The complete genome sequence and analysis of vB_VorS-PVo5, a Vibrio phage infectious to the pathogenic bacterium Vibrio ordalii ATCC-33509
© The Author(s). 2016
Received: 25 October 2015
Accepted: 3 May 2016
Published: 4 July 2016
The bacterium Vibrio ordalii is best known as the causative agent of vibriosis outbreaks in fish and thus recognized for generating serious production losses in aquaculture systems. Here we report for the first time on the isolation and the genome sequencing of phage vB_VorS-PVo5, infectious to Vibrio ordalii ATCC 33509. The features as well as the complete genome sequence and annotation of the Vibrio phage are described; vB_VorS-PVo5 consists of a lineal double stranded DNA totaling ~ 80.6 Kb in length. Considering its ability to lyse Vibrio ordalii ATCC 33509, the phage is likely to gain importance in future aquaculture applications by controlling the pathogen and as such replacing antibiotics as the treatment of choice.
The Chilean coast is characterized by the Humboldt Current System, a cold, low-salinity ocean current that is considered one of the most productive marine ecosystems on Earth. Cold, nutrient rich waters are constantly upwelled into the photic zone providing sustenance for primary producers [1, 2]. This environment offers highly favorable natural conditions for the growth of heterotrophic bacteria , e.g., for Vibrio species, known as the main pathogenic bacterial group in Chilean aquaculture [4, 5]. One of the most frequently isolated marine Vibrio species in the salmon industry is Vibrio ordalii which has been described as highly pathogenic for larvae reared in hatcheries [6–9], and therefore significantly contributes to production losses. In order to prevent diseases and control infections in fish farms, the intensive use of a wide variety of antimicrobials is applied. However, the poor management of such treatments, e.g., the use of antibiotics in discrete doses as a prophylactic therapy [10, 11], has caused enormous damage to the environment [12–14]. Moreover, the increasing development of antimicrobial resistance in natural bacterial communities [10, 15, 16], calls for stricter regulations of antibiotic use . As a consequence, the interest in phage therapy as an alternative control for bacteria in aquaculture systems has recently re-gained momentum [18–21].
In our quest to find a natural control for V. ordalii in aquaculture, we focused our research on isolating phages potentially effective against the pathogen, surveying various marine sources, e.g., sea water, sediment and intertidal filter organisms. Amongst others we tested the filter-feeding Perumytilus purpuratus (Lamarck, 1819), an intertidal mussel common to the northern Chilean coast and a promising source organism for phages as it uptakes and concentrates local microbiota in its gut system. We succeeded in isolating and identifying vB_VorS-PVo5 a novel marine phage belonging to the family Siphoviridae that causes lytic infections in the bacterium V. ordalii ATCC 33509 and therefore qualifies as a potent future candidate to control one of the most harmful bacteria in the aquaculture industry. The whole genome sequence of the phage was sequenced on an Illumina MiSeq platform and is described here, presenting the first report of an isolated and sequenced phage that infects the marine bacterium Vibrio ordalii .
Classification and features
The Vibrio phage vB_VorS-PVo5 belongs to the Siphoviridae, a family of double-stranded DNA viruses in the order Caudovirales, and forms ~2-mm diameter plaques when infecting V. ordalii type strain ATCC 33509.
Genome sequencing information
Genome project history
Classification and general features of Vibrio phage vB_VorS-PVo5
Domain: viruses, dsDNA viruses, no RNA phage
(Type) strain: vB_VorS-PVo5 (KT345706)
Icosahedral head with a long tail
pH range; Optimum
35 % NaCl (w/v)
Obligate intracellular parasite of Vibrio ordalii
Lytic virus of Vibrio ordalii
Jan 25 2014
Gene calling method
GenBank Date of Release
November 03 2015
Source Material Identifier
Personal culture collection
Growth conditions and genomic DNA preparation
The phage multiplication was performed applying the double-layer agar plates method [26, 27]. Vibrio ordalii cells (conc. 5E + 06 cells/mL) in the soft layer of Tryptone Soy Agar, (Oxoid, UK) were subjected to six serial dilutions (1:10–1:1000000) of phage and the lysis plaques formed were counted to determine the total number of phage. For the DNA extraction each of three plates were inoculated with 1000 PFU phage and incubated for 16 h at 25 °C. Viral particles were re-suspended in 4 mL phage buffer  and incubated for 4 h with intervals of gentle shaking at 30 min. Subsequently the supernatant was transferred into 15 mL falcon tubes, a 1 mL chloroform solution was added, gently shaken for 30 s and centrifuged at 5000 rpm for 5 min. The product was filtered through 0.22 μm nitrocellulose filters (Merck-Millipore, Germany) to eliminate bigger cells. For the elimination of all external genomic content, DNase I (Thermo-Fisher, Germany) and RNase-A (Thermo-Fisher, Germany) were added at a final concentration of 5 units mL−1 each, for an incubation time of 30 min at 37 °C. Subsequently the flocculant PEG-80 was added at a 4:10 ratio and incubated overnight at 4 °C. Viral particles were pelleted in a centrifugation step at 10000 × g for 1 h at 4 °C. The supernatant was removed, the pellet dried for ~5 min under a sterile hood, a 50 μl of 10 units mL−1 Proteinase K (Thermo-Fisher, Germany) and phage buffer mix added, and incubated at 50 °C for 30 min to inactivate nucleases. The genomic DNA of the vB_VorS-PVo5 phage was extracted with a Phage DNA Isolation Kit (Norgen Biotek Corp., Canada), and evaluated and quantified with a UV–VIS spectrophotometer (BioTek Epoch, USA). In order to confirm the type of nucleic acid extracted, the product was digested separately in 1 unit/mL DNAse I and RNAse A, respectively. DNAse I, as opposed to RNAse A, degraded the extract, confirming the organism to be a DNA-containing phage.
