A novel Siphoviridae family Phage BS5, which infects Pseudoalteromonas atlantica, was isolated from the surface waters of the Yellow Sea. Morphological study by transmission electron microscopy revealed that the novel phage belongs to Siphoviridae. The complete genome sequence of PBS5 contained a linear, double-strand 39949-bp DNA molecule with a G + C content of 40.6% and 65 putative open reading frames. Twelve conserved domains were detected by BLASTP in NCBI, and of these the functions of 5 were known. The genome was grouped into four modules as follows: phage structure, phage packaging, DNA replication and regulation, and some additional functions.16 S rDNA sequence analysis was also applied to identify the host bacteria. After initial characterization of bacteriophage PBS5, it was found that the optimal pH was 7.0, the optimal temperature was 30 °C, and the burst size was about 95 virions per cell. This information will provide an important benchmark for further research on the interaction between bacteriophages and their hosts.
Viruses are the most abundant organisms in the ocean and contain the highest genetic diversity in marine ecosystems . Viruses play an integral role in the marine ecosystem where they outnumber of all other life forms by at least an order of magnitude .The total abundance of viruses in the ocean is apparently more than 1030  as they are found at an abundance of up to 2.5 × 108 cells per milliliter in surface waters . Every living organism seems to be susceptible to infection by viruses. Because they are so abundant and dynamic, bacteriophages are also critically importance to marine biogeochemical cycles [5–7]. So far, only a few sequenced phages have been found infecting marine heterotrophic bacteria [8–10]. In order to investigate the influence of bacteriophages on bacteria by lysing bacterial cells, we need to isolate, proliferate, and purify phage-host systems .
The marine genus Pseudoalteromonas is a member of Gammaproteobacteria, which is widespread from surface waters to deep-sea sediments . Gammaproteobacteria may comprise more than 30% of all marine bacterioplankton . Pseudoalteromonas is heterotrophic and plays an important role in the decomposition of particulate organic matter (POM) since it can produce large quantities of extracellular enzymes [14–16]. Relatively few Pseudoalteromonas phages have been studied so far [13, 15, 17].
To understand the interaction between phages and their Pseudoalteromonas host, Pseudoalteromonas phage BS5 from the Yellow Sea was isolated and characterized. Complete genome sequencing of phage BS5 was also undertaken.
Materials and Methods
The surface seawater sample, from which the bacteriophage BS5 and its host bacteria Pseudoalteromonas atlantica was isolated, was collected on January 27, 2016 from a depth of 0.5 m in the Yellow Sea of China at location 36°07ʹN, 123°94ʹE. The water sample was stored at 4 ℃ after collection [15, 18, 19].
Isolation and Identification of Bacteria Strains
The host bacteria strain Pseudoalteromonas atlantica was isolated using serial dilution and incubated in liquid Zobell medium at 28 ℃ [5, 12, 15]. 16S rRNA gene sequencing was used for the molecular identification. Phylogenetic analysis, based on the neighbor-joining algorithm, was conducted using MEGA software version 6.0 .
The seawater sample was filtered through 0.2 µm pore size, low protein-binding PVDF filters (Millipore) to remove the bacteria, and phytoplankton. The phage was isolated by the standard double-layer agar method as described by Mathias et al. . Plaque was picked for more than three times and suspended in SM buffer [100 mM NaCl, 8 mM MgSO4, 50 mM TrisHCl (pH 7.5)], and the phage was stored at 4 ℃ [15, 18].
Morphology Study by Transmission Electron Microscopy
One-Step Growth Curve Assay
The one-step growth curve, which suggested the burst size (Burst size = the number of phages produced /infected bacterium), and the latent period of the phage was carried out by the double-layer agar plate method, described by Mathias et al. . Experiments were repeated three times.
pH Stability and Thermal stability
A double-layer agar assay was performed to examine the pH stability of the phage BS5 in the range 3.0–12.0. To study the thermal stability of phage, the phage suspension was incubated at different temperatures (−20~80 ℃) for 2 h by double-layer agar method.
Genome Sequencing and Bioinformatic Analysis
Phage DNA extraction was performed using a TIANamp Virus DNA Kit (TIANGEN). Purified PBS5 genomic DNA was sequenced using Illumina Miseq 2 × 300 paired-end sequence methods. An ABI 3730 automated DNA sequencer was used to complete the sequencing. Gaps between remaining contigs were closed via a Gapcloser and GapFiller using purified genomic DNA as the template. Genome annotations were analyzed using RAST (http://rast.nmpdr.org/). Nucleotide sequences and protein sequences were scanned for homologs using BLAST search of the updated GeneBank database. (http://blast.ncbi.nlm.nih.gov/) [15, 21, 22].
Genome Sequence Accession Number
The complete genome sequence of phage BS5 was given an accession number KX365748 after it had been submitted to NCBI.
Identification of the Bacteria Strain
One host bacteria of phage BS5 was isolated from the Yellow Sea. The 16S rRNA gene sequence of the the PBS5 bacterial host BBS5 showed 99.86% homology to P. atlantica. Phylogenetic analysis, based on the neighbor-joining algorithm, was conducted using MEGA software version 6.0  (Fig. 1).
