- Email: [email protected]
Isolation, characterization and genome sequencing of the novel phage SL25 from the Yellow Sea, China
Marine Genomics xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Isolation, characterization and genome sequencing of the novel phage SL25 from the Yellow Sea, China Zhaoyang Liua,1, Huifang Lia,1, Min Wanga,b,⁎, Yong Jianga,b,⁎, Qingwei Yanga, Xinhao Zhoua, Zheng Gonga, Qian Liua, Hongbing Shaoa a b
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Pseudoalteromonas phage Siphoviridae Complete genome
Outnumbering all other biological entities on earth, bacteriophages play critical roles in structuring microbial communities. However, only a small number of phages have so far been reported. In this study, a novel Pseudoalteromonas phage, SL25, was isolated from the yellow sea, China. Transmission electron microscope observations showed that phage has an icosahedral head, 100 ± 1 nm in diameter, and a tail with a length of 150 ± 1 nm, and should be grouped into the Siphoviridae family. To better understand the genetic diversity of this phage, the complete genome was characterized. It consists 29,130-bp-length double-stranded DNA with a GC content of 41.04% and is predicted to have 61 open reading frames (ORFs) with an average length of 504 nucleotides. This study adds a new Siphoviridae phage to the marine bacteriophage dataset that could potentially infect Pseudoalteromonas. It also provides useful data for further molecular research on the interaction mechanism between bacteriophages and their hosts.
1. Introduction Pseudoalteromonas, affiliated with the order Alteromonadales (Bowman and Mcmeekin, 2005; Euzéby, 2005) of the Gammaproteobacteria (Euzéby, 2005; Garrity et al., 2005), is a genus of heterotrophic, Gram-negative marine bacteria (Gauthier and Gauthier, 1995) with a single polar flagellum. Members of the genus Pseudoalteromonas are generally found in eukaryotic hosts (Holmström and Kjelleberg, 1999), associated with marine animals (i.e., tunicates and mussels) (Ivanova et al., 1998) and marine algae (Egan et al., 2001). The genus is potentially involved in complex ecological networks across trophic levels, and is ecologically and evolutionarily influenced by phages. Viral particles outnumber bacteria in the world's oceans by a factor of about 10:1 and are thought to enforce diversity at the base of the microbiological community by restraining overgrowth of any particularly successful microbial species and returning their biomolecules as nutrients into the water through lysis (Wommack et al., 1992; Weinbauer, 2004). As the largest source of genetic material on the planet (Suttle, 2007), viruses are thought to be the major vehicle for gene transfer in the ocean. Pseudoalteromonas phages have been shown to represent a significant group of phages in the ocean (Wichels et al., 1998; Duhaime
⁎
1
et al., 2011). The genomes of Pseudoalteromonas phages are small (3–300 kb) compared with their hosts (1000–13, 000 kb) and typically contain abundant unknown gene content. The majority of the open reading frames (ORFs) (over 60%) is hypothetical proteins (unique in public sequence databases) or conserved hypothetical proteins (similar only to other unknown proteins). As of April 2017, only 22 complete Pseudoalteromonas phage genomes have been deposited in GenBank. In this report, we report the biological properties and genome sequence of the Pseudoalteromonas phage SL25, isolated from the Yellow Sea. The genome of SL25 was also analyzed for its relationships with other known Pseudoalteromonas phages. 2. Data description Seawater was collected from the Yellow Sea, during the spring of 2016, filtered through 0.22 m–pore-size membranes (Millipore) (Hardies et al., 2013) and checked for the presence of phages by using the double-agar layer method (0.5% low-melt top agar) to obtain the plaques (Hyman and Abedon, 2010). Each plaque was cored, plaquepurified three times and resuspended in 500 μL of sterile SM buffer (100 mMNaCl, 81.2 mM MgSO4. 7H2O, 50 mM Tris-HCl (pH 7.5), 0.01% gelatin) (Duhaime et al., 2011). Purified bacteriophage lysate was
Corresponding authors. E-mail addresses: [email protected] (M. Wang), [email protected] (Y. Jiang). These authors contributed equally to this study
http://dx.doi.org/10.1016/j.margen.2017.09.008 Received 12 September 2017; Received in revised form 27 September 2017; Accepted 27 September 2017 1874-7787/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, Z., Marine Genomics (2017), http://dx.doi.org/10.1016/j.margen.2017.09.008
