus spp., alongside significant contributions from Candidatus Pelagibacter spp., Candidatus Actinomarina spp., and a broad spectrum of other species. At the class level, this shallower sample was marked by the presence of Unclassified organisms, Cyanophyceae, Alphaproteobacteria, Gammaproteobacteria, Actinomycetia, and Flavobacteriia, indicating a complex, stratified microbial community. Collectively, these findings contribute to the broader understanding of microbial diversity and the distribution of EVproducing organisms across different oceanic depths.