Introduction

Cyanophages have been shown to be a key component of aquatic microbial communities because of their abundance, ubiquity, and potential impact on the microbial loop (Huang et al., 2012). Cyanophage–cyanobacterium interactions may have important implications for global biogeochemical cycles (Sullivan et al., 2003; Bailey et al., 2004; Paul and Sullivan, 2005). Cyanophages also mediate the horizontal transfer of genetic material between host microbes, and thereby the genetic diversity of microorganisms is affected (Mann, 2003).

All known cyanophages belong to three families: Myoviridae, Siphoviridae, and Podoviridae. Cyanosiphoviruses are a group of viruses that infect cyanobacteria, and receive much less attention than cyanomyoviruses and cyanopodoviruses (Huang et al., 2012). To date, only eight cyanosiphovirus genomes (S-CBS1, S-CBS2, S-CBS3, S-CBS4, P-SS2, KBS2A, A-HIS1, and A-HIS2) have been reported (Wang and Chen, 2008; Sullivan et al., 2009; Huang et al., 2012; Ponsero et al., 2013; Chan et al., 2015). The first genome of a cyanosiphovirus, P-SS2, isolated from Atlantic slope waters, infecting the Prochlorococcus S.W. Chisholm et al host MIT9313, was described in 2009 (Sullivan et al., 2009). Five more cyanosiphoviruses (SCBS1, S-CBS2, S-CBS3, S-CBS4 and KBS2A), infecting Synechococcus strains and isolated from Chesapeake Bay, were reported by Huang (Huang et al., 2012) and Ponsero (Ponsero et al., 2013). Recently, two cyanosiphoviruses, A-HIS1 and A-HIS2, which infect a Caryochloris marina H. Miyashita & M. Chihara strain MBIC11017, were isolated from reef waters off Heron Island, Australia (Chan et al., 2015). The genome sizes of the aforementioned eight cyanosiphoviruses range from 30kb to 108kb with 40 to 105 ORFs.

The large terminase subunit (TerL), a protein responsible for phage DNA packaging, is essential for double-stranded DNA (dsDNA) phages. For cyanophages, phylogenetic clustering of TerL not only reflects the relative genetic conservation among the three cyanophage families, but also supports the separation of four subtypes of cyanosiphoviruses (Huang et al., 2012; Chan et al., 2015).

Here we sequenced the genome of a dsDNA cyanosiphovirus S-ESS1 infecting marine Synechococcus isolated from the East China Sea, and analyzed the phylogenetic relationship to the known marine cyanosiphoviruses by building the phylogenetic tree of the TerL.

1 Materials and Methods

1.1 Phage isolation and purification

Synechococcus sp. SJ01 was isolated from the East China Sea by using sterilized artificial seawater media (Zhang et al., 2013), and the strain was identified as Synechococcus sp WH8102, according to its partial 16S rRNA sequence (Accession: BX569694.1). Lytic cyanosiphovirus S-ESS1 that can infect Synechococcus sp. SJ01 was isolated from coastal water samples of the East China Sea (33°44’38″N, 122°25’44″E) by a serial dilution method (Middelboe et al., 2010). Phage purification was performed using sucrose density gradient centrifugation. The lysate was initially centrifuged at 4°C, 10,000 g for 1 h to remove cells (Eppendorf 5810R centrifuge equipped with a 20270 rotor and several 125-mL centrifuge tubes) before the supernatant was centrifuged at 4°C, 120,000 g for 2 h to precipitate phages (Beckman L-100XP ultracentrifuge equipped with an SW28Ti rotor and several 38.5-mL centrifuge tubes); then five sucrose steps of 20%, 30%, 40%, 50%, and 60% were used for centrifugation at 4°C, 450,000 g for 3 h (Beckman SW60Ti rotor and several 4-mL centrifuge tubes). A visible band at the 50% step was collected and washed twice by centrifugation at 4°C and 250,000 g (Beckman SW41Ti rotor and several 13.2-mL centrifuge tubes) for 1.5 h to obtain purified phage particles.

1.2 TEM observation cyanophage

A total of 20 µL of sucrose density gradient purified phage concentrate was transferred to 200 mesh Formvar carbon-coated copper grids and then negatively stained with 2% sodium phosphotungstate (pH 7.0). The grids were viewed using a Hitachi 3H-7000FA TEM.

1.3 DNA extraction, genome sequencing, and ORF annotation

DNA was prepared from sucrose density gradient-purified phages following the method of Wilson (Wilson et al., 1993). Sequencing of the phage DNA was performed using Illumina’s Hiseq 2000 (sequencing platform produced by Illumina company) platform to generate 2 G of average 2×100 bp original data and then spliced using software velvet (Version 1.2.07) by Sangon Biotech company. The genomic sequence was then subjected for ORF prediction by ORF finder (ORF finder: https://www.ncbi.nlm.nih.gov/orffinder/). Next, predicted ORFs were considered as hypothetical proteins and function annotations were assigned when BLASTP E-values were ≤ 0.001 (Huang et al., 2012) (BLASTP: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&BLAST_SPEC=&LINK_LOC=blasttab).

1.4 Phylogenetic tree of TerL

A neighbor-joining phylogenetic tree based on the TerL was built by MEGA5.1(http://www.megasoftware.net/). Distance analyses were used to test the bootstrap support. A heuristic search with 1000 bootstrap replications was conducted in this analysis.