Marine Genomics xxx (xxxx) xxx–xxx
Z. Liu et al.
using Velvet (Version 1.2.07). Data gaps were filled using GapCloser (Version: 1.12) and GapFiller (Version: 1–11) (Gong et al., 2017). The genome of phage SL25, was found to be a linear, double-stranded 29,130-bp DNA molecule, with a GC content of 41.04%. No tRNA gene was found in the genome. Sixty one ORFs, with an average length of 504 nucleotides each, were finally predicted from the genome sequence of phage SL25 using a combination of Glimmer (http://ccb.jhu.edu/software/glimmer/index. shtml) and RAST (http://rast.nmpdr.org). ORFs were analyzed by considering ATG, GTG and TTG as possible initiation codons. All three stop codons, TAA, TGA, and TAG, are used in the phage SL25 sequence, with TAA being the most frequent (Liu et al., 2016). The predicted ORF sequences were translated using EMBOSS Transeq (Version: 6.5.7). Nucleotide sequences were compared to the NCBI (http://www.ncbi. nlm.nih.gov) reference genomic sequence database using BLASTX algorithm, and the predicted proteins were compared to the NCBI nonredundant (nr) protein database to predict their functions using the BLASTP algorithm (Huang et al., 2013; Li et al., 2016; Wang et al., 2015). When all 61 predicted ORFs were subjected to functional annotation, only 23 (37.7%) could be assigned to specific functions (evalue < 10− 5), while another 38 (62.3%) were predicted to encode for hypothetical proteins, which is probably due to the inadequacy of the information on bacteriophage genomes. All the predicted ORFs in phage SL25 could be classified into five functional groups, including phage structure (ORF 7, ORF 10, ORF 11, ORF 12, ORF 14, ORF 15, ORF 20, ORF 22), packaging (ORF 1, ORF 21, ORF 23, ORF 24, ORF 25), DNA replication and nucleotide metabolism (ORF 40, ORF 41, ORF 42, ORF 49, ORF 50, ORF 51), lysis (ORF 58) and additional functions (Fig. 2a). Proteins encoded by DNA replication and nucleotide metabolism were mostly based on the existence of conserved functional domains in each protein, in addition to sequence similarities to other proteins in public databases. The bacteriophage origin was also demonstrated by the BLASTP (evalue < 10− 5) results. High similarities were found to marine Pseudoalteromonas phages belonging to the Siphoviridae family, i. e. Pseudoalteromonas phage PHS3 (GenBank accession number KX912252), Pseudoalteromonas phage PHS21 (GenBank accession number KY379511), Pseudoalteromonas phage BS5 (GenBank accession number NC_031917), Pseudoalteromonas phage Pq0 (GenBank accession number NC_029100) (Wang et al., 2016; Meng et al., 2017). These results suggest that Pseudoalteromonas phage SL25 is closely related to Pseudoalteromonas phage PHS3, which was found to have 20 (32.79%) common genes (including 5 genes about DNA replication and nucleotide metabolism) with SL25. The established measure for grouping into a Siphoviridae genus is 40% of proteins matching within a 75 bit score by BLASTP (Lavigne et al., 2008) but PHS3 falls outside that threshold. Therefore, phage SL25 has been designated as a new bacteriophage. Seventeen common genes were also found in Pseudoalteromonas phage PHS21. However, SL25 shows no similarity with Pseudoalteromonas phage PH1, whose host was also Pseudoalteromonas marina Mano4 (T) (Liu et al., 2016). The SL25 genome was also compared to the genome of PHS3 to obtain insight into the similarities and differences between these two phages that infect the same host strain using BLASTx (Altschul et al., 1990). The figure was drawn using Easyfig (Sullivan et al., 2011) (Fig. 2b). Not surprisingly, the PHS3 genome was similar to the SL25 genome, especially in the structural proteins and the lysis protein. The synteny of the two phage genomes was also shown in the structure module, including the terminase large subunit and tail and the major capsid protein. The major difference between the two phages lies in the packaging proteins. This pattern of similarity, which was biased highly toward the structural module, suggests that the two phages may show different aspects during packaging within host cells. The terminase large subunit (TerL), one of marker proteins for phages, was used to infer phylogenetic position and type of phage SL25, and the phylogenetic trees based on the neighbor–joining and
