Highly diverse flavobacterial phages isolated from North Sea spring blooms

Abstract

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.

Fig. 1. Flavophage detection during the 2018 spring phytoplankton bloom, as inferred from phage isolation and metagenome read mapping.

Upper panel: Results are presented in the context of chlorophyll a concentration (green), total bacterial cell numbers (black line), Bacteroidetes numbers (orange line), phage numbers by transmission electron microscopy (TEM, black bar), and phage numbers by epifluorescence light microscopy (LM, gray bar). Lower panel: Phage isolation is shown for different time points (x axis, Julian days), with the phage identity verified by sequencing (black dots) or not determined (gray dots). Most of the isolations were done by enrichment, and some by direct plating (asterisks). Phage detection in metagenomes was performed by read mapping, with a 90% read identity threshold for phages in the same species (full red circles) and 70% read identity threshold for related phages (dashed red circles). Alternating gray shading of isolates indicates phages belonging to the same family.

Fig. 2. Phylogenetic tree (consensus between RAxML and neighbor-joining) of the 16S rRNA gene from all bacterial strains used to enrich for phages in 2017 and 2018 (red), plus reference genomes.

Red squares indicate successful phage isolation.

Fig. 3. VirClust hierarchical clustering of the new dsDNA flavophages and their relatives (reduced dataset), based on intergenomic distances calculated using the protein cluster content.

For an extended tree, including the larger ICTV dataset, see SI file 3. 1. Hierarchical clustering tree. Two support values, selective inference (si, [99]) and approximately unbiased (au, [100]), are indicated at branching points (si/au) only for the major clades (see SI file 1 Figs. 6 and 7 for all support values). The tree was cut into smaller viral genome clusters (VGCs) using a 0.9 distance threshold. Each VGC containing our flavophages was proposed here as a new family. Exceptions were made for the “Aggregaviridae” and “Forsetiviridae”, for which only part of the VGC were included in the families, to exclude some genomes with lower support values. Each VGC is framed in a rectangle in 2 and 3. 2. Silhouette width, measures how related is a virus with other viruses in the same VGCs. Similarity to other VGCs is indicated by values closer to -1 (red). Similarity to viruses in the same VGC is indicated by values closer to 1 (green). 3. Distribution of the protein clusters (PCs) in the viral genomes. 4. Genome length (bps). 5. Fraction of proteins shared with other viruses (dark gray), based on protein assignment to PCs. 6. Virus names, with flavophages isolated in this study marked in red. 7. Genus assignment. Gray bars indicate already defined genera by the ICTV with the following names from top to bottom: Silviavirus, Labanvirus, Unahavirus, Pippivirus, Lillamyvirus, Muminvirus, Lillamyvirus, Helsingorvirus, and Incheonvirus. 8. Habitat. 9. Host association, as determined by: 9a) BlastN; 9b) CRISPR spacers; 9c) WIsH with host database GEM. 10. Life style genes. 11. TEM images of the new flavophages, uranyl acetate negative staining. Scale bar in each TEM image has 50 nm 12. Newly proposed families. Full name of environmental phages in “Winoviridae” and unclassified: AP013511.1_Uncultured_Mediterranean_phage_uvMED,_group_G21,_isolate__uvMED-CGR-C117A-MedDCM-OCT-S32-C49 and, AP013358.1_Uncultured_Mediterranean_phage_uvMED,_group_G1,_isolate__uvMED-CGR-U-MedDCM-OCT-S27-C45, respectively.

Fig. 4. VirClust hierarchical clustering of the new ssDNA flavophages and their relatives, based on intergenomic distances calculated using the protein cluster content.

1. Hierarchical clustering tree. Two support values, selective inference (si [99]) and approximately unbiased (au [100]) are indicated at branching points (si/au) only for the major clades (see SI file 1 Figs. 16 and 17 for all support values). The tree was cut into smaller viral genome clusters (VGCs) using a 0.9 distance threshold. Each VGC is framed in a rectangle in 2 and 3. 2. Silhouette width, measures how related is a virus with other viruses in the same VGCs. Similarity to other VGCs is indicated by values closer to -1 (red). Similarity to viruses in the same VGC is indicated by values closer to 1 (green). 3. Distribution of the protein clusters (PCs) in the viral genomes. 4. Genome length (bps). 5. Fraction of proteins shared with other viruses (dark gray), based on protein assignment to PCs. 6. Virus names, with flavophages isolated in this study marked in red. 7. TEM image of the new flavophage, uranyl acetate negative staining. Scale bar in TEM image has 50 nm. 8. Family (ICTV). 9. Kingdom (ICTV). 10. Realm (ICTV). Lighter colors in columns 8–10 represent phages not recognized by the ICTV, but by publications.

Fig. 5. Genome maps of phage isolates with color-coded gene annotations.

Genomes of one representative of each new phage species are shown and depicted at the same scale to allow phage genome size comparisons. Due to its small genome Omtje is not at the same scale. Genes are color-coded by annotation and specific functional annotated proteins are indicated at genomes. Alternating gray shading indicates phages of the same host genus.