Polar freshwater cyanophage S-EIV1 represents a new widespread evolutionary lineage of phages

The ISME Journal (2015) 9, 2046–2058 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Polar freshwater cyanophage S-EIV1 represents a new widespread evolutionary lineage of phages C Chénard1, AM Chan1, WF Vincent2 and CA Suttle1,3,4 1 Department of Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, British Columbia, Canada; 2Département de Biologie and Centre d’études nordiques (CEN), Laval University, Quebec City, Quebec, Canada; 3Departments of Botany, and Microbiology & Immunology, University of British Columbia, Vancouver, British Columbia, Canada and 4Canadian Institute for Advanced Research, Toronto, Ontario, Canada Cyanobacteria are often the dominant phototrophs in polar freshwater communities; yet, the phages that infect them remain unknown. Here, we present a genomic and morphological characterization of cyanophage S-EIV1 that was isolated from freshwaters on Ellesmere Island (Nunavut, High Arctic Canada), and which infects the polar Synechococcus sp., strain PCCC-A2c. S-EIV1 represents a newly discovered evolutionary lineage of bacteriophages whose representatives are widespread in aquatic systems. Among the 130 predicted open reading frames (ORFs) there is no recognizable similarity to genes that encode structural proteins other than the large terminase subunit and a distant viral morphogenesis protein, indicating that the genes encoding the structural proteins of S-EIV1 are distinct from other viruses. As well, only 19 predicted coding sequences on the 79 178 bp circularly permuted genome have homology with genes encoding proteins of known function. Although S-EIV1 is divergent from other sequenced phage isolates, it shares synteny with phage genes captured on a fosmid from the deep-chlorophyll maximum in the Mediterranean Sea, as well as with an incision element in the genome of Anabaena variabilis (ATCC 29413). Sequence recruitment of metagenomic data indicates that S-EIV1-like viruses are cosmopolitan and abundant in a wide range of aquatic systems, suggesting they have an important ecological role. The ISME Journal (2015) 9, 2046–2058; doi:10.1038/ismej.2015.24; published online 27 March 2015 Introduction Cyanobacteria are often the dominant phototrophs in polar and subpolar lakes (Vincent, 2000) and can account for 450% of the phytoplankton chlorophyll a in northern lakes (Bergeron and Vincent, 1997). In meromictic lakes in the High Arctic (Van Hove et al., 2008) and Antarctica (Rankin et al., 1997) planktonic cyanobacteria occur at abundances of up to 104 and 106 cells ml  1, respectively. Most planktonic polar cyanobacteria are related to Synechococcus spp. and fall within two groups that contain brackish and freshwater representatives from different latitudes (Van Hove et al., 2008). Arctic and Antarctic cyanobacteria may be mostly cosmopolitan, generalist taxa rather than endemic specialists, but this will require ongoing genomic analysis to fully resolve (Jungblut et al., 2010). Despite the ecological importance of cyanobacteria in polar Correspondence: C Suttle, Earth, Ocean & Atmospheric Sciences, Microbiology & Immunology, Botany and the Canadian Institute fo, University of British Columbia, 6339 Stores Road Vancouver, Vancouver, British Columbia, Canada V6T 1Z4. E-mail: Received 6 February 2014; revised 2 January 2015; accepted 6 January 2015; published online 27 March 2015 freshwaters, and the long history of research on freshwater cyanophages (Safferman and Morris, 1963, 1964), there were no polar freshwater cyanophage systems in culture of which we were aware. This provided motivation to isolate a cyanophagehost system from high-arctic freshwaters. In marine systems, titers of cyanophages infecting Synechococcus spp. can be in excess of 105 ml  1, and vary with temperature, salinity and host abundance (Waterbury and Valois, 1993; Suttle and Chan, 1993; 1994), and are estimated to remove up to a few percent of the Synechococcus population each day (Suttle and Chan, 1993; Suttle, 1994). Historically, cyanophages have been classified into the families Myoviridae, Siphoviridae and Podoviridae based largely on tail morphologies that are either contractile, non-contractile and flexible, or short and non-contractile, respectively (Suttle, 2000; Nelson, 2004; Lavigne et al., 2012). Representatives of all three families have been isolated from seawater (Wilson et al., 1993; Suttle and Chan, 1993; Waterbury and Valois, 1993; Sullivan et al., 2003) and freshwater (Safferman and Morris, 1963, 1964; Adolph and Haselkorn, 1971; Yoshida et al., 2006; Liu et al., 2007). Host-range studies have revealed that some cyanophages have broad host ranges and Genomic analysis of a new group of cyanophages C Chénard et al 2047 are able to infect strains that are distantly related (Suttle and Chan, 1993; Waterbury and Valois, 1993) or that even belong to different genera (Sullivan et al., 2003). Nonetheless, the mosaic architecture of phage genomes, including structural genes that result in similar morphology but that appear to have a different evolutionary origin (Sabehi et al., 2012), increasingly call into question the use of a Linnaeanbased hierarchical classification, including morphology, as a basis for classifying phage (Lawrence et al., 2002; Nelson, 2004). Many sequenced genomes exist for cyanophages that infect marine Synechococcus spp. ( Chen and Lu, 2002; Mann et al., 2005; Millard et al., 2009; Sullivan et al., 2010; Huang et al., 2012; Sabehi et al., 2012) and Prochlorococcus spp. (Sullivan et al., 2005, 2009; Sullivan et al., 2010; Labrie et al., 2013), as well as one from a myovirus that infects both genera (Weigele et al., 2007). Most cyanophage isolates are myoviruses with genome size ranges from 161 to 252 kb, and share core genes involved in virion structure, DNA replication and host-derived genes with T4-like phage (Mann et al., 2005; Millard et al., 2009; Sullivan et al., 2010) A number of sequenced marine cyanophages are also podoviruses, having genomes between 42 kb and 47 kb, and sharing similar genome architecture, as well as core genes with T7-like phages, including genes involved in virion structure, DNA replication and that are host-derived (Chen and Lu, 2002; Sullivan et al., 2005; Labrie et al., 2013). The few sequenced marine cyanophages that are siphoviruses have genome sizes between 30 and 108 kb (Sullivan et al., 2005; Huang et al., 2012; Ponsero et al., 2013), and, although divergent from other siphoviruses, they share several functional genes with lambda-like phages. The comparatively limited data for freshwater cyanophages reveal that most are not closely related to their marine counterparts. Of the five sequenced cyanophages that infect Microcystis aeruginosa (Yoshida et al., 2008), Phormidium foveolarum (Liu et al., 2007, 2008), Planktothrix agardhii (Gao et al., 2012) and Synechococcus spp. (Dreher et al., (...truncated)