2 Results

2.1 Siphovirus morphology

Using liquid dilution cultures and sucrose density gradient centrifugation, we isolated and purified a lytic marine Synechococcus siphovirus S-ESS1, infecting strain SJ01 (Figure 1). The morphology of negatively stained S-ESS1, as observed with TEM, revealed an icosahedral capsid that was ~65 nm in diameter and exhibited a long tail (~210 nm long and ~20 nm in diameter) (Figure 2).

2.2 Gene content and ORF information

The complete sequence of the Synechococcus siphovirus S-ESS1 genome can be accessed under the GenBank accession no. KY249644. The linearly assembled dsDNA genome of S-ESS1 was 60,362 bp in length, and the G+C content of S-ESS1 was 60.9%. Fifty-six of 282 ORFs in the S-ESS1 genome had recognizable homologues by BLASTP, among which 20 are hypothetical proteins that having unknown function and 36 had ascribed functions (Table 1). No similarity was found to ORFs of known cyanosiphoviruses, and no tRNA sequence was identified in the S-ESS1 genomes. Cyanosiphovirus S-ESS1 was most similar to cyanosiphovirus KBS2A (Ponsero et al., 2013) among the known eight cyanosiphoviruses in terms of gene capacity and ORF content (Table 2). However, the predicted genetic distribution of cyanosiphovirus S-ESS1 was similar to that of cyanosiphovirus PSS2 (Sullivan et al., 2009), with a shared structural gene in the middle, and replication and metabolism-related genes at both ends of the DNA (Figure 3).

2.3 TerL phylogenetic tree

From TerL phylogenetic tree (Figure 4), cyanosiphovirus S-ESS1 had far genetic distance from known cyanosiphoviruses but, unexpectedly, had a relatively close genetic distance with the E. coli bacteriophage T7 and a Ruegeria bacteriophage DSS3-P1.

3 Discussion

S-ESS1 had an icosahedral capsid and a long non-contractile tail, which are often seen in siphoviruses. S-ESS1 was morphologically similar to S-CBS4, a cyanosiphovirus infecting marine Synechococcus CB0101, which also had an isometric head (~72 nm) and a long flexible tail (~200 nm) (Huang, 2012).

Although cyanosiphovirus S-ESS1 has some similarity with cyanosiphoviruses KBS2A and PSS2 in terms of sequence length, ORF capacity, and gene distribution, the predicted ORFs of S-ESS1showed no homology with the other eight reported cyanosiphovirus genomes (including KSB2A and PSS2) (Sullivan et al., 2009; Huang et al., 2012; Ponsero et al., 2013; Chan et al., 2015). In other words, S-ESS1 showed very obvious genetic diversity from known cyanosiphoviruses. Moreover, only 36 of the 56 predicted ORFs have ascribed functions by BLASTP, which means that there is still much genetic information remaining to be described.

According to the sequence of TerL, cyanosiphovirus S-ESS1 was more related to E. coli bacteriophage T7 and Ruegeria phage DSS3-P1 compared to other cyanosiphoviruses, and the extremely low value for the specific node branching of the tree stated that S-ESS1 was not closely related to the eight known cyanosiphoviruses. The TerL protein-based phylogeny showed that cyanosiphoviruses could fall into three distantly related phyletic groups among the five-known marine siphoviruses (S-CBS1, S-CBS2, S-CBS3, S-CBS4 and P-SS2) by 2012 (Huang et al., 2012). And the latest reported cyanosiphoviruses, A-HIS1 and A-HIS2, became the fourth distantly related phyletic group (Chan et al., 2015). Here, the distant phylogenetic kinship between S-ESS1 and the other eight cyanosiphoviruses, together with their different ORF annotations, reconfirms that S-ESS1 can be classified into a new fifth cyanosiphovirus subtype.

4 Conclusions

Our study aims to identify and classify the cyanophage we isolated from East China Sea. This cyanophage turn out to belong to long-tailed cyanophage which relatively receive less attention and coverage in the current study. From the genome length and content S-ESS1 is defined as a new number of the 8 reported cyanosiphoviruses, but from the TerL phylogenetic tree S-ESS1 have hardly gene homology with reported 8 cyanosiphovirus, thus greatly enriching the genetic diversity of existing cyanosiphovirus. Those results reveal that more work worth investing in cyanosiphoviruses’ diversity research. Since different virus–host lifestyles (i.e. broad host vs. narrow host, lytic vs. lysogenic) pose different selection pressures on gene acquisition between virus and host (Huang et al., 2012), and S-ESS1 (as well as its host) was the only cyanosiphovirus isolated from the East China Sea (western part of the Pacific Ocean), this suggests that the regional difference might be the reason for such congeneric genetic variation.

Authors' contributions

HY performed data analyses and wrote the first draft of the manuscript with input from all co-authors. ZY and HY conducted the experiments. ZYJ and CK performed data analyses and wrote the final version of the manuscript. CK conceived of the study.

Acknowledgements

We thank National Science Foundation of China [grant numbers 31370148, 31200385], Science and Technology Support Program of Hubei province (Grant number: 2014BCB037) and Key Science and Technology Program of Wuhan (Grant number: 2014060101010061) for finical supporting, and Dr. Ge Xingyi from the Wuhan Institute of Virology (WIV) of the Chinese Academy of Sciences (CAS) for help in the analysis of the genome.

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