High-throughput cultivation of heterotrophic bacteria during a north sea spring bloom and genomic insights into the genus winogradskyella

Abstract

The coastal waters surrounding the Helgoland Island, located in the German bight in the North Sea, experience a yearly spring phytoplankton bloom in parallel with the sunlight and temperature increase. These blooming phenomena induce secondary blooms of planktonic bacteria that trigger the succession of distinct bacterial clades. In this thesis, we focused on the study of the bloom-related microbial diversity and dynamics by means of culture-dependent (tandem approach using Whole Cell MALDI-TOF MS and 16S rRNA gene phylogenetic reconstruction of recovered isolates) and culture-independent (16S rRNA gene amplicon sequencing, genomic and metagenomic data) methodologies. The culture-dependent approach consisted of a high-throughput cultivation in solid agar plates applied to recover a collection of over 5,000 heterotrophic bacteria linked to the spring bloom waters of Helgoland during 2016. To this purpose, eight different media varying in carbon content and the chemical nature of the organic matter were used. Most of the 4,136 strains identified affiliated with Alphaproteobacteria (40.6%), Gammaproteobacteria (26.9%), Bacteroidetes (13.3%) and Actinobacteria (6.7%). The effort yielded many novel isolates as of the 271 operational phylogenetic units (OPUs) identified, 13 were likely to represent novel genera and 143 novel species. The collection of cultivated isolates of the present thesis led to the isolation of 41 strains affiliated with the genus Winogradskyella belonging to the Flavobacteriia class of the Bacteroidetes phylum. It is known that Flavobacteriia play a major role in the organic matter remineralization of the oceans especially for their capability of complex organic matter degradation such as the algal-derived biomass from spring blooms. Among the abovementioned Winogradskyella isolates that represented ten species we sequenced 15 genomes to investigate their taxonomy and ecology. The phylogenetic, genomic and phenotypic analysis of the isolates revealed that, among the ten different co-existing species in North Sea waters, eight represented novel taxa that we could formally describe. In addition, fifteen Metagenome-Assembled genomes (MAGs) were recovered from samples taken in the same location in the years 2010-2012 and 2016. All MAGs belonged to a unique species, but from a different phylogenetic lineage than the cultures and was classified as a new Candidatus species. With the genome and metagenome information we analysed their genomic and metabolic characteristics, with especial focus on the capability to degrade algal derived polysaccharides. The genome sequences formed two major lineages, the first mainly represented by cultivated organisms characterized by larger genomes ~4.3 Mb (± 0.4 Mb) and a high abundance and diversity of Polysaccharide Utilization Locis (PULs). By contrast, the second lineage was mostly represented by MAGs of ~2.3 Mb per genome and some isolates that showed genomes between ~3.0 – 3.5 Mb. This lineage, a part from having smaller genomes, showed less variety and number of PULs, revealing even both lineages show degradation capabilities they seem to occupy different niches and have different roles in the environment.