Archives of Virology (2020) 165:219–222

https://doi.org/10.1007/s00705-019-04432-5

ANNOTATED SEQUENCE RECORD

Genomic characterization of bacteriophage pEt‑SU, a novel

phiKZ‑related virus infecting Edwardsiella tarda
Sang Guen Kim1 · Sib Sankar Giri1 · Saekil Yun1 · Hyoun Joong Kim1 · Sang Wha Kim1 · Jung Woo Kang1 · Se Jin Han1 ·
Jun Kwon1 · Jin Woo Jun2 · Woo Taek Oh1 · Se Chang Park1

Received: 20 May 2019 / Accepted: 11 September 2019 / Published online: 16 October 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Abstract
A bacteriophage infecting Edwardsiella tarda (named pEt-SU) was isolated from freshwater collected in Chung-ju, South
Korea. The whole genome of pEt-SU was 276,734 bp in length, representing the first giant phage infecting Edwardsiella
reported to date. A total of 284 putative open reading frames were predicted and annotated. Morphology and genome analyses
verified that pEt-SU may be distantly related to the phiKZ-like phages, a well-known giant myovirus. The findings in this
study provide new insights into the phages infecting E. tarda ads well as fundamental data for the study of giant phages.

Edwardsiella tarda is a Gram-negative, intracellular, motile
bacterium that is frequently found in aquatic environments.
E. tarda infections, known as edwardsiellosis, are com-
mon in economically important fish species and represent a
severe economic threat to the aquaculture industry [1]. This
organism can also infect a wide variety of animals, including
humans, cetaceans, reptiles, and birds [2–5]. Owing to this
broad host range, prevention and protection strategies are
needed for both animals and humans. However, the intracel-
lular parasitic nature of E. tarda makes chemotherapeutic
treatment and development of vaccines against the disease
difficult. Indeed, the efficacy of vaccination for edwardsiel-
losis remains controversial [reviewed in reference 6].
The majority of research on edwardsiellosis to date has
focused its impact on the aquaculture industry. Bacterio-
phages (phages), viruses of bacteria, that show potential
Handling Editor: Johannes Wittmann.
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0070​5-019-04432​-5) contains
supplementary material, which is available to authorized users.
* Se Chang Park
parksec@snu.ac.kr
1
Laboratory of Aquatic Biomedicine, College of Veterinary
Medicine and Research Institute for Veterinary Science,
Seoul National University, Seoul 08826, Republic of Korea
2
Department of Aquaculture, Korea National College
of Agriculture and Fisheries, Kongjwipatjwi‑ro, Wansan‑gu,
Jeonju‑si, Jeollabuk‑do, Republic of Korea
protective effects against edwardsiellosis have been isolated
[7–10]. As a result, the prophylactic effect was verified for
loaches (Misgurnus anguillicaudatus) by immersion, and for
olive flounder (Paralichthys olivaceus) and red sea bream
(Pagrus major) by intraperitoneal injection [7]. However,
the therapeutic effect of these phages has not yet been clearly
demonstrated, regardless of its administration route (injec-
tion, oral, or immersion), due to the intracellular activity of
the E. tarda [8].
To better understand phage-host interactions, we isolated
a novel giant Edwardsiella phage and sequenced its whole
genome. Phage isolation was performed using freshwater
and seawater, and the lytic phage (named pEt-SU) was
detected from a freshwater sample (Chung-ju, South Korea,
36°50’39.0” N 127°59’06.1” E E) via a spot assay using E.
tarda NUF251 (from diseased olive flounder) as a host. A
double-layer overlay plaque assay was performed to confirm
the presence of lytic phages, and a small plaque (~ 0.1 mm)
was purified by isolating a single plaque with a sterile straw
five times to ensure that the isolated phages were descend-
ants of a single virion. The phages were cultured using tryp-
tic soy broth and agar (Becton Dickinson, Franklin Lakes,
NJ) at 25 °C. The morphology of pEt-SU was examined
using a Talos L120C (FEI, Czech) microscope operated at
120 kV, and images are shown in Fig. 1a and b. The average
sizes of phages were obtained by measuring five separate
virions. Phage pEt-SU has an icosahedral head of 121 ± 2.2
nm in diameter, a contractile tail 211.9 ± 1.5 nm in length,
and a baseplate on the end of the tail.

