Cultivable Winogradskyella species are genomically distinct from the sympatric abundant candidate species.

Winogradskyella is a genus within the phylum Bacteroidetes with a clear marine origin. Most members of this genus have been found associated with marine animals and algae, but also with inorganic surfaces such as sand. In this study, we analyzed genomes of eleven species recently isolated from surface seawater samples from the North Sea during a single spring algae bloom. Corresponding metagenomes yielded a single Candidatus species for this genus. All species in culture, with the exception of W. ursingii, affiliated with a Winogradskyella lineage characterized by large genomes (~4.3 ± 0.4 Mb), with high complexity in their carbohydrate and protein degradation genes. Specifically, the polysaccharide utilization loci (PULs) were diverse within each individual strain, indicating large substrate versatility. Although present in the North Sea, the abundances of these strains were at, or below, the detection limit of the metagenomes. In contrast, the single species, classified as Candidatus W. atlantica, to which all North Sea MAGs belonged, affiliated with a lineage in which the cultivated representatives showed small genomes of ~3.0-3.5 Mb, with the MAGs having ~2.3 Mb. In Ca. W. atlantica, genome streamlining has apparently resulted in the loss of biosynthesis pathways for several amino acids including arginine, methionine, leucine and valine, and the PUL loci were reduced to a single one for utilizing laminarin. This as-yet uncultivated species seems to capitalize on sporadically abundant substrates that are released by algae blooms, mainly laminarin. We also suggest that this streamlined genome might be responsible for the lack of growth on plates for this Candidatus species, in contrast to growth of the less abundant but coexisting members of the genus.

High-throughput cultivation of heterotrophic bacteria during a spring phytoplankton bloom in the North Sea.

On-going studies of phytoplankton-bacterioplankton interactions at the long-term ecological research site Helgoland Roads have indicated that many of the heterotrophic bacterial taxa have not yet been cultivated. A high-throughput approach combining whole cell matrix-assisted laser desorption ionization - time of flight mass spectroscopy with 16S rRNA gene sequencing was applied to the spring bloom of 2016. Aiming at an assessment of cultivability during a spring bloom, cultivation on solid marine media had to be used since dilution to extinction would not have been feasible for a high-throughput approach, as performed in this study. A total of 5023 isolates were obtained from nine weekly samples on eight different solid media between the early-bloom and post-bloom periods. Most of the 4136 strains identified affiliated with Bacteroidetes (13.3%), Gammaproteobacteria (26.9%), Alphaproteobacteria (40.6%) and Actinobacteria (6.7%). Of the 271 operational phylogenetic units (OPUs) identified, 13 are likely to represent novel genera and 143 novel species. A comparison with 16S rRNA gene tag data indicated that most of the isolates were rather rare in surface waters, with the exception of five OPUs affiliating with Rhodobacteraceae, Polaribacter, Psychromonas and Pseudoalteromonas. The effort yielded many novel isolates, yet most of the abundant heterotrophic bacteria still remained elusive. The large strain collection obtained will not only provide insights into the succession of the cultivable fraction of the bacterioplankton, but also enable fine-tuned taxonomic and physiological follow-up studies for improving our knowledge on heterotrophic bacteria in North Sea waters.

Taxonomic study of nine new Winogradskyella species occurring in the shallow waters of Helgoland Roads, North Sea. Proposal of Winogradskyella schleiferi sp. nov., Winogradskyella costae sp. nov., Winogradskyella helgolandensis sp. nov., Winogradskyella vidalii sp. nov., Winogradskyella forsetii sp. nov., Winogradskyella ludwigii sp. nov., Winogradskyella ursingii sp. nov., Winogradskyella wichelsiae sp. nov., and Candidatus "Winogradskyella atlantica" sp. nov.

Evaluation of bacterial succession with cultivation-dependent strategies during a spring phytoplankton bloom in the North Sea led to the isolation of 41 strains that affiliated with the genus Winogradskyella. Fifteen of the strains were selected for a taxonomic study after discarding clonal cultures. A thorough phylogenetic, genomic and phenotypic analysis of the isolates indicated that they represented eight new species that coexisted in North Sea waters. Molecular data revealed the existence of an as yet uncultivated novel species recurrently binned from the North Sea metagenomes. The metagenome-assembled genomes (MAGs) of this new Winogradskyella were used to classify it as a new Candidatus species. This study represented a new example of the use of the tandem approach of whole cell mass spectrometry linked to 16S rRNA gene sequencing in order to facilitate the discovery of new taxa by high-throughput cultivation, which increases the probability of finding more than a single isolate for new species. In addition, we demonstrated the reasons for classifying MAGs representing recurrently retrieved heterotrophic species that evade cultivation even after an important high-throughput effort. The taxonomic study resulted in the classification of eight new species and one new Candidatus species of the genus Winogradskyella for which we propose the names W. schleiferi sp. nov., W. costae sp. nov., W. helgolandensis sp. nov., W. vidalii sp. nov., W. forsetii sp. nov., W. ludwigii sp. nov., W. ursingii sp. nov., W. wichelsiae sp. nov., and Candidatus "W. atlantica" sp. nov.