A New Globally Distributed Phage Group Infecting Rhodobacteraceae in Marine Ecosystems

The ISME Journal https://doi.org/10.1038/s41396-019-0362-7 ARTICLE Cobaviruses – a new globally distributed phage group infecting Rhodobacteraceae in marine ecosystems 1 2 2 2 3 1 Vera Bischoff ● Boyke Bunk ● Jan P. Meier-Kolthoff ● Cathrin Spröer ● Anja Poehlein ● Marco Dogs ● 4 2 3 2 2 1 Mary Nguyen ● Jörn Petersen ● Rolf Daniel ● Jörg Overmann ● Markus Göker ● Meinhard Simon ● 1 1 Thorsten Brinkhoff ● Cristina Moraru Received: 7 July 2018 / Revised: 15 January 2019 / Accepted: 22 January 2019 © International Society for Microbial Ecology 2019. This article is published with open access Abstract Bacteriophages are widely considered to influence bacterial communities, however most phages are still unknown or not studied well enough to understand their ecological roles. We have isolated two phages infecting Lentibacter sp. SH36, affiliated with the marine Roseobacter group, and retrieved similar phage genomes from publicly available metagenomics databases. Phylogenetic analysis placed the new phages within the Cobavirus group, in the here newly proposed genus Siovirus and subfamily Riovirinae of the Podoviridae. Gene composition and presence of direct terminal repeats in 1234567890();,: 1234567890();,: cultivated cobaviruses point toward a genome replication and packaging strategy similar to the T7 phage. Investigation of the genomes suggests that viral lysis of the cell proceeds via the canonical holin-endolysin pathway. Cobaviral hosts include members of the genera Lentibacter, Sulfitobacter and Celeribacter of the Roseobacter group within the family Rhodobacteraceae (Alphaproteobacteria). Screening more than 5,000 marine metagenomes, we found cobaviruses worldwide from temperate to tropical waters, in the euphotic zone, mainly in bays and estuaries, but also in the open ocean. The presence of cobaviruses in protist metagenomes as well as the phylogenetic neighborhood of cobaviruses in glutaredoxin and ribonucleotide reductase trees suggest that cobaviruses could infect bacteria associated with phototrophic or grazing protists. With this study, we expand the understanding of the phylogeny, classification, genomic organization, biogeography and ecology of this phage group infecting marine Rhodobacteraceae. Introduction Viruses infecting bacteria and archaea are abundant in Supplementary information The online version of this article (https:// marine systems and are major drivers of biogeochemical doi.org/10.1038/s41396-019-0362-7) contains supplementary material, which is available to authorized users. cycles, through their ability to lyse cells, express auxiliary metabolic genes (AMG) and mediate horizontal gene * Cristina Moraru transfer [1–3]. Marine habitats contain abundant and [email protected] extremely diverse viral communities, the majority of which – 1 Institute for Chemistry and Biology of the Marine Environment, remains uncultivated [4 6]. University of Oldenburg, Carl-von-Ossietzky-Str. 9 -11, D-26111 Marine Rhodobacteraceae are an important group of Oldenburg, Germany heterotrophic bacteria in the marine environment. They are 2 Leibniz-Institut DSMZ—Deutsche Sammlung von known under the operational term “Roseobacter group” [7] Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7 B, D- and their phages are known as roseophages. Marine Rho- 38124 Braunschweig, Germany dobacteraceae show high metabolic versatility, metabolize 3 Georg-August-University Göttingen, Institute of Microbiology and a wide variety of organic compounds, degrade dimethyl- Genetics, Department of Genomic and Applied Microbiology & sulfoniopropionate, perform anoxygenic photosynthesis Göttingen Genomics Laboratory, Grisebachstr. 8, D-37077 – Göttingen, Germany and produce various secondary metabolites [7 9]. Rhodo- bacteraceae are present in diverse marine habitats (pelagic, 4 Centre for Marine Bio-Innovation, School of Biological, Earth and Environmental Sciences, The University of New South Wales, sediment, surface-associated) and are most abundant in Kensington, NSW 2052, Australia coastal temperate to polar regions [10–13]. Members of V. Bischoff et al. Rhodobacteraceae typically increase in abundance during Materials and methods algal blooms, where they dominate the active bacterial community, utilize algal exudates and lysates and poten- Phage enrichments and isolation of ICBM1 and tially engage in both mutualistic and pathogenic interactions ICBM2 phages with the algae [14–18]. Amongst the roseobacters, Lentibacter is a genus relevant in coastal and estuarine Surface seawater was collected from multiple stations waters, where its relative abundance can be up to 30% (53.978 8.059; 53.937 7.806; 53.896 7.535; 53.840 7.255; of the bacterial community [19], and has been repeatedly 53.793 6.997) in the southern