Table 1 Classification, general features, and genome sequencing information for phage SL25 according to the MIxS recommendations. Item
Description
Classification
Domain: unassigned (ds DNA viruses) Order Caudovirales Family Siphoviridae Isometric capsid with a long non-contractile tail MF370965 Virus The Yellow Sea, China 36°06′ N, 120°32′ E 0.3 m February 2016 Temperate shelf and sea biome Coastal water body Sea water Illumina Miseq 1 Velvet Finish (complete)
Particle shape Submitted to GenBank Investigation type Geographic location Latitude and longitude Depth Collection date Environment (biome) Environment (feature) Environment (material) Sequencing method Number of contigs Assembly method Finishing quality
stored in SM buffer at 4 °C for a few months or further processing (Khawaja et al., 2016) (Table 1). The host bacterial strain was isolated from the same place, and incubated in liquid Zobell medium at 28 °C. The 16S rRNA sequence of the SL25 bacterial host showed a 99.7% homology to Pseudoalteromonas marina Mano4 (T) (Accession number AY563031) (Li et al., 2016). The morphology of the isolated phage was obtained after negative staining with 2% uranyl acetate using Transmission Electron Microscopy (JEOL-1200 EX, Japan) at 100 kV (Liu et al., 2016). The results showed that phage SL25 belongs to the family Siphoviridae, a group of dsDNA bacteriophages characterized by icosahedral heads and long non-contractile tails. The head diameter and tail length of phage SL25 were approximately 100 ± 1 nm and 150 ± 1 nm, respectively (Fig. 1). Phage DNA extraction was performed by Sangon Biotech Co (Shanghai, China). Purified phage genomic DNA was sequenced using IlluminaMiseq 2 × 300 paired-end sequence methods. Then, the raw data were filtered and clean reads were assembled in a single contig
Fig. 1. Transmission electron micrograph of Pseudoalteromonas phage SL25, the scale bar is 50 nm.
2
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Fig. 2. Genome analysis of Pseudoalteromonas phage SL25. (a) Genome map of SL25 and functional annotation of the predicted proteins. (b) Genome-wide comparison of phages SL25 and PHS3. Genome regions showing similarity were searched using tBLASTx and the matches satisfying length and e-value cutoffs were indicated by the grey rectangle according to the color scale on the right.
environment” (No. CHINARE-2012-01-05 till 2021-12-31).
maximum likelihood algorithm were conducted using the genetic analysis software MEGA (Version 7.0.18) (Saitou and Nei, 1987). Phylogenetic analysis of the TerL amino acid sequence grouped phage SL25 together with Pseudoalteromonas phage PHS21 in one clade (Fig. S1). In this study, a novel Siphoviridae phage that could infect Pseudoalteromonas was isolated and identified. It will provide important information for assessing the role of viruses in marine ecosystems.
References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215 (3), 403–410. Bowman, J.P., Mcmeekin, T.A., 2005. Alteromonadales, ord. nov.. In: Bergey's Manual® of Systematic Bacteriology, pp. 443–491. Duhaime, M.B., Wichels, A., Waldmann, J., Teeling, H., Glöckner, F.O., 2011. Ecogenomics and genome landscapes of marine Pseudoalteromonas phage H105/1. ISME J. 5, 107. Egan, S., Holmström, C., Kjelleberg, S., 2001. Pseudoalteromonas ulvae sp. nov., a bacterium with antifouling activities isolated from the surface of a marine alga. Int. J. Syst. Evol. Microbiol. 51, 1499. Euzéby, J., 2005. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Int. J. Syst. Evol. Microbiol. 55, 983–985. Garrity, G.M., Bell, J.A., Lilburn, T., 2005. Class ii. Betaproteobacteria class. nov. In: Bergey's Manual® of Systematic Bacteriology. 2005. pp. 93–98. Gauthier, G., Gauthier, M.R., 1995. Phylogenetic analysis of the genera alteromonas, shewanella, and moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera, Alteromonas (emended) and Pseudoalteromonas gen nov, and proposal of twelve new species combinations. Int. J. Syst. Bacteriol. 45, 755. Gong, Z., Wang, M., Yang, Q., Li, Z., Xia, J., Gao, Y., 2017. Isolation and complete genome sequence of a novel Pseudoalteromonas phage PH357 from the Yangtze river estuary. Curr. Microbiol. 