Morphology of Phage BS5
The purified phage BS5 was examined by transmission electron microscopy (Fig. 2). The transmission electron microscope image showed that phage BS5 belongs to family Siphoviridae, which had an icosahedral head (with diameter of 54 ± 1-nm) and a long non-flexible tail (125 ± 10-nm).
One-Step Growth Curve Assay
One-step growth curve indicates that phage BS5 has a latent period of approximately 80 min, a rise period of 20 min, and a burst size of about 95 virions per cell (Fig. 3).
The pH stability test showed that the tolerance range of the phage is from 4 to 9, and the optimal pH is 7. The response function showed that the biological activity of the phage was stable between pH 5 and 9 (Fig. 4).
The thermal stability test showed that biological activity generally remained high at temperatures between −20 ~ 35 ℃, but it decreased sharply at temperatures above 40 ℃ (Fig. 5).
Genome Sequencing and Bioinformatic Analysis
The genome of PBS5 consists of a linear, double-stranded 39,949-bp DNA molecule with a GC content of 40.6%, and no tRNA genes. The phage genome, of which the coding ratio is 30.8%, has 65-bp protein-coding genes. And, the average length is 189.71-bp, including the minimum length gene 37-bp and the maximum length gene 869-bp. Sixty-five putative open reading frames (ORF) were detected in the 12,331-bp coding genes, and of these, 47 belonged to the plus strand and 18 belonged to the minus strand. Twenty-two conserved domains were detected by BLASTP in NCBI, and of these 5 were functionally known and 17 were unknown (Fig. 6). The genome was grouped into four modules as follows: phage structure, phage packaging, DNA replication and regulation, and some additional functions (Table 1). Thirty-six ORFs were found to match with various phages, including Vibrio phage, Serratia phage, Pseudoalteromonas phage, Escherichia phage, Paracoccus phage, Burkholderia phage, and Shewanella sp. phage. Sixteen ORFs were found to match with different bacteria, including Pseudomonas, Sporosarcina, Moraxella, Halomonas, Vibrio, Escherichia coli, Xenorhabdus, Algiphilus Acinetobacter, and Lactococcus. One of the 16 ORFs contained a sequence that was matched with the Pseudoalteromonas atlantica of phage BS5.
In the present study, a novel bacteriophage, infecting Pseudoalteromonas atlantica was isolated and purified from the Yellow Sea. According to the study of characterization and genomics, the phage BS5 belongs to the Siphoviridae family.The bacteriophage plaques became larger after a few days incubation, demonstrating that there might be secondary lysis function of endolysin [20, 24, 25]. From the one-step growth curve, it can be seen that phage BS5 has a long latent period.
The genome sequence analysis in this study adds new content to the phage library, which is useful to further molecular phage-host system research. According to the analysis of the phage origin (e < 10−5), Pseudoalteromonas phage H105/1 was found to share 10 genes with phage BS5, including three conserved domains (tape_meas_nterm, ERFandHTH_XRE) and two functional sequences (the DNA single-strand annealing proteins and Helix-turn-helix XRE-family like proteins). Pseudoalteromonas Phage H103 was also found to share 5 common genes with phage BS5, and Pseudoalteromonas phage Pq0 was found to share 7 common genes with phage BS5.Pseudoalteromonas phage TW1 and Pseudoalteromonas phage RIO separately share one common gene with phage BS5. The genomic analysis of PBS5 suggests that it shows homology with other phages Pseudoalteromonas phages, which indicates that the genome sequence of PBS5 provides further basic information to the Pseudoalteromonas phage library. It is noteworthy that some phage genome sequences were found to share common genes with bacteria. This demonstrates that PBS5 may be a lysogenic phage as PBS5 was induced into prophage . Among the 65 predicted ORFs, 5 of them were found to match with Pseudoalteromonas; furthermore, one of the five was found to be its host bacteria Pseudoalteromonas atlantica, from which it can be infered that during the process of the phage replication, a part of the bacteria’s genes were integrated into the phage genome. The capsid proteins of a new phage are highly conserved , and in this study, both ORF2 and ORF3 are related to phage putative head morphogenesis protein. The phage packaging protein includes ORF8, ORF33, and ORF35. ORF8 was identified as ATP-dependent chaperone ClpB, which can help polypeptide assembly in the cell . ORF11, ORF44, ORF46, ORF55, ORF58, and ORF64 were identified as different enzymes. Hydrolase is the key element of bacteriolysis. Helicase is a protein involved in DNA unwinding and nucleic acid metabolism, and it plays an important part in the survival and development of cells . PAPS reductase enzymes are involved in sulfate assimilation. ORF64 is identified as a terminase small subunit. It is reported that terminase is involved in DNA translocation, which comprises large and small subunits . And electron microscopy shows that small submit can self-assemble into a stable ring .
