bioRxiv preprint doi: https://doi.org/10.1101/2021.05.20.444936; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Highly diverse flavobacterial phages isolated from North Sea 2 spring blooms 3 Nina Bartlau1, Antje Wichels2, Georg Krohne3, Evelien M. Adriaenssens4, Anneke 4 Heins1, Bernhard M. Fuchs1, Rudolf Amann1*, Cristina Moraru5* 5 *corresponding authors 6 Affiliations 7 (1) Max Planck Institute for Marine Microbiology, Bremen, Germany 8 (2) Alfred-Wegner Institute Helmholtz Centre for Polar and Marine Research, 9 Biologische Anstalt Helgoland, Germany 10 (3) Imaging Core Facility, Biocenter, University of Würzburg, Germany 11 (4) Quadram Institute Bioscience, Norwich Research Park, Norwich, UK 12 (5) Institute for Chemistry and Biology of the Marine Environment, University of 13 Oldenburg, Germany 14 15 Addresses of corresponding authors 16 Cristina Moraru ([email protected]) 17 Institute for Chemistry and Biology of the Marine Environment, 18 Postbox 2503, Carl-von-Ossietzky –Str. 9 -11, 26111 Oldenburg, Germany 19 TEL: 0049 441 798 3218 20 21 Rudolf Amann ([email protected]) 22 Max Planck Institute for Marine Microbiology, 23 Celsiusstr. 1, 28359 Bremen, Germany 24 TEL: 0049 421 2028 930 25 Key words 26 Marine flavophages, Helgoland, virus counts, phage isolation, phage genomes 27 Running Title 28 North Sea flavophages 29 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.20.444936; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 30 Abstract 31 It is generally recognized that phages are a mortality factor for their bacterial 32 hosts. This could be particularly true in spring phytoplankton blooms, which are known 33 to be closely followed by a highly specialized bacterial community. We hypothesized that 34 phages modulate these dense heterotrophic bacteria successions following phytoplankton 35 blooms. In this study, we focused on Flavobacteriia, because they are main responders 36 during these blooms and have an important role in the degradation of polysaccharides. A 37 cultivation-based approach was used, obtaining 44 lytic flavobacterial phages 38 (flavophages), representing twelve new species from two viral realms. Taxonomic 39 analysis allowed us to delineate ten new phage genera and ten new families, from which 40 nine and four, respectively, had no previously cultivated representatives. Genomic 41 analysis predicted various life styles and genomic replication strategies. A likely 42 eukaryote-associated host habitat was reflected in the gene content of some of the 43 flavophages. Detection in cellular metagenomes and by direct-plating showed that part of 44 these phages were actively replicating in the environment during the 2018 spring bloom. 45 Furthermore, CRISPR/Cas spacers and re-isolation during two consecutive years 46 suggested that, at least part of the new flavophages are stable components of the 47 microbial community in the North Sea. Together, our results indicate that these diverse 48 flavophages have the potential to modulate their respective host populations. 49 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.20.444936; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 50 Introduction 51 Marine bacteriophages outnumber their hosts by one order of magnitude in 52 surface seawater and infect 10-45% of the bacterial cells at any given time (1-3). They 53 have a major impact on bacterioplankton dynamics. This impact can be density 54 dependent (4) and take many forms. By lysing infected cells, viruses decrease the 55 abundance of their host population, shifting the dominant bacterial population, and 56 recycling the intracellular nutrients inside the same trophic level (5). By expressing 57 auxiliary metabolic genes, phages likely enhance the metabolic capabilities of the 58 virocells (6, 7). By transferring pieces of host DNA, they can drive bacterial evolution 59 (8). By blocking superinfections with other phages (9), they can protect from immediate 60 lysis. Potentially phages even influence carbon export to the deep ocean due to 61 aggregation of cell debris resulted from cell lysis, called the viral shuttle (10, 11). Marine 62 phages modulate not only their hosts, but also the diversity and function of whole 63 ecosystems. This global impact is reflected in a high phage abundance (12) and diversity 64 (13, 14). 