1–8. Hardies, S.C., Hwang, Y.J., Hwang, C.Y., Jang, G.I., Cho, B.C., 2013. Morphology, physiological characteristics, and complete sequence of marine bacteriophage ϕRIO-1 infecting Pseudoalteromonas marina. J. Virol. 87, 9189–9198. Holmström, C., Kjelleberg, S., 1999. Marine Pseudoalteromonas, species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol. Ecol. 30, 285–293. Huang, G., Le, S., Peng, Y., Zhao, Y., Yin, S., Zhang, L., et al., 2013. Characterization and genome sequencing of phage ABP1, a new phiLMV-like virus infecting multidrugresistant Acinetobacter baumannii. Curr. Microbiol. 66, 535–543. Hyman, P., Abedon, S.T., 2010. Bacteriophage host range and bacterial resistance. Adv. Appl. Microbiol. 70, 217. Ivanova, E.P., Kiprianova, E.A., Mikhailov, V.V., Levanova, G.F., Garagulya, A.D., Gorshkova, N.M., et al., 1998. Phenotypic diversity of Pseudoalteromonas citrea from different marine habitats and emendation of the description. Int. J. Syst. Bacteriol. 48 (Pt 1), 247. Khawaja, K.A., Abbas, Z., Rehman, S.U., 2016. Isolation and characterization of lytic
Genome sequence accession number The complete genome sequence of bacteriophage SL25 is available in the GenBank database under accession number MF370965. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.margen.2017.09.008. Acknowledgments We are grateful to the research vessel Dong Fang Hong 2, for providing the seawater samples. The research was funded by the National Natural Science Foundation of China (Nos. 41076088, 41676178 and 31500339), the National Key Basic Research Program of China (973Program, Grant No: 2013CB429704), China Postdoctoral Science Foundation (Grant Nos. 2015 M570612 and 2016T90649), Fundamental Research Funds for the Central University of Ocean University of China (Grant Nos. 201762017, 201564010, 201562018, 201512013 and 201512008), the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No.2016ASKJ14), Global change and Air-Sea interface project supported by State Oceanic Administration People's republic of China “cruises for an oceanographic survey of South-central Western Pacific in winter” (No. GASI-02-PACST-MSwin), and project supported by the Special Foundation from Chinese Arctic and Antarctic Administration “Marine biodiversity in the surrounding waters of Antarctic and investigation of ecological 3
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Bioinformatics 27, 1009–1010. Suttle, C.A., 2007. Marine viruses-major players in the global ecosystem. Nat. Rev. Microbiol. 5, 801–812. Wang, D.B., Sun, M.Q., Shao, H.B., Li, Y., Meng, X., Liu, Z.Y., 2015. Characterization and genome sequencing of a Novel bacteriophage PH101 infecting Pseudoalteromonas marina BH101 from the Yellow Sea of China. Curr. Microbiol. 71, 594–600. Wang, D.B., Li, Y., Sun, M.Q., Huang, J.P., Shao, H.B., Xin, Q.L., et al., 2016. Complete genome of a novel Pseudoalteromonas phage PHq0. Curr. Microbiol. 72, 81–87. Weinbauer, M.G., 2004. Ecology of prokaryotic viruses. FEMS Microbiol. Rev. 28, 127–181. Wichels, A., Biel, S.S., Gelderblom, H.R., Brinkhoff, T., Muyzer, G., Schütt, C., 1998. Bacteriophage diversity in the North Sea. Appl. Environ. Microbiol. 64, 4128–4133. Wommack, K.E., Hill, R.T., Kessel, M., Russek-Cohen, E., Colwell, R.R., 1992. Distribution of viruses in the Chesapeake Bay. Appl. Environ. Microbiol. 58, 2965–2970.
phages tse1-3 against enterobacter cloacae. Open Life Sci. 11, 287–292. Lavigne, R., Seto, D., Mahadevan, P., Ackermann, H.W., Kropinski, A.M., 2008. Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using blastp-based tools. Res. Microbiol. 159, 406–414. Li, Y., Wang, M., Liu, Q., ng, X., Wang, D., Ma, Y., et al., 2016. Complete genomic sequence of bacteriophage H188: a Novel Vibrio kanaloae phage isolated from Yellow Sea. Curr. Microbiol. 72, 628. Liu, Z., Wang, M., Meng, X., Li, Y., Wang, D., Jiang, Y., 2016. Isolation and genome sequencing of a novel Pseudoalteromonas phage PH1. Curr. Microbiol. 1–7. Meng, X., Wang, M., You, S., Wang, D., Li, Y., Liu, Z., 2017. Characterization and complete genome sequence of a novel Siphoviridae bacteriophage BS5. Curr. Microbiol. 1–6. Saitou, N., Nei, M., 1987. The neighbor-joining method-a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sullivan, M.J., Petty, N.K., Beatson, S.A., 2011. Easyfig: a genome comparison visualizer.
