Characterization and Complete Genome Sequence of a Novel Siphoviridae Bacteriophage BS5

Abstract

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.

Introduction

Viruses are the most abundant organisms in the ocean and contain the highest genetic diversity in marine ecosystems [1]. Viruses play an integral role in the marine ecosystem where they outnumber of all other life forms by at least an order of magnitude [2].The total abundance of viruses in the ocean is apparently more than 1030 [3] as they are found at an abundance of up to 2.5 × 108 cells per milliliter in surface waters [4]. 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 [57]. So far, only a few sequenced phages have been found infecting marine heterotrophic bacteria [810]. In order to investigate the influence of bacteriophages on bacteria by lysing bacterial cells, we need to isolate, proliferate, and purify phage-host systems [11].

The marine genus Pseudoalteromonas is a member of Gammaproteobacteria, which is widespread from surface waters to deep-sea sediments [12]. Gammaproteobacteria may comprise more than 30% of all marine bacterioplankton [13]. 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 [1416]. 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

Sampling

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 [8].

Phage Isolation

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. [20]. 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

The purified phage particles were studied by transmission electron microscope (JEOLJEM-1200EX, Japan) at 100 KV and at a magnification 400,000× [15, 19].

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. [20]. 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.

Results

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 [23] (Fig. 1).

Fig. 1
figure1

Neighbor-joining tree for selected bacteria constructed from the 16S rDNA sequence. Bacteria BS5 seems to be highly related to Pseudoalteromonas atlantica IAM 12927(T). (T) indicates a type strain

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).

Fig. 2
figure2

Transmission electron microscopic image of phage BS51. Bar represents 0.05% estimated sequenced sequence change

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).

Fig. 3
figure3

One-step growth curve of phage BS5

pH Stability

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).

Fig. 4
figure4

pH stability of phage BS5

Temperature Stability

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).

Fig. 5
figure5

Temperature stabilityof phage BS5

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.

Fig. 6
figure6

Full genome of bacteriophage BS5

Table 1 Genomic annotation of Pseudoalteromonas Phage PH101 and conserved domains detected (e < 10−5)

Discussion

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 [26]. 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 [19], 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 [27]. 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 [28]. 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 [29]. And electron microscopy shows that small submit can self-assemble into a stable ring [29].

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.

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Acknowledgements

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).

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Correspondence to Min Wang or Yong Jiang.

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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

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Keywords

  • Phage
  • Genomic
  • Siphoviridae family Phage
  • Yellow Sea