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.

Abstract

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.

Introduction

Winogradskyella is a genus of the phylum Bacteroidetes classified within the family Flavobacteriaceae [5] with the type species W. thalassocola [37]. Currently, the genus consists of 34 classified species as listed in https://lpsn.dsmz.de/genus/winogradskyella [45], and has been emended on multiple occasions [4], [16], [38], [71]. The members of the genus have a very versatile phenotype, since they appear as non-sporulating rod or coccoid Gram-negative cells, which form colonies with diverse morphologies and yellow-, orange-, or salmon-colored pigmentation, but without flexirubins. Cells are either non-motile or motile by gliding, while some can form network-like structures; they are strictly aerobic or facultatively anaerobic and chemoorganotrophic with a NaCl range between 0.0–10% (w/v). Depending on the species, strains grow at a broad range of temperatures between 4 and 44 °C.

Members of Winogradskyella have been isolated from marine environments, including plankton (e.g. [18]) and sediments (e.g. [19]). Many strains have originated from samples associated with marine invertebrates, such as sponges (e.g. [56]), corals [13], mollusks (e.g. [41]), and echinoderms (e.g. [40]), while others came from marine vertebrates, such as tuna [62], algae (e.g. [22]) or inert surfaces of marine origin (e.g. [63]). Winogradskyella spp. have been reported to be polysaccharide degraders that have a very diverse arsenal of enzymes for dealing with complex and diverse macromolecules, especially polysaccharides. In addition, some species of the genus have been reported to have biotechnological relevance, for example, for their antifouling effect [7], or for the genetic engineering of their rhodopsins [9].

Cultivation-dependent strategies of bacterial succession during a spring phytoplankton bloom in the North Sea led to approximately 5000 strains being isolated in pure culture from the shallow waters of Helgoland Roads, located in the North Sea German Bight [2]. From among the more than 365 species detected in the North Sea survey, 41 isolates were identified as members of the genus Winogradskyella, and they were selected for a genomic study (Alejandre-Colomo, unpublished results). 16S rRNA gene-based phylogenetic reconstruction affiliated the new strains with nine different clades, several of them representing new species due to their unique phylogenetic position and genetic divergence from their closest relatives. After dereplication using DNA fingerprinting techniques, the genomes of 15 representative strains were sequenced and compared, and eight of the pure culture clades and two metagenome-assembled genome (MAG) collections represented as yet unclassified genomospecies of Winogradskyella. The present study therefore represented another good example of the use of the tandem approach of cultivation - MALDI-TOF MS–16S rRNA gene sequencing [64], which overcomes the lack of multiple strains in species descriptions [8], [53], [61]. The tandem approach consists of using whole cell biomass taken directly from fresh cultures for analysis using MALDI-TOF mass spectrometry, which provides simple profiles reflecting to a large extent the major ribosomal proteins [67]. Clustering based on similarity values between profiles allows the grouping of cultures into species or groups of closely related species. To identify each cluster, only a few representative members of each group need to have their 16S rRNA gene sequenced, and therefore identified. This process reduces costs significantly and therefore allows a significant increase in the number of strains tested, thus enhancing the chances of isolating multiple strains of the same taxon [67]. In this current study, we formally propose new specific names for eight new species of the genus Winogradskyella that have been isolated in a high-throughput cultivation survey [2], and the classification of a new Candidatus member of the genus that corresponded to the most abundantly retrieved genotype of the genus in German Bight waters using culture-independent methods, and that evaded cultivation.