Complete Genome Sequence of Enterococcus faecalis CAUM157 Isolated from Raw Cow’s Milk

Abstract

Enterococcus faecalis CAUM157, isolated from raw cow’s milk, is a Gram-positive, facultatively anaerobic, and non-spore-forming bacterium capable of inhabiting a wide range of environmental niches. E. faecalis CAUM157 was observed to produce a two-peptide bacteriocin that had a wide range of activity against several pathogens, including Listeria monocytogenes, Staphylococcus aureus, and periodontitis-causing bacteria. The whole genome of E. faecalis CAUM157 was sequenced using the PacBio RS II platform, revealing a genome size of 2,972,812 bp with a G+C ratio of 37.44%, assembled into two contigs. Annotation analysis revealed 2,830 coding sequences, 12 rRNAs, and 61 tRNAs. Further, in silico analysis of the genome identified a single bacteriocin gene cluster.

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pISSN 2733-4554
eISSN 2733-4562
J. Dairy Sci. Biotechnol. 2020;38(3):142-145
https://doi.org/10.22424/jdsb.2020.38.3.142
142｜J Dairy Sci Biotechnol Vol. 38, No. 3 https://www.ejmsb.org
ARTICLE
Received: August 28, 2020
Re vised: August 31, 2020
Accepted: August 31, 2020
*Corresponding author :
Geun-Bae Kim
Department of Animal Science and
Technology, Chung-Ang University,
Anseong, Korea
Tel : +82-31-670-3027
Fax : +82-31-676-5986
E-mail : kimgeun@cau.ac.kr
Copyright © 2020 Korean Society of
Dairy Science and Biotechnology.
This is an Open Access article distributed
under the terms of the Creative Commons
Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/3.0)
which permits unrestricted non-commercial
use, distribution, and reproduction in any
medium, provided the original work is
properly cited.
ORCID
Arxel G. Elnar
https://orcid.org/0000-0002-2716-4924
Sang-Dong Lim
https://orcid.org/0000-0002-1500-4413
Geun-Bae Kim
https://orcid.org/0000-0001-8531-1104
Complete Genome Sequence of
Enterococcus faecalis
CAUM157 Isolated from Raw Cow’s Milk
Arxel G. Elnar
1
, Sang-Dong Lim
2
, and Geun-Bae Kim
1*
1Department of Animal Science and Technology, Chung-Ang University, Anseong, Korea
2Korea Food Research Institute, Wanju, Korea
Abstract
Enterococcus faecalis
CAUM157, isolated from raw cow’s milk, is a Gram-positive, faculta-
tively anaerobic, and non-spore-forming bacterium capable of inhabiting a wide range of
environmental niches.
E. faecalis
CAUM157 was observed to produce a two-peptide
bacteriocin that had a wide range of activity against several pathogens,
including
Listeria
monocytogenes
,
Staphylococcus aureus
,
and periodontitis-causing bacteria. The whole
genome of
E. faecalis
CAUM157 was sequenced using the PacBio RS II platform, revealing
a genome size of 2,972,812 bp with a G+C ratio of 37.44%, assembled into two contigs.
Annotation analysis revealed 2,830 coding sequences, 12 rRNAs, and 61 tRNAs. Further,
in
silico
analysis of the genome identified a single bacteriocin gene cluster.
Keywords
Enterococcus faecalis
, whole genome, bacteriocin, raw milk
Antimicrobial drug resistance and the rapid emergence of resistant bacterial species
continue to be a growing concern as it threatens the effectivity of traditional antimicro -
bial compounds against its target pathogens [1,2]. This evolutionary arms race demands
the identification and development of novel antimicrobial compounds that have the
potential to substitute antibiotic use and other similar applications. Bacteriocins, which
are ribosomal-synthesized peptides produced by bacteria to inhibit the growth of closely
related strains [2,3], are potential alternatives to traditional antibiotics [4].
Enterococcus
faecalis
CAUM157, a Gram-positive, facultatively anaerobic, non-spore-forming cocci
[5], was observed to produce an antimicrobial protein with potent activity against a wide
range of bacteria including periodontal disease-causing pathogens,
Prevotella intermedia
and
Fusobacterium nucleatum
[6], and
Listeria monocytogenes
,
and
Staphylococcus
aureus
(unpublished data). Whole-genome sequencing of strain CAUM157 was performed
to further investigate the molecular characteristics of the bacteriocin gene structure.
E. faecalis
CAUM157 was isolated from raw cow’s milk obtained from a local farm
in Anseong, Korea. Strain CAUM157 was routinely grown in de Man, Rogosa, and Sharpe
(MRS, Difco Laboratory, USA) broth supplemented with 1% L-cysteine at 37℃. The
genomic DNA was extracted from 12 h cultures using QIAamp PowerFecal DNA Kit
(Qiagen, Germany) according to the manufacturer’s instructions. Genomic DNA of strain
CAUM157 was sent to ChunLab (Korea) and sequenced using the Pacific Biosciences
(PacBio, USA) RSII Single Molecule Real-Time (SMRT) platform with 20 kb SMRTbell ™
template library. The PacBio reads were assembled
de novo
using the PacBio SMAR
Analysis ver. 2.3.0 program. Genome annotation was performed with the Rapid Annota -
tion using Subsystem Technology (RAST) using default parameters [7] and CLgenomics ™

