Study of diversity and function of the marine key group Vis6 with cultivation-independent methods

Abstract

Phytoplankton in the ocean represents about 0.2% of all photosynthetic biomass on Earth, but is responsible for half of the global CO2 fixation. A large proportion of this produced organic matter is funneled through the microbial loop towards higher trophic levels by heterotrophic microorganisms. These have consequently a great impact on the global carbon cycle, the CO2 concentration in the atmosphere and ultimately our climate. Phytoplankton blooms are short-termed increases in phytoplankton biomass, followed by secondary blooms of heterotrophic bacteria. Taxonomic and ecological studies at the long term ecological research station Helgoland Roads, North Sea, revealed recurring patterns of bacteria on taxonomic and functional level. Members of the phylum Bacteroidetes and in particular of the class Flavobacteriia are among the major responders to phytoplankton blooms. Analyses with both cultivation and cultivation-independent techniques, based on genome sequencing and gene annotations, can reveal the role of individual groups in the degradation of phytoplankton derived organic matter. The focus of this work was the development of a culture-independent pipeline for the targeted access and the subsequent functional genome characterization of bacterial species from the environment. This pipeline combines fluorescence in situ hybridization (FISH) with fluorescence activated cell sorting (FACS) and subsequent genome sequencing. A taxon-specific FISH probe hybridizes to the target cells which are subsequently enriched by cell sorting based on the fluorescence signal. Finally, the DNA of the sorted cells is amplified and sequenced. The development of the pipeline involved an optimization of the recently developed hybridization chain reaction (HCR)-FISH method and the assessment of various cell fixatives concerning their influence on HCR-FISH signal intensity and the quality of genome sequencing and assembly. The developed pipeline was successfully applied on a planktonic seawater sample targeting the so far uncharacterized clade Vis6. This member of the Cryomorphaceae within the Flavobacteriia is recurring during spring phytoplankton blooms off the coast of Helgoland. The description of Vis6 as a novel candidate genus Candidatus Abditibacter with three species was enabled by the combination of the sequences retrieved from targeted sorting pipeline with additional data of North Sea metagenomes and abundance data based on the 16S rRNA identity of Vis6. The growth of Candidatus Abditibacter spp. is likely based on the utilization of phytoplankton-derived polysaccharides and proteins. In the future, the developed pipeline has the potential to be used on low abundance species from highly complex samples, where previous attempts with metagenomics have been unsuccessful so far.