65 Phages are also well known for modulating bacterial communities in temperate 66 coastal oceans. Here, the increase in temperature and solar radiation in spring induces the 67 formation of phytoplankton blooms, which are often dominated by diatoms (15), and are 68 globally important components of the marine carbon cycle. These ephemeral events 69 release high amounts of organic matter, which fuels subsequent blooms of heterotrophic 70 bacteria. Flavobacteriia belong to the main responders (16, 17) and their increase is 71 linked to the release of phytoplankton derived polysaccharides (18, 19). These 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.20.444936; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 polysaccharides are produced by microalgae as storage compounds, cell wall building 73 blocks, and exudates (20-22). This highly complex organic matter is likely converted by 74 the Flavobacteriia to low molecular weight compounds and thus they are important for 75 the carbon turnover during phytoplankton blooms (18, 23-25). Recurrent genera like 76 Polaribacter, Maribacter, and Tenacibaculum succeed each other in a highly dynamic 77 fashion (19). This bacterial succession is likely triggered by the availability and quality of 78 substrates such as polysaccharides (18, 19), yet it cannot be fully understood without 79 considering mortality factors such as grazing by protists, and viral lysis. Grazing by 80 protists is mostly size dependent (26), whereas viral lysis is highly host-specific (27). 81 Based on the availability of suitable host bacteria, marine phages can be obtained 82 with standard techniques. Over the years notable numbers of phages infecting marine 83 Alphaproteobacteria (e.g. (28, 29)), Gammproteobacteria (e.g. (30)), and Cyanobacteria 84 (e.g. (31-36)) have been isolated. Despite the importance of Flavobacteriia as primary 85 degraders of high molecular weight algal derived matter only few marine flavobacterial 86 phages, to which we refer in the following as flavophages, have been characterized. This 87 includes several Cellulophaga phage isolates from the Baltic Sea, covering all phage 88 morphotypes in the realm Duplodnaviria and also two different phage groups in the 89 realm Monodnaviria (27, 37). Phages were also isolated for members of the genera 90 Polaribacter (38), Flavobacterium (39, 40), Croceibacter (41) or Nonlabens (42) and 91 included eight tailed phages, one having a myoviral and the rest having a siphoviral 92 morphology. However, the coverage of the class Flavobacteriia and the diversity of 93 marine flavophages remains low. With the exception of the Cellulophaga phages, most of 94 the other flavophages have only been briefly characterized in genome announcements. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.20.444936; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 95 In the context of a large project investigating bacterioplankton successions during 96 North Sea spring bloom season, we isolated and characterized new flavophages, with the 97 purpose of assessing their ecological impact and diversity. In total, more than 100 phage 98 isolates were obtained, sequenced, annotated, and classified. This diverse collection is 99 here presented in the context of virus and bacterioplankton abundances. Metagenomes 100 obtained for Helgoland waters of different size fractions were mapped to all newly 101 isolated flavophage genomes, testing the environmental relevance of the flavophage 102 isolates. This study indicates that flavophages are indeed a mortality factor during spring 103 blooms in temperate coastal seas. Furthermore it provides twelve novel phage-host 104 systems of six genera of Flavobacteriia, doubling the number of known hosts. 105 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.20.444936; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 106 Material and methods 107 Sampling campaigns 108 Surface water samples were taken off the island Helgoland at the long term 109 ecological research station Kabeltonne (54° 11.3’ N, 7° 54.0’ E). The water depth was 110 fluctuating from 7 to 10 m over the tidal cycle. In 2017, a weekly sampling was 111 conducted over five weeks starting on March 14 (Julian days 73-106) and covered the 112 beginning of a spring phytoplankton bloom. In 2018, a weekly sampling was conducted 113 over eight weeks starting on March 29 and ending on May 24 (Julian days 88-145). It 114 covered the full phytoplankton bloom. Additional measurements were performed to 115 determine the chlorophyll concentration, total bacterial cell counts, absolute 116 Bacteroidetes cell numbers and total virus abundances (SI file 1 text). Viruses were 117 counted both by epifluorescence microscopy of SYBR Gold stained samples and by 118 transmission electron microscopy (TEM) of uranyl acetate stained samples (SI file 1 119 text). The Bacteroidetes cell numbers were determined by 16S rRNA targeted 120 fluorescence in situ hybridization (FISH) with specific probes (SI file 1 text).