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Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Isolation, characterization and genome sequencing of the novel phage SL25 from the Yellow Sea, China Zhaoyang Liua,1, Huifang Lia,1, Min Wanga,b,⁎, Yong Jianga,b,⁎, Qingwei Yanga, Xinhao Zhoua, Zheng Gonga, Qian Liua, Hongbing Shaoa a b
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Pseudoalteromonas phage Siphoviridae Complete genome
Outnumbering all other biological entities on earth, bacteriophages play critical roles in structuring microbial communities. However, only a small number of phages have so far been reported. In this study, a novel Pseudoalteromonas phage, SL25, was isolated from the yellow sea, China. Transmission electron microscope observations showed that phage has an icosahedral head, 100 ± 1 nm in diameter, and a tail with a length of 150 ± 1 nm, and should be grouped into the Siphoviridae family. To better understand the genetic diversity of this phage, the complete genome was characterized. It consists 29,130-bp-length double-stranded DNA with a GC content of 41.04% and is predicted to have 61 open reading frames (ORFs) with an average length of 504 nucleotides. This study adds a new Siphoviridae phage to the marine bacteriophage dataset that could potentially infect Pseudoalteromonas. It also provides useful data for further molecular research on the interaction mechanism between bacteriophages and their hosts.
1. Introduction Pseudoalteromonas, affiliated with the order Alteromonadales (Bowman and Mcmeekin, 2005; Euzéby, 2005) of the Gammaproteobacteria (Euzéby, 2005; Garrity et al., 2005), is a genus of heterotrophic, Gram-negative marine bacteria (Gauthier and Gauthier, 1995) with a single polar flagellum. Members of the genus Pseudoalteromonas are generally found in eukaryotic hosts (Holmström and Kjelleberg, 1999), associated with marine animals (i.e., tunicates and mussels) (Ivanova et al., 1998) and marine algae (Egan et al., 2001). The genus is potentially involved in complex ecological networks across trophic levels, and is ecologically and evolutionarily influenced by phages. Viral particles outnumber bacteria in the world's oceans by a factor of about 10:1 and are thought to enforce diversity at the base of the microbiological community by restraining overgrowth of any particularly successful microbial species and returning their biomolecules as nutrients into the water through lysis (Wommack et al., 1992; Weinbauer, 2004). As the largest source of genetic material on the planet (Suttle, 2007), viruses are thought to be the major vehicle for gene transfer in the ocean. Pseudoalteromonas phages have been shown to represent a significant group of phages in the ocean (Wichels et al., 1998; Duhaime
⁎
1
et al., 2011). The genomes of Pseudoalteromonas phages are small (3–300 kb) compared with their hosts (1000–13, 000 kb) and typically contain abundant unknown gene content. The majority of the open reading frames (ORFs) (over 60%) is hypothetical proteins (unique in public sequence databases) or conserved hypothetical proteins (similar only to other unknown proteins). As of April 2017, only 22 complete Pseudoalteromonas phage genomes have been deposited in GenBank. In this report, we report the biological properties and genome sequence of the Pseudoalteromonas phage SL25, isolated from the Yellow Sea. The genome of SL25 was also analyzed for its relationships with other known Pseudoalteromonas phages. 2. Data description Seawater was collected from the Yellow Sea, during the spring of 2016, filtered through 0.22 m–pore-size membranes (Millipore) (Hardies et al., 2013) and checked for the presence of phages by using the double-agar layer method (0.5% low-melt top agar) to obtain the plaques (Hyman and Abedon, 2010). Each plaque was cored, plaquepurified three times and resuspended in 500 μL of sterile SM buffer (100 mMNaCl, 81.2 mM MgSO4. 7H2O, 50 mM Tris-HCl (pH 7.5), 0.01% gelatin) (Duhaime et al., 2011). Purified bacteriophage lysate was
Corresponding authors. E-mail addresses: [email protected] (M. Wang), [email protected] (Y. Jiang). These authors contributed equally to this study
http://dx.doi.org/10.1016/j.margen.2017.09.008 Received 12 September 2017; Received in revised form 27 September 2017; Accepted 27 September 2017 1874-7787/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, Z., Marine Genomics (2017), http://dx.doi.org/10.1016/j.margen.2017.09.008
