Particle-associated bacteria in seawater dominate the colony-forming microbiome on ZoBell marine agar.

Planktonic particle-associated bacteria comprise particle-attached and motile free-living cells. These groups were obtained by settlement in Imhoff cones. Dilution plating on marine agar 2216 (ZoBell marine agar) and microscopic counts indicated a cultivability of 0.7% (0.4%-1.2%) of bacteria in coastal seawater collected at Helgoland Roads, North Sea. Particle-associated bacteria presented a minority population in seawater, but had a larger cultivability of 25% (0.9%-100%) for populations collected by settlement of particles and 5.7% (0.9%-24%) for populations collected by filtration. Partial 16S rRNA gene sequences indicated that 84% of the cultured taxa were either enriched in particle-associated microbiomes or only found in these microbiomes, including Sulfitobacter and other Rhodobacteraceae, Pseudoalteromonas, Psychromonas, Arcobacter and many Flavobacteriaceae. Illumina-based 16S rRNA V3V4 amplicon sequences of plate communities revealed that nearly all operational taxonomic units had a cultivated and described strain in close phylogenetic proximity. This suggested that decades of strain isolation from seawater on ZoBell marine agar had achieved a very good coverage of cultivable genera abundant in nature. The majority belonged to particle-associated bacteria, complementing observations that abundant free-living seawater bacteria often require cultivation conditions closer to their natural habitat like liquid cultivation in oligotrophic medium.

Niche partitioning of the ubiquitous and ecologically relevant NS5 marine group.

Niche concept is a core tenet of ecology that has recently been applied in marine microbial research to describe the partitioning of taxa based either on adaptations to specific conditions across environments or on adaptations to specialised substrates. In this study, we combine spatiotemporal dynamics and predicted substrate utilisation to describe species-level niche partitioning within the NS5 Marine Group. Despite NS5 representing one of the most abundant marine flavobacterial clades from across the world's oceans, our knowledge on their phylogenetic diversity and ecological functions is limited. Using novel and database-derived 16S rRNA gene and ribosomal protein sequences, we delineate the NS5 into 35 distinct species-level clusters, contained within four novel candidate genera. One candidate species, "Arcticimaribacter forsetii AHE01FL", includes a novel cultured isolate, for which we provide a complete genome sequence-the first of an NS5-along with morphological insights using transmission electron microscopy. Assessing species' spatial distribution dynamics across the Tara Oceans dataset, we identify depth as a key influencing factor, with 32 species preferring surface waters, as well as distinct patterns in relation to temperature, oxygen and salinity. Each species harbours a unique substrate-degradation potential along with predicted substrates conserved at the genus-level, e.g. alginate in NS5_F. Successional dynamics were observed for three species in a time-series dataset, likely driven by specialised substrate adaptations. We propose that the ecological niche partitioning of NS5 species is mainly based on specific abiotic factors, which define the niche space, and substrate availability that drive the species-specific temporal dynamics.

Highly diverse flavobacterial phages isolated from North Sea spring blooms.

It is generally recognized that phages are a mortality factor for their bacterial hosts. This could be particularly true in spring phytoplankton blooms, which are known to be closely followed by a highly specialized bacterial community. We hypothesized that phages modulate these dense heterotrophic bacteria successions following phytoplankton blooms. In this study, we focused on Flavobacteriia, because they are main responders during these blooms and have an important role in the degradation of polysaccharides. A cultivation-based approach was used, obtaining 44 lytic flavobacterial phages (flavophages), representing twelve new species from two viral realms. Taxonomic analysis allowed us to delineate ten new phage genera and ten new families, from which nine and four, respectively, had no previously cultivated representatives. Genomic analysis predicted various life styles and genomic replication strategies. A likely eukaryote-associated host habitat was reflected in the gene content of some of the flavophages. Detection in cellular metagenomes and by direct-plating showed that part of these phages were actively replicating in the environment during the 2018 spring bloom. Furthermore, CRISPR/Cas spacers and re-isolation during two consecutive years suggested that, at least part of the new flavophages are stable components of the microbial community in the North Sea. Together, our results indicate that these diverse flavophages have the potential to modulate their respective host populations.

Cultivation of particle-associated heterotrophic bacteria during a spring phytoplankton bloom in the North Sea.

Seawater contains free-living and particle-attached bacteria. Only a small fraction is cultivable on plates. As free-living and particle-associated bacteria differ in their physiological traits, their cultivability on plates may coincide with particle association. Using filtration and Imhoff sedimentation cones, particles were collected during a spring phytoplankton bloom off Helgoland (North Sea) in order to obtain particle-associated bacteria as inocula. Direct dilution plating resulted in 526 strains from 3 µm filtration retentates and 597 strains from settled particles. Motile Gammaproteobacteria from the genera Pseudoalteromonas, Shewanella, Psychrobacter, Vibrio and Colwellia, as well as particle-attached Flavobacteriia affiliating with the genera Tenacibaculum and Gramella, were frequently isolated. As a result, a diverse collection comprised of 266 strains was deposited. Two strains were most likely to represent novel genera and 78 strains were probably novel species. Recently, a high-throughput cultivation study from the same site using seawater as an inoculum had retrieved 271 operational phylogenetic units (OPUs) that represented 88% of the 4136 characterized strains at the species level. A comparison of 16S rRNA gene sequences revealed that the collection obtained matched 104 of the 271 seawater OPUs at the species level and an additional 113 at the genus level. This large overlap indicated a significant contribution of particle-associated bacteria to the cultivable microbiome from seawater. The presence of 49 genera not identified in the larger seawater study suggested that sample fractionation was an efficient strategy to cultivate rare members of the planktonic microbiome. The diverse collection of heterotrophic bacteria retrieved in this study will be a rich source for future studies on the biology of particle-associated bacteria.

Particle Collection in Imhoff Sedimentation Cones Enriches Both Motile Chemotactic and Particle-Attached Bacteria.

Marine heterotrophic microorganisms remineralize about half of the annual primary production, with the microbiomes on and around algae and particles having a major contribution. These microbiomes specifically include free-living chemotactic and particle-attached bacteria, which are often difficult to analyze individually, as the standard method of size-selective filtration only gives access to particle-attached bacteria. In this study, we demonstrated that particle collection in Imhoff sedimentation cones enriches microbiomes that included free-living chemotactic bacteria and were distinct from particle microbiomes obtained by filtration or centrifugation. Coastal seawater was collected during North Sea phytoplankton spring blooms, and the microbiomes were investigated using 16S rRNA amplicon sequencing and fluorescence microscopy. Enrichment factors of individual operational taxonomic units (OTUs) were calculated for comparison of fractionated communities after separation with unfractionated seawater communities. Filtration resulted in a loss of cells and yielded particle fractions including bacterial aggregates, filaments, and large cells. Centrifugation had the lowest separation capacity. Particles with a sinking rate of >2.4 m day-1 were collected in sedimentation cones as a bottom fraction and enriched in free-living chemotactic bacteria, i.e., Sulfitobacter, Pseudoalteromonas, and Vibrio. Subfractions of these bottom fractions, obtained by centrifugation, showed enrichment of either free-living or particle-attached bacteria. We identified five distinct enrichment patterns across all separation techniques: mechano-sensitive and mechano-stable free-living bacteria and three groups of particle-attached bacteria. Simultaneous enrichment of particle-attached and chemotactic free-living bacteria in Imhoff sedimentation cones is a novel experimental access to these groups providing more insights into the diversity, structure, and function of particle-associated microbiomes, including members of the phycosphere.