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Fig. 1  Transmission electron micrograph and genome map of pEt-
SU. a. pEt-SU virion with contracted tail. b. pEt-SU virion with
extended tail. Scale bar = 100 nm. c. The ORFs are categorized and
colored based on their predicted function. Blue represents predicted
structural and packaging-related proteins, yellow represents predicted
Total DNA of pEt-SU was extracted as described pre-
viously [11] and sequenced using an Illumina HiSeq 2500
platform (Illumina, San Diego, CA, USA), and a total of
11,993,048 reads (1,211,287,848 bases) were obtained. The
reads were assembled using CLC Genomics Workbench
v6.5.1 at Genotech (Daejeon, South Korea). The open read-
ing frames (ORFs) were predicted using GeneMarkS and
confirmed using the Rapid Annotation using Subsystem
Technology (RAST) server [12, 13]. The tRNA was detected
using tRNAscan-SE v2.0 [14], and annotation was carried
out using protein BLAST in the NCBI nr database and visu-
alized with DNA plotter [15].
The complete genome sequence of pEt-SU was 276,734
bp in length with a GC content of 43.2%. The genome con-
tained one tRNA gene and 284 ORFs, which are mainly
located on the positive strand (245 ORFs; 85.9%), with only
40 ORFs (14.1%) located on the negative strand. The pre-
dicted genes were analyzed and classified according to their
function into the following six categories: nucleotide regula-
tion (e.g., non-virion DNA-dependent RNA polymerase sub-
unit beta’, putative DNA polymerase, putative tubulin-like
13
S. G. Kim et al.
nucleotide-regulation-related proteins, red represents predicted lysis-
related proteins, green represents predicted proteins with additional
function, black represents predicted tRNA, and grey represents pre-
dicted hypothetical proteins. Scale units are base pairs
protein), structure and packaging (e.g., putative virion struc-
tural protein, tail length tape measure protein, putative ter-
minase large subunit), lysis (e.g., putative endolysin), tRNA,
additional function, and hypothetical function (Fig. 1c). Of
these categories, ORFs related to structure and packaging
clustered together (e.g., 38,145–98,304 or 233,010–247,130
bases). pEt-SU does not possess lysogeny-related genes
such as integrase, CI repressor, and CII regulatory protein.
Most of the predicted genes were conserved with those of
phiKZ-like phages, which are well-known giant myoviruses,
including the phiKZ internal capsid protein, several RNA
polymerase beta subunits, and putative tubulin-like protein
(Table S1 in Online Resource). These results indicate that
phage pEt-SU has a lytic life cycle, making it a suitable
therapeutic agent.
Comparative genome analysis was performed, which
revealed substantial divergence of the nucleotide sequence
of pEt-SU among phages infecting Edwardsiella spe-
cies. However, the genome organization of pEt-SU was
highly conserved with that of phage OBP infecting Pseu-
domonas fluorescens, another member of the phiKZ-like
Genomic characterization of bacteriophage pEt-SU
phages. Nucleotide sequence comparisons were done
using EMBOSS stretcher [16], and the highest sequence
similarity was found for the phiKZ-like phages (OBP
[17, 18]: 48.2% identity), followed by the T4-like phages
(PEi20, PEi26, and T4 [19]: 41.7, 41.6, and 40.4% identity,
respectively), eiAU-like phages (eiAU [20] and eiAU-183:
13.2% identity), phiPLPE-like phages (GF2 [10], Edno5
[21], MSW-3 [9, 22], PEi21 [23], and phiPLPE [24]: 13.3,
12.7, 13.2, 13.3, and 14.6% identity, respectively), and
KF-1-like phages (IW-1 [22, 25] and KF-1 [25]: 12.9%
identity). General information about the phages analyzed
in this study is summarized in Table S2 (Online Resource).
A whole-genome dot plot comparison using Gepard [26]
revealed a close relationship, with a diagonal pattern form-
ing five clusters within each phage group (pEt-SU and
OBP, T4-like phages, eiAU-like phages, phiPLPE-like
phages, and KF-1-like phages; Fig. 2a). Phage pEt-SU,
newly isolated in this study, produced a strong diagonal
line when compared with phage OBP; however, no pat-
terns were observed with other phages, such as PEi 20
and MSW-3, infecting Edwardsiella species. Similarly,
phylogenetic analysis performed using VICTOR [27] dem-
onstrated the same relationships of a total of 14 phages