North Sea, during a phyto- isolated from algal blooms in different geographical loca- plankton bloom in March 2015, on board of the cruise ship tions [20, 21]. RV Heincke. Further, the seawater from each station was In contrast to the large diversity of marine Rhodo- filtered on board through 0.7 µm filters, 47 mm in diameter bacteraceae, which includes currently over 70 genera [22], (GTTP filters, Millipore). To prevent clogging, the filters only a few roseophages, infecting seven genera, have been were exchanged every 2 liters. The seawater from all sta- isolated so far. More than half of the cultivated roseophages tions was pooled, transported to the laboratory and stored at belong to the N4-like phage group [23–28] and 4 °C in the dark. Two phage enrichments (S1 and S2), each recent taxonomic reevaluation suggests that they likely form of 100 ml, were set up by mixing 90 ml of freshly filtered a subfamily within Podoviridae [29]. Other roseophages (Nalgene rapid-flow, 0.2 µm, PES membrane, Thermo- have been classified in the Podoviridae or Siphoviridae Scientific) seawater with 10 ml of 10x ASW (see SI file S1 families [30–38]. text) and 2.1 ml of exponentially growing host culture The SIO1 phage infecting Roseobacter sp. SIO67 was the Lentibacter sp. SH36 (final OD600 = 0.006). Two controls first described roseophage and the first sequenced marine were prepared in parallel. The first, a positive control (PC) phage [30]. Highly similar phages (>96% nucleotide identity) for host growth, consisted of 100 ml 1x ASW and 2.1 ml of were isolated twelve years later from the same geographic exponentially growing host culture (final OD600 = 0.006). area, providing evidence that marine viruses can exist as The second, a negative control (NC) for growth of seawater discrete populations over long periods of time [39]. The SIO1 bacteria contaminants, which might have passed through the phage belongs to the Podoviridae and is distantly related to 0.2 µm filter, consisted of 90 ml freshly filtered (0.2 µm) the T7 phage [30], but has no RNA polymerase. A close seawater and 10 ml 10x ASW. Bacterial growth was mon- relative of SIO1 is the roseophage P12053L infecting Celer- itored by measuring the optical density at 600 nm (Beck- ibacter marinus IMCC12053 [33, 40]. Hardies et al. [41] mann DU520, USA). The cultures were incubated at 20 °C assigned SIO1 and Vibrio phage VpV262 to a T7 supergroup. and 100 rpm overnight, until the enrichment cultures This supergroup includes T7 related phages with an RNA showed signs of cell lysis. Lysis was indicated by polymerase (the Autographivirinae subfamily), and also decreasing OD in S1 and S2 cultures compared to the phages without the RNA polymerase. Hardies et al. [40] positive control and by the presence of cell debris in S1 and showed that all podoviruses, except those in the Picovirinae S2. The cell free phage lysates from the S1 and S2 subfamily, have a tail structure related to T7 and propose their enrichments were used to isolate the ICBM1 and ICBM2 placement within the transient tail structural homology phages, respectively, by plaque assays and single plaque group, with the following subgroups: the T7 subgroup picking. To determine the host range, 94 strains (SI file S1 (the Autographivirinae), the epsilon15 subgroup, the phage Table S1) covering the phylogenetic diversity of Rhodo- RIO-1 subgroup (including SIO1 phage), the P22 subgroup, bacteraceae were challenged with ICBM1 and ICBM2 the N4 subgroup, and a new subgroup represented by Pseu- phages, at three different temperatures (15, 20, and 28 °C), domonas phage F116. using the spot and plaque assay. For long-term storage, the In this study, two novel roseophage species infecting phages were infected into the host cells (before cell lysis), Lentibacter sp. SH36 were isolated and sequenced. The resuspended in glycerol and kept at −80 °C. Further details recruitment from sequence data sets of similar genomes, on phage isolation, host range determination and long-term including that of SIO1 and of environmental phages, has led storage procedures are found in SI file S1 text. to the delineation of a new group of phages which we termed the “Cobavirus” group. Furthermore, we carried out ICBM1 and ICBM2 phage lysates a comprehensive analysis of the phylogeny, classification, genomic organization, host range and ecology of the To obtain a high amount of ICBM1 and ICBM2 phage Cobavirus group, infecting also two other roseobacter biomass for TEM and genome sequencing, two subsequent genera. A survey of its global biogeography showed that infection cultures of Lentibacter sp. SH36 with phage cobaviruses occur mainly in coastal but also in oceanic ICBM1 or ICBM2 were performed. For the first round of water masses from temperate to tropical oceans.

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