Whole genome sequence analysis of Ligilactobacillus agilis C7 isolated from pig feces revealed three bacteriocin gene clusters

Jeong Min Yoo1, Remilyn M. Mendoza1, In-Chan Hwang1, Dae-Kyung Kang1,*
Author Information & Copyright
1Department of Animal Resources Science, Dankook University, Cheonan 31116, Korea
*Corresponding author: Dae-Kyung Kang, Department of Animal Resources Science, Dankook University, Cheonan 31116, Korea., Tel: +82-41-550-3655, E-mail:

© Copyright 2022 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: May 27, 2022; Revised: Jun 13, 2022; Accepted: Jun 20, 2022

Published Online: Sep 30, 2022


We here report the whole genome sequence of Ligilactobacillus agilis C7 with anti-listerial activity, which was isolated from pig feces. The genome size of L. agilis C7 (~ 3.0 Mb) is relatively larger compared with other L. agilis strains. L. agilis C7 carries three bacteriocin gene clusters encoding garvicin Q, salivaricin A, and Blp family class II bacteriocin. Garvicin Q and salivaricin A are reported to be active against Listeria monocytogenes and Micrococcus luteus, respectively, as well as against other Gram-positive bacteria. Meanwhile, the bacteriocin encoded in the blp cassette was shown to be active against pneumococci, mediating intraspecies competition. This report highlights the potential of L. agilis C7 for the production of bacteriocins inhibiting pathogenic bacteria.

Keywords: Ligilactobacillus agilis; Genome; Bacteriocin; Anti-listerial


Ligilactobacillus agilis, commonly isolated from animals, has been used as a representative species for the study of the motility of lactic acid bacteria, because it has motility features that are uncommon in lactobacilli [1,2]. Very recently, bacterial culture and cell-free supernatant of L. agilis 32 isolated from pig manure were reported to inhibit the growth of Enterotoxigenic Escherichiacoli 10 (ETEC 10), one of the causative agents of post-weaning diarrhea in piglets [3]. In this announcement, we report the genome of L. agilis C7, which inhibits Listeria monocytogenes (unpublished data), a frequent contaminant of many foods including dairy and meat products, and the three bacteriocin gene clusters encoding garvicin Q, salivaricin A, and a Blp family class II bacteriocin.

L. agilis C7, which showed antibacterial activity against L. monocytogenes ATCC 19114 in the agar-well diffusion assay (unpublished data), was isolated from piglet fecal samples. L. agilis C7 was cultured in de Man, Rogosa and Sharpe (MRS) broth and streaked on MRS agar. MRS agar plates were incubated at 37°C for 24 h. Genomic DNA was extracted from the C7 strain using the conventional phenol–chloroform protocol and was sent to Macrogen (Seoul, Korea) for whole-genome sequencing. The PacBio Sequel System (Pacific Biosciences, Menlo Park, CA, USA) and Illumina Platform (Illumina, San Diego, CA, USA) technologies were used to generate long and short sequence reads, respectively. PacBio sequencing generated a total of 2,235,708,853 bases with 237,414 total long reads (subreads), while Illumina sequencing generated a total of 800,163,663 bases and 5,301,328 paired-end reads after filtering. The Microbial Assembly Application was used for de novo assembly of the PacBio reads. Pilon (v1.21) was used to improve the initial assembly by mapping the Illumina reads, generating a more accurate assembly. The genome assembly of L. agilis C7 has a total size of 2,961,932 base pair (bp) with four contigs. Quality statistics were generated using QUAST (v5.0.2) and Benchmarking Universal Single-Copy Orthologs (BUSCO) (v5.3.2). QUAST reported the largest contig and N50 length of 1,727,998 bp and a guanine + cytosine (G + C) content of 40.15%, while BUSCO reported 124 complete and single-copy BUSCOs. Average Nucleotide Identity, ANIb option (pyani v0.2.10) identified L. agilis with > 95% sequence identity to publicly available L. agilis genomes.

Annotation of L. agilis C7 revealed 93 transfer RNAs, 24 ribosomal RNAs, 1 transfer-messenger RNA, 7 repeat regions, 2946 coding sequences, and 3116 genes (Table 1). BAGEL4 [4] identified two bacteriocin gene clusters encoding the bovicin 255 peptide variant and salivarin A, while AntiSMASH [5] mapped RiPP-like bacteriocin IIc (Fig. 1). The genome size of L. agilis C7 is relatively larger (~3.0 Mb) than those of other L. agilis strains (~2.0–2.5 Mb) in the NCBI database. Mobile genetic elements such as an integrative and conjugative element (~255 kb) and the 17 prophage regions (7 intact prophages) were also mapped to the genome of L. agilis C7 via VRprofile [6].

Table 1. Genome features of Ligilactobacillus agilis C7
Genome size (bp) 2,961,932
No. of contigs 4
N50 1,727,998
GC content (%) 40.15
CDS 2,946
Genes 3116
tRNA 93
Misc RNA 52
rRNA 24
Repeat region 7
tmRNA 1

bp, base pair; GC, guanine + cytosine; CDS, coding sequences.

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Fig. 1. Genome map of Ligilactobacillus agilis C7. Marked features from outside going in: CDS on the forward strand, CDS on the reverse strand (highlighted in yellow and red within the CDS region are the bacteriocin regions for garvicin Q and Blp family class II, and salivaricin A, respectively), tRNAs, rRNAs, repeat regions, GC content, and GC skew. bp, base pair; CDS, coding sequences; GC, guanine + cytosine.
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Blastp analyses of the bovicin 255 peptide variant, salivaricin A, and the RiPP-like bacteriocin IIc revealed 100% identity to garvicin Q family class II bacteriocin from other L. agilis strains, 62.5% identity to type A2 lanthipeptide from Streptococcus pyogenes, and 51.46% identity to Blp family class II bacteriocin from S. hyointestinalis, respectively. Garvicin Q is a subclass IId bacteriocin that was initially isolated from Lactococcus garvieae BCC 43578 and has been reported to be active against L. monocytogenes and several bacterial species belonging to the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, and Pediococcus [7]. Meanwhile, salivaricin A, which is active against Micrococcus luteus [8], is among the lantibiotics produced by strains of S. salivarius, although genetic variants have also been found in S. pyogenes [8,9]. Lantibiotics are ribosomally synthesized molecules that are heat-stable and reported to have therapeutic potential in treating infectious diseases. On