clustered in the five groups as observed in the dot plot
whole-genome comparison (Fig. 2b). For more-detailed
comparison, the conserved genes between pEt-SU and
other phages examined in this study were compared using
Coregenes (75% threshold; [28]), as shown in Table S3
(Online Resource). A total of 217 genes (76.41%) of pEt-
SU were homologous to the ones in OBP, which are the
Fig. 2  Comparative genome analysis of pEt-SU. a. Dot plot showing sequence similarity generated in Gepard (word size, 10). b. Phylogenetic
tree generated in VICTOR with settings recommended for prokaryotic viruses
221
core genes of phages, such as those required for nucleotide
regulation, structure, packaging, and lysis. By contrast, the
genes unique to pEt-SU encode hypothetical proteins that
require further study to elucidate their specific functions.
There were few genes conserved with other phage clusters
(0–14), confirming that these phages are highly divergent
from pEt-SU according to different comparison methods.
Generally, members of the genus Phikzvirus are charac-
terized by a genome size of 280 – 316 kb and low GC con-
tent (36% for phiKZ), and they are specific for members
of the bacterial genus Pseudomonas. Both morphologic
and genomic evidence indicate that pEt-SU is most likely
a distant relative of phiKZ-like phage OBP having high
GC content (43.46%). Therefore, we propose Edwardsiella
virus pEt-SU to be a member of a new species within the
Phikzvirus in the family Myoviridae. Currently, only 11
whole-genome sequences of Edwardsiella-specific phages
are available in NCBI GenBank database, and pEt-SU is
the first giant phage found to infect members of the genus
Edwardsiella. Thus, these complete genome data of pEt-
SU will provide a fundamental resource for gaining a bet-
ter understanding of novel Edwardsiella viruses and new
treatments for edwardsiellosis.
Nucleotide sequence accession number The complete
genome sequence of phage pEt-SU was submitted to Gen-
Bank under accession number MK689364.
Acknowledgements  This research was supported by the Cooperative
Research Program of the Center for Companion Animal Research
(PJ0139852019) of the Rural Development Administration, Republic of
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222
Korea, and the Basic Science Research Program through the National
Research Foundation of Korea (NRF), funded by the Ministry of Edu-
cation (2017R1C1B2004616).
Compliance with ethical standards  improved detection of transfer RNA genes in genomic sequence.
Conflict of interest  The authors declare that they have no conflict of
interest.
Ethical approval  This article does not contain studies with human par-
ticipants or animals performed by any of the authors.
References
1. Xu T, Zhang XH (2014) Edwardsiella tarda: an intriguing prob-
lem in aquaculture. Aquaculture 431:129–135
2. Hirai Y, Asahata-Tago S, Ainoda Y, Fujita T, Kikuchi K (2015)
Edwardsiella tarda bacteremia. A rare but fatal water-and food-
borne infection: review of the literature and clinical cases from a
single centre. Can J Infect Dis Med Microbiol 26:313–318
3. Lee K, Kim HK, Park SK, Sohn H, Cho Y, Choi YM, Jeong DG,
Kim JH (2018) First report of the occurrence and whole-genome
characterization of Edwardsiella tarda in the false killer whale
(Pseudorca crassidens). J Vet Med Sci 80:1041–1046
4. Shin DM, Hossain S, Wimalasena S, Heo GJ (2016) Antimicrobial
resistance and virulence factors of Edwardsiella tarda isolated
from pet turtles. Pak Vet J 37:85–89
5. Miniero Davies Y, Xavier de Oliveira MG, Paulo Vieira Cunha
M, Soares Franco L, Pulecio Santos SL, Zanolli Moreno L, Tulio
de Moura Gomes V, Zanolli Sato MI, Schiavo Nardi M, Micke
Moreno A, Becker Saidenberg A, de Sa RM (2018) Edwardsiella
tarda outbreak affecting fishes and aquatic birds in Brazil. Vet Q
38:99–105
6. Park SB, Aoki T, Jung TS (2012) Pathogenesis of and strategies
for preventing Edwardsiella tarda infection in fish. Vet Res 43:67
7. Nakai T (2010) Application of bacteriophages for control of infec-
tious diseases in aquaculture. In: Sabour PM, Griffiths MW (eds)
Bacteriophages in the control of food- and waterborne pathogens.
ASM Press, Washington, DC, pp 257–272
8. Wu JL (1982) Isolation and application of a new bacteriophage,