In this study, the characterization and genomic analysis of PBS5 was performed. The genome sequence of PBS5 shows that PBS5 is a novel phage with some similarities to PH105/1, PH103 and PHq0.
Suttle CA (2007) Marine viruses–major players in the global ecosystem. Nat Rev Microbiol 5(10): 801–812
Lang AS et al (2009) RNA viruses in the sea. Fems Microbiol Rev 33(2):295–323
Lorenz T (2014) Hatfull et al 2013
Bergh Ø et al (1989) High abundances of viruses found in aquatic environments. ApplEnvirMicrobiol. Nature 340(6233):467–468
Comeau AM, Suttle CA (2007) Distribution, genetic richness and phage sensitivity of Vibrio spp. from coastal British. Columbia. Environ Microbiol 9(7):1790–1800
Lara E et al (2015) Life-style and genome structure of marine Pseudoalteromonas siphovirus b8b isolated from the northwestern Mediterranean sea. PLoS ONE 10(1):e0114829–e0114829
Suttle CA, Chan AM (1994) Dynamics and distribution of cyanophages and their effect on marine Synechococcus spp. Appl Environ Microbiol 60(9):3167–3174
Kang I et al (2013) Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans. Proc Nat Acad Sci USA 110(30):12343–12348
Qin QL et al (2011) Comparative genomics reveals a deep-sea sediment-adapted life style of Pseudoalteromonas sp. SM9913. Isme J 5(2):274–284
Zhao Y et al (2013) Abundant SAR11 viruses in the ocean. Nature 494(7437):357–360
Altamirano A, Lara A (2010) Deforestation in temperate ecosystems of pre-Andean range of south-central Chile. Bosque 31(1):53–64
Zhou MY et al (2009) Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea. Microb Ecol 58(3):582–590
Wang DB et al (2016) Complete genome of a novel Pseudoalteromonas, Phage PHq0. Curr Microbiol 72(1):81–87
Chen XL et al (2003) Two different proteases produced by a deep-sea psychrotrophic bacterial strain, Pseudoaltermonas sp. SM9913. Marine Biol 143(5):989–993
Duhaime MB et al (2011) Ecogenomics and genome landscapes of marine Pseudoalteromonas phage H105/1. Isme J 5(1):107–121
Wang DB et al (2015) Characterization and genome sequencing of a novel bacteriophage PH101 infecting Pseudoalteromonas marina BH101 from the Yellow Sea of China. Curr Microbiol 71(5):594
Belin LJ et al (2013) An oncolytic vaccinia virus expressing the human sodium iodine symporter prolongs survival and facilitates SPECT/CT imaging in an orthotopic model of malignant pleural. Mesothelioma. Surgery 154(3):486–495
Duran AE et al (2002) Removal and inactivation of indicator bacteriophages in fresh waters. J Appl Microbiol 92(2):338–347
Li Y et al (2016) Complete genomic sequence of bacteriophage H188: a novel Vibrio kanaloae phage isolated from Yellow Sea”. Curr Microbiol 72(5):628–633
Huang G et al (2013) Characterization and genome sequencing of phage Abp1, a new phiKMV-like virus infecting multidrug-resistant Acinetobacter baumannii. Curr Microbiol 66(6):535–543
Bai Q et al (2013) Characterization and genome sequencing of a novel bacteriophage infecting Streptococcus agalactiae, with high similarity to a phage from Streptococcus pyogenes. Arch Virol 158(8):1733–1741
Hyman P, Abedon ST (2010) Bacteriophage host range and bacterial resistance. Adv Appl Microbiol 70:217–248
Middelboe M, Chan AM, Bertelsen SK (2010) Isolation and life-cycle characterization of lytic viruses infecting heterotrophic bacteria and cyanobacteria. Man Aquat Viral Ecol 2010:149–180
Loessner MJ, Wendlinger G, Scherer S (1995) Heterogeneous endolysins in Listeria monocytogenes, bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes. Mol Microbiol 16(6):1231–1241
Loessner MJ, Schneider A, Scherer S (1995) A new procedure for efficient recovery of DNA, RNA, and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin. Appl Environ Microbiol 61(3):1150–1152
Jiang SC, Paul JH (1998) Significance of lysogeny in the marine environment: studies with isolates and a model of lysogenic phage production. Microbial Ecol 35(35):235–243
Ellis J (1987) Proteins as molecular chaperones. Nature 328(6129):378–379
Gorbalenya AE, Koonin EV (1993) Helicases: amino acid sequence comparisons and structure-function relationships. Curr Opin Struct Biol 3(3):419–429
Němeček D et al (2008) Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit”. J Mol Biol 383(3):494–501
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 (No. 41076088 and 31500339) and China Postdoctoral Science Foundation (No.2015M5Z0612).
Conflict of interest
The authors declare that they have no competing interests.
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Meng, X., Wang, M., You, S. et al. Characterization and Complete Genome Sequence of a Novel Siphoviridae Bacteriophage BS5. Curr Microbiol 74, 815–820 (2017). https://doi.org/10.1007/s00284-017-1221-2
- Siphoviridae family Phage
- Yellow Sea