Genome sequencing and assembly
The genome was sequenced on an Illumina MiSeq platform at the MR-DNA Laboratory (Shallowater, TX). The library for each sample was prepared using a Nextera DNA Sample Preparation Kit (Illumina), following the manufacturer’s instructions. 2 × 130-bp paired-end reads allowed for an estimate of 50.000 output sequences of 287 bp length with 45,367 reads remaining after the quality filtering. The assemblage of quality-filtered reads was executed for the complete genome sequence, using the pipeline by MR-DNA and resulted in an average coverage of 130 fold. A single contig of 80,578 bp corresponding to the linear genome was assembled using NGEN (DNASTAR ®) by MR-DNA.
The prediction of open reading frames and the comparative analysis were performed combining two methods: the PHAST server  and Glimmer 2.1 . For the assignment of protein functions to ORFs a combination of an automatic and a manual method was used, i.e., the PHAST server and BLASTp against the NCBI non-redundant database. Only homologues with E-values <1e-5 were present in the annotations. The tRNA genes were searched using the tRNAscan-SE 1.21 tool  and TMHMM , and SignalP  were used to predict transmembrane helices and signal peptides, respectively.
% of the Totala
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
% of Totala
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Mobilome: Prophage, Transposons
Insights from the genome sequence
Phage reproduction may involve either a lytic or a lysogenic cycle with some viruses being capable of performing both. In the lysogenic cycle the viral genome will integrate with host DNA and replicate along with it, whereas the lytic phage will destroy the host cell immediately after the replication, breaking bacterial cells open and allowing the phage progeny to find new hosts to infect. Due to quickly destroying the bacterial cells, lytic phages are more suitable for phage therapy, and the genes coding for the production of endolysin are the key evidence for the phage’s lytic characteristics [36, 37]. A BLASTP comparison of the endolysin sequences displayed a similarity of 94 % and 46 % with the Vibrio phage pVp-1 and Vibrio phage Phi-3, respectively. Furthermore, the enzyme destroys bacterial cell walls and has therefore been discussed for its use as an anti-infective to control pathogens [36, 38].
The specificity of the phage vB_VorS-PVo5 has been tested on Vibrio anguillarum , the most closely related species to Vibrio ordalii  and another Chilean strain of Vibrio ordalii , isolated from scallops, and whereas vB_VorS-PVo5 infected both Vibrio ordalii species it did not lyse Vibrio anguillarum . More research has to be done in order to test the effect of vB_VorS-PVo5 phage and its endolysin as a therapy, however, the fact that only Vibrio ordalii strains were lysed indicates that vB_VorS-PVo5 might be highly species specific and may therefore prove to be a very promising candidate for phage therapy against Vibrio ordalii .
Here we report for the first time on the isolation and genome sequencing of vB_VorS-PVo5 a novel phage that belongs to the family of the Siphoviridae and is capable of lysing the pathogen Vibrio ordalii ATCC 33509. The lytic character of the phage, together with the first indication of its specificity for Vibrio ordalii strains indicates the potential for its future use in aquaculture applications, controlling the pathogen either by using the phage or its endolysin.
We would like to thank Roberto Bastías for providing microbial strains. This work was funded by the following grants: Project CORFO FIC-R 13IDL2-18530 to RA, and Beca de Doctorado CONICYT FIC-R N°21092008 and Beca de Apoyo de Tesis, Programa de Doctorado en Ciencias Aplicadas, Mención Sistemas Marinos Costeros 2013–2014 to AE.
AE conceived the study, participated in all samplings, performed sample preparation, conducted all molecular genetic studies and drafted the manuscript. PM participated in samplings, culture maintenance, sample preparation and molecular genetic studies. CS participated in samplings, culture maintenance, sample preparation and molecular genetic studies. JC assisted in drafting the manuscript. RA participated in the experimental design, coordinated the project and assisted in drafting the manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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