ET-1, which infect Edwardsiella tarda, the pathogen of edwards-
iellosis. Rep Fish Dis Res 4:8–17
9. Yasuike M, Sugaya E, Nakamura Y, Shigenobu Y, Kawato Y,
Kai W, Nagai S, Fujiwara A, Sano M, Kobayashi T, Nakai T
(2013) Complete genome sequence of a novel myovirus which
infects atypical strains of Edwardsiella tarda. Genome Announc
1:e00248-12
10. Yasuike M, Nishiki I, Iwasaki Y, Nakamura Y, Fujiwara A, Sug-
aya E, Kawato Y, Nagai S, Kobayashi T, Ototake M, Nakai T
(2015) Full-genome sequence of a novel myovirus, GF-2, infect-
ing Edwardsiella tarda: comparison with other Edwardsiella myo-
viral genomes. Arch Virol 160:2129–2133
11. Kim SG, Jun JW, Giri SS, Yun S, Kim HJ, Kim SW, Kang JW,
Han SJ, Jeong D, Park SC (2019) Isolation and characterisation of
pVa-21, a giant bacteriophage with anti-biofilm potential against
Vibrio alginolyticus. Sci Rep 9:6284
12. Besemer J, Lomsadze A, Borodovsky M (2001) GeneMarkS: a
self-training method for prediction of gene starts in microbial
genomes. Implications for finding sequence motifs in regulatory
regions. Nucleic Acids Res 29:2607–2618
13. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA,
Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ,
13
S. G. Kim et al.
Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D,
Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O,
Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid
annotations using subsystems technology. BMC Genom 9:75
14. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for
Nucleic Acids Res 25:955–964
15. Carver T, Thomson N, Bleasby A, Berriman M, Parkhill J (2009)
DNAPlotter: circular and linear interactive genome visualization.
Bioinformatics 25:119–120
16. Rice P, Longden I, Bleasby A (2000) EMBOSS: the european
molecular biology open software suite. Trends Genet 16:276–277
17. Cornelissen A, Hardies SC, Shaburova OV, Krylov VN, Mattheus
W, Kropinski AM, Lavigne R (2012) Complete genome sequence
of the giant virus OBP and comparative genome analysis of the
diverse ϕKZ-related phages. J Virol 86:1844–1852
18. Shaburova OV, Hertveldt K, de la Cruz DMA, Krylov SV, Plete-
neva EA, Bourkaltseva MV, Lavigne R, Volckaert G, Krylov VN
(2006) Comparison of new giant bacteriophages OBP and Lu11
of soil pseudomonads with bacteriophages of the ϕKZ-supergroup
of Pseudomonas aeruginosa. Russ J Genet 42:877–885
19. Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, Rüger
W (2003) Bacteriophage T4 genome. Microbiol Mol Biol Rev
67:86–156
20. Carrias A, Welch TJ, Waldbieser GC, Mead DA, Terhune JS, Liles
MR (2011) Comparative genomic analysis of bacteriophages spe-
cific to the channel catfish pathogen Edwardsiella ictaluri. Virol
J 8:6
21. Katharios P, Kalatzis PG, Kokkari C, Pavlidis M, Wang Q (2019)
Characterization of a highly virulent Edwardsiella anguillarum
strain isolated from Greek aquaculture, and a spontaneously
induced prophage therein. Front Microbiol 10:141
22. Kawato Y, Nakai T (2012) Infiltration of bacteriophages from
intestinal tract to circulatory system in goldfish. Fish Pathol
47:1–6
23. Yasuike M, Kai W, Nakamura Y, Fujiwara A, Kawato Y, Hassan
ES, Mahmoud MM, Nagai S, Kobayashi T, Ototake M, Nakai T
(2014) Complete genome sequence of the Edwardsiella ictaluri-
specific bacteriophage PEi21, isolated from river water in Japan.
Genome Announc 2:e00228-14
24. Leblanc C, Caumont-Sarcos A, Comeau AM, Krisch HM (2009)
Isolation and genomic characterization of the first phage infecting
Iodobacteria: ϕPLPE, a myovirus having a novel set of features.
Environ Microbiol Rep 1:499–509
25. Yasuike M, Sugaya E, Nakamura Y, Shigenobu Y, Kawato Y, Kai
W, Fujiwara A, Sano M, Kobayashi T, Nakai T (2013) Complete
genome sequences of Edwardsiella tarda-lytic bacteriophages
KF-1 and IW-1. Genome Announc 1:e00089-12
26. Krumsiek J, Arnold R, Rattei T (2007) Gepard: a rapid and sen-
sitive tool for creating dotplots on genome scale. Bioinformatics
23:1026–1028
27. Meier-Kolthoff JP, Göker M (2017) VICTOR: genome-based phy-
logeny and classification of prokaryotic viruses. Bioinformatics
33:3396–3404
28. Turner D, Reynolds D, Seto D, Mahadevan P (2013)
CoreGenes3.5: a webserver for the determination of core genes
from sets of viral and small bacterial genomes. BMC Res Notes
6:140
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