Marine Genomics xxx (xxxx) xxx–xxx
Z. Liu et al.
using Velvet (Version 1.2.07). Data gaps were filled using GapCloser (Version: 1.12) and GapFiller (Version: 1–11) (Gong et al., 2017). The genome of phage SL25, was found to be a linear, double-stranded 29,130-bp DNA molecule, with a GC content of 41.04%. No tRNA gene was found in the genome. Sixty one ORFs, with an average length of 504 nucleotides each, were finally predicted from the genome sequence of phage SL25 using a combination of Glimmer (http://ccb.jhu.edu/software/glimmer/index. shtml) and RAST (http://rast.nmpdr.org). ORFs were analyzed by considering ATG, GTG and TTG as possible initiation codons. All three stop codons, TAA, TGA, and TAG, are used in the phage SL25 sequence, with TAA being the most frequent (Liu et al., 2016). The predicted ORF sequences were translated using EMBOSS Transeq (Version: 6.5.7). Nucleotide sequences were compared to the NCBI (http://www.ncbi. nlm.nih.gov) reference genomic sequence database using BLASTX algorithm, and the predicted proteins were compared to the NCBI nonredundant (nr) protein database to predict their functions using the BLASTP algorithm (Huang et al., 2013; Li et al., 2016; Wang et al., 2015). When all 61 predicted ORFs were subjected to functional annotation, only 23 (37.7%) could be assigned to specific functions (evalue < 10− 5), while another 38 (62.3%) were predicted to encode for hypothetical proteins, which is probably due to the inadequacy of the information on bacteriophage genomes. All the predicted ORFs in phage SL25 could be classified into five functional groups, including phage structure (ORF 7, ORF 10, ORF 11, ORF 12, ORF 14, ORF 15, ORF 20, ORF 22), packaging (ORF 1, ORF 21, ORF 23, ORF 24, ORF 25), DNA replication and nucleotide metabolism (ORF 40, ORF 41, ORF 42, ORF 49, ORF 50, ORF 51), lysis (ORF 58) and additional functions (Fig. 2a). Proteins encoded by DNA replication and nucleotide metabolism were mostly based on the existence of conserved functional domains in each protein, in addition to sequence similarities to other proteins in public databases. The bacteriophage origin was also demonstrated by the BLASTP (evalue < 10− 5) results. High similarities were found to marine Pseudoalteromonas phages belonging to the Siphoviridae family, i. e. Pseudoalteromonas phage PHS3 (GenBank accession number KX912252), Pseudoalteromonas phage PHS21 (GenBank accession number KY379511), Pseudoalteromonas phage BS5 (GenBank accession number NC_031917), Pseudoalteromonas phage Pq0 (GenBank accession number NC_029100) (Wang et al., 2016; Meng et al., 2017). These results suggest that Pseudoalteromonas phage SL25 is closely related to Pseudoalteromonas phage PHS3, which was found to have 20 (32.79%) common genes (including 5 genes about DNA replication and nucleotide metabolism) with SL25. The established measure for grouping into a Siphoviridae genus is 40% of proteins matching within a 75 bit score by BLASTP (Lavigne et al., 2008) but PHS3 falls outside that threshold. Therefore, phage SL25 has been designated as a new bacteriophage. Seventeen common genes were also found in Pseudoalteromonas phage PHS21. However, SL25 shows no similarity with Pseudoalteromonas phage PH1, whose host was also Pseudoalteromonas marina Mano4 (T) (Liu et al., 2016). The SL25 genome was also compared to the genome of PHS3 to obtain insight into the similarities and differences between these two phages that infect the same host strain using BLASTx (Altschul et al., 1990). The figure was drawn using Easyfig (Sullivan et al., 2011) (Fig. 2b). Not surprisingly, the PHS3 genome was similar to the SL25 genome, especially in the structural proteins and the lysis protein. The synteny of the two phage genomes was also shown in the structure module, including the terminase large subunit and tail and the major capsid protein. The major difference between the two phages lies in the packaging proteins. This pattern of similarity, which was biased highly toward the structural module, suggests that the two phages may show different aspects during packaging within host cells. The terminase large subunit (TerL), one of marker proteins for phages, was used to infer phylogenetic position and type of phage SL25, and the phylogenetic trees based on the neighbor–joining and