Enterococcus faecalis CAUM157 Genome
https://www.ejmsb.org J Dairy Sci Biotechnol Vol. 38, No. 3 ｜143
ver. 1.55 software. Transfer RNAs (tRNAs) were identified using tRNAscan-SE ver. 1.3.1
[8]. Ribosomal RNAs (rRNAs) and non-coding RNAs were identified using INFERNAL ver.
1.1.3 software with Rfam 12.0 database [9]. Functional annotation of protein-coding se-
quences (CDSs) was performed using the PRODIGAL ver. 2.6.2 software [10] and compared
to protein databases (SwissProt, KEGG, SEED, EggNOG) using USEARCH ver. 8.1 [11].
The complete genome of
E. faecalis
CAUM157 (Fig. 1) has a length of 2,972,812 bp with
a G+C content of 37.44% assembled into 2 contigs with an N
50
value of
2,913,602 bp.
The genome contains 2,830 coding genes, 12 rRNAs, and 61 tRNAs (Table 1).
Putative bacteriocin-encoding gene clusters were determined
in silico
using the
BAGEL4 software tool [12] which revealed 1 area of interest (AOI) in contig
CM157.00001, corresponding to the bacteriocin genes. Two open reading frames (ORFs)
encoding the core peptides of bacteriocin MR10A (E value, 2×10
–
27
) and MR10B (E value,
1×10
–
27
) were detected. The amino acid sequences of the two core peptides deduced
from the putative bacteriocin gene share a similar sequence and lack the YGNGVXC
motif characteristic of the leader sequence, which suggests that the bacteriocin is of
class IIB
–
leaderless two-peptide bacteriocin [13]. Additionally, the amino acid
sequence exhibited a high degree of similarity with a previously described bacteriocin
(MR10A and MR10B) from
E. faecalis
[14]. The corresponding sequences are homologous
with the plasmid-encoded enterocins L50A and L50B described in
E. faecium
L50 [15]
except for a conservative change (Glu 38 to Asp) in MR10A and two residue change (Thr
Fig. 1. Circular genome map of Enteroccocus faecalis CAUM157. Circles represent the following
characteristics from the outermost circle to the center: (1) contig information, (2) coding sequences
on forward strand, (3) coding sequences on reverse strand, (4) transfer RNAs (tRNAs) and
ribosomal RNAs (rRNAs), (5) GC skew, and (6) GC ratio.

Elnar et al.
144｜J Dairy Sci Biotechnol Vol. 38, No. 3 https://www.ejmsb.org
9 to Ala, and Leu 15 to Phe) in MR10B [16]. Furthermore, genes encoding for
self-immunity and ABC-transport system (efflux RND transporter, ABC transporter
ATP-binding protein, and ABC transporter permease) were detected downstream of the
putative bacteriocin genes.
Nucleotide Sequence Accession Number
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank
under the accession JACSYK000000000. The version described in this paper is version
JACSYK010000000.
Conflict of Interest
The authors declare no potential conflict of interest.
Acknowledgments
This work was supported by the Korea Institute of Planning and Evaluation for
Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bio
Industry Technology Development Program, funded by the Ministry of Agriculture, Food
and Rural Affairs (MAFRA) (318091-03-1-HD030).
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Table 1. Genome features of Enterococcus faecalis CAUM157
Attribute Value
Genome size (bp) 2,972,812
GC content (%) 37.4
No. of contigs 2
Total genes 2,903
Protein-coding gene 2,830
tRNA 61
rRNA 12
Plasmids 0
GenBank accession no. JACSYK000000000

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