ANNOTATED SEQUENCE RECORD
Genomic annotation for the temperate phage EFC-1, isolated
from Enterococcus faecalis KBL101
Bo Hyun Yoon
Received: 24 April 2014 / Accepted: 19 October 2014 / Published online: 31 October 2014
Ó Springer-Verlag Wien 2014
Abstract The temperate phage EFC-1 was newly isolated
from a mitomycin-C-induced lysate of Enterococcus fae-
calis KBL101. EFC-1 has an isometric head and a long tail.
The phage belongs to the family Siphoviridae according to
its genomic structure and morphology. The phage EFC-1
has 40,286 base pairs of double-stranded DNA and a G?C
content of 35.05 %. Bioinformatic analysis of the phage
revealed 60 putative open reading frames (ORFs). The
genome of the temperate phage EFC-1 was not signiﬁ-
cantly similar to that of previously reported bacteriophages
from E. faecalis.
Enterococcus faecalis is a gram-positive coccus that
commonly inhabits the intestines, oral cavity, and vaginal
tract of most animals . Enterococcus faecalis can be
used as a starter for fermented dairy food and in probiotics.
However, the bacterium can also cause endocarditis, uri-
nary tract infections and other infections in humans . In
this study, E. faecalis KBL101was isolated from raw milk
and identiﬁed by 16S rRNA-based sequence analysis .
The temperate phage EFC-1 was identiﬁed as a pro-
phage in the E. faecalis KBL101 chromosome after it was
isolated from a mitomycin C (MMC)-induced lysate of the
bacterium. The phage was extracted and puriﬁed by
ultracentrifugation in a CsCl gradient with densities of 1.1
to 1.7 g/ml at 25,000 rpm and 4 °C for 16 h (Beckman
SW28 rotor) . CsCl-puriﬁed phage samples were neg-
atively stained with 2 % (w/v) aqueous uranyl acetate (pH
4.0) on copper grids covered with a ﬁlm of carbon-Form-
var, ionized, and examined by transmission electron
microscopy (Hitachi H-7500, Japan) at an accelerating
voltage of 80 kV. We found that EFC-1 has an isometric
head and a long tail and can therefore be classiﬁed as a
member of the large family Siphoviridae  (Fig. 1).
The genomic DNA was isolated from puriﬁed phage
particles by phenol extraction as described previously .
The genome sequence was generated by random sequenc-
ing using a GS FLX pyrosequencer and was assembled
using GS De Novo Assembler V. 2.9. Putative open
reading frames (ORFs) were analyzed with Gene-
Mark.hmm for Prokaryotes, V. 3.19 (http://opal.biology.
gatech.edu/hmmchoice.html). Annotation and func-
tional analysis of the predicted ORFs were performed using
the protein Basic Local Alignment Search Tool (BLASTp)
at the National Center for Biotechnology Information
(NCBI) . The t-RNA was identiﬁed using the tRNAscan-
SE program (http://lowelab.ucsc.edu/tRNAscan-SE/).
Bioinformatic analysis of the EFC-1 genome revealed
40,286 base pairs (bp) with a G?C content of 35.05 % and
one putative tRNA gene speciﬁc for methionine (bp
759-832). Using BLASTn, the complete nucleotide
sequence of the EFC-1 genome showed 95 % identity to
the uncharacterized prophage region of E. faecalis
DENG1. (Supplementary Material 1). Sixty putative ORFs
were identiﬁed in the genome, including ﬁve that had a
leucine as the start codon and one that started with a valine
residue. The predicted proteins of nine of the ORFs were in
the opposite orientation on the complementary strand.
Thirty-one ORFs were similar to genes with annotated
functions in the GenBank database. Twenty-eight ORFs
were similar to uncharacterized proteins, and one ORF did
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-014-2263-4) contains supplementary
material, which is available to authorized users.
B. H. Yoon Á H.-I. Chang (&)
College of Life Sciences and Biotechnology, Korea University,
145 Anam-Ro, Sungbuk-Gu, Seoul, Korea
Arch Virol (2015) 160:601–604