Table 1 Classification, general features, and genome sequencing information for phage SL25 according to the MIxS recommendations. Item
Description
Classification
Domain: unassigned (ds DNA viruses) Order Caudovirales Family Siphoviridae Isometric capsid with a long non-contractile tail MF370965 Virus The Yellow Sea, China 36°06′ N, 120°32′ E 0.3 m February 2016 Temperate shelf and sea biome Coastal water body Sea water Illumina Miseq 1 Velvet Finish (complete)
Particle shape Submitted to GenBank Investigation type Geographic location Latitude and longitude Depth Collection date Environment (biome) Environment (feature) Environment (material) Sequencing method Number of contigs Assembly method Finishing quality
stored in SM buffer at 4 °C for a few months or further processing (Khawaja et al., 2016) (Table 1). The host bacterial strain was isolated from the same place, and incubated in liquid Zobell medium at 28 °C. The 16S rRNA sequence of the SL25 bacterial host showed a 99.7% homology to Pseudoalteromonas marina Mano4 (T) (Accession number AY563031) (Li et al., 2016). The morphology of the isolated phage was obtained after negative staining with 2% uranyl acetate using Transmission Electron Microscopy (JEOL-1200 EX, Japan) at 100 kV (Liu et al., 2016). The results showed that phage SL25 belongs to the family Siphoviridae, a group of dsDNA bacteriophages characterized by icosahedral heads and long non-contractile tails. The head diameter and tail length of phage SL25 were approximately 100 ± 1 nm and 150 ± 1 nm, respectively (Fig. 1). Phage DNA extraction was performed by Sangon Biotech Co (Shanghai, China). Purified phage genomic DNA was sequenced using IlluminaMiseq 2 × 300 paired-end sequence methods. Then, the raw data were filtered and clean reads were assembled in a single contig
Fig. 1. Transmission electron micrograph of Pseudoalteromonas phage SL25, the scale bar is 50 nm.
2
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Fig. 2. Genome analysis of Pseudoalteromonas phage SL25. (a) Genome map of SL25 and functional annotation of the predicted proteins. (b) Genome-wide comparison of phages SL25 and PHS3. Genome regions showing similarity were searched using tBLASTx and the matches satisfying length and e-value cutoffs were indicated by the grey rectangle according to the color scale on the right.
environment” (No. CHINARE-2012-01-05 till 2021-12-31).
maximum likelihood algorithm were conducted using the genetic analysis software MEGA (Version 7.0.18) (Saitou and Nei, 1987). Phylogenetic analysis of the TerL amino acid sequence grouped phage SL25 together with Pseudoalteromonas phage PHS21 in one clade (Fig. S1). In this study, a novel Siphoviridae phage that could infect Pseudoalteromonas was isolated and identified. It will provide important information for assessing the role of viruses in marine ecosystems.
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Genome sequence accession number The complete genome sequence of bacteriophage SL25 is available in the GenBank database under accession number MF370965. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.margen.2017.09.008. Acknowledgments We are grateful to the research vessel Dong Fang Hong 2, for providing the seawater samples. The research was funded by the National Natural Science Foundation of China (Nos. 41076088, 41676178 and 31500339), the National Key Basic Research Program of China (973Program, Grant No: 2013CB429704), China Postdoctoral Science Foundation (Grant Nos. 2015 M570612 and 2016T90649), Fundamental Research Funds for the Central University of Ocean University of China (Grant Nos. 201762017, 201564010, 201562018, 201512013 and 201512008), the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No.2016ASKJ14), Global change and Air-Sea interface project supported by State Oceanic Administration People's republic of China “cruises for an oceanographic survey of South-central Western Pacific in winter” (No. GASI-02-PACST-MSwin), and project supported by the Special Foundation from Chinese Arctic and Antarctic Administration “Marine biodiversity in the surrounding waters of Antarctic and investigation of ecological 3
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