Ecogenomics of uncultured marine prokaryotes

Abstract

In the last few decades, novel approaches have been applied to the study of marine microorganism aiming to retrieve taxa that escape isolation in culture. Culture independent methodologies, together with high-throughput sequencing and extensive oceanographic sampling, have provided insight into a previously unknown taxonomic and functional diversity of marine microbes. Marine microbes play a fundamental role in nutrient cycling and climate regulation at a planetary scale. Thus, it is of paramount importance to define their taxonomic classification, distribution patterns, habitat preferences and functional properties in the ocean. Linking taxonomy with function has been a challenge in Microbial Ecology, and in the recent years two alternatives have been developed towards this end. Single Cell Genomics allows the sequencing of individual genomes from environmental samples (Single Amplified Genomes, SAGs) and genome reconstruction from metagenomes allows building genomes from the whole community’s DNA content (Metagenomic Assembled Genomes, MAGs). In the present dissertation, I have retrieved SAGs and MAGs from underexplored areas like the North Indian Ocean and the Arctic Ocean. The North Indian Ocean is subject to seasonal upwelling events that provide surface waters with fresh nutrients, resulting in phytoplankton blooms. Such high primary productivity in the surface waters results in heterotrophic metabolism in the subsurface, by prokaryotes that feed on the products released by primary producers. Such high heterotrophic activity consumes the available oxygen, and together with physical processes than prevent water mixing, generates an oxygen-depleted layer in the water column: the Oxygen Minimum Zone (OMZ). These water layers are predicted to increase due to global warming and have caught the attention of microbial ecologists as they are rich in microbes involved in the cycling of nitrogen and several microaerophilic and anaerobic metabolisms. Even though the North Indian Ocean has one of the most intense and large OMZs, little is known about the prokaryotic diversity of this environment. With Single Cell Genomics I was able to retrieve 98 SAGs of a novel species in the genus Kordia and after genetically screening them for microdiversity patterns, ten were selected for complete sequencing. The ten genomes were co-assembled together and manually curated for the generation of a reference, almost complete, draft genome. I described the novelty of this species based on multiple phylogenies and comparative genomics with the other described species of the genus Kordia. I also defined the functional potential and niche preference of the novel species combining its functional annotation with its distribution in the different metagenomes of the water column of origin, that included multiple depths and size fractions. The Arctic Ocean has a huge impact in climate regulation of our Planet and is currently being affected severely by global warming. The prokaryotic diversity of its waters has been assessed in sporadic sampling events, mostly focused on a specific season or geographic extension. In the present work I have built 3550 bins from Arctic metagenomes from different regions and seasons that are representative of almost half of the genetic content of the community. Of these, 530 can be classified as MAGs due to their medium and high-quality features and include a majority of novel taxa, especially at the species level but also at higher taxonomic ranks like Class in the case of Bacteria. I have studied their implications for the Arctic’s carbon cycle, their distribution patterns and habitat preferences, and have defined habitat generalists and specialists that can serve as future sentinels of climate change in the Arctic. Overall, this dissertation provides new insights into the taxonomic and functional diversity of uncultured taxa, and proposes new methodologies to improve genome assembly and quality controls in meta-omic mappings