Microbial growth and metabolism are the major cause of seafood spoilage. And Shewanella species is a typical specific spoilage organism that is capable of degrading nitrogenous substances into amine, sulfides, and organic acids, producing unpleasant flavors and odors [1, 12]. The Shewanella species are the major spoilage flora in the iced marine fish such as large yellow croaker (Pseudosciaena crocea)  and bighead carp (Aristichthys nobilis) , that posed large economic losses. Therefore, taking measures to control growth of Shewanella species is necessary for extending the preservation life of seafood during low temperature storage. Phages could infect bacteria with high specificity, and it has demonstrated potential as antibacterial drugs [4, 9]. However, phages infecting Shewanella spp. had largely been unexplored. In this study, we have sequenced and analyzed the complete genome of a newly isolated Shewanella baltica phage vB_Sb_QDWS. Bioinformatic analysis indicated that vB_Sb_QDWS is a new member of the family Siphoviridae and might belong to a novel phage lineage.
The bacterial strain used in this study was Shewanella baltica OS155, which was grown on LB medium at 25 °C, in a shaking incubator. And it was also used as the host for isolation of phage vB_Sb_QDWS. The isolation and purification of phage vB_Sb_QDWS collected from wastewater samples in Qingdao were done according to the procedures described previously [7, 15]. Phage vB_Sb_QDWS could produce small plaques on S. baltica OS155 lawns grown on LB soft agar at 25 °C (Fig. 1A). To examine the morphology of phage vB_Sb_QDWS, the purified phage particles were observed using a JEM-2000EX transmission electron microscope (TEM) (JEOL, Tokyo, Japan). TEM results revealed that vB_Sb_QDWS has an icosahedral head (70 ± 2 nm) connected to a tail (235 ± 5 nm). Based on the morphology features, vB_Sb_QDWS was designated as a member of the family Siphoviridae (Fig. 1B).
Genomic DNA was purified using a Bacteria DNA Kit (OMEGA) according to the manufacturer’s instructions. Whole-genome sequencing and assembly were performed by Shanghai Biozeron Biothchnology Co., Ltd. (Shanghai, China.) on the Illumina Hiseq paired-end platform. Phage vB_Sb_QDWS was found to have a double-stranded DNA genome with a length of 47,902 bp and an overall G+C content of 63.16% (Fig. 2). Using the GeneMark server (http://topaz.gatech.edu/GeneMark/genemarks.cgi) and the RAST server (http://rast.nmpdr.org/rast.cgi), we identified 69 open reading frames (ORFs) and predicted 49 putative protein coding genes in the genome, 20 of which were functionally assigned by searching against the non-redundant protein database with BLASTp (http://blast.ncbi.nlm.nih.gov/). These functionally assigned proteins involved in DNA packaging and replication, head and tail morphogenesis and host lysis (Table S1). We identified genes for the host nuclease inhibitor protein (ORF 8), endolysin (ORF 20), lysis protein (ORF 33), terminase large subunit (ORF 47), baseplate protein (ORF 27/29), tail fiber protein (ORF 25) and portal protein (ORF 46). No tRNA genes were searched by using tRNAscan-SE .
To assess the phylogenetic relationship of vB_Sb_QDWS to known phages, a proteomic tree was generated in MEGA7.0  using the neighbor-joining method based on the terminase large subunit (ORF 47) sequence (Fig. 3). Eight Shewanella phages vary significantly in phage size, GC content and protein amount were selected for phylogenetic analysis. Shewanella phage S0112, Spp001, 3/49, 1/44 and SppYZU05 are from siphoviruses, while 1/41, SFCi1, SppYZU01 are from Myoviridae [3, 6, 11, 13, 14]. Results showed vB_Sb_QDWS is quite different from other Shewanella phages that have been reported, which formed an independent cluster. However, vB_Sb_QDWS and Escherichia phage Rac-SA53 (ALP46869.1) were grouped into a clade. Though, they share low similarity (query coverage, 99%; identity, 39.5%) with each other according to BLASTp results. BLASTn analysis of the whole genome sequence also showed almost no similarity between Shewanella phage vB_Sb_QDWS and other phages in the NCBI database. Based on its unique phenotype and phylogeny, vB_Sb_QDWS is considered to represent a novel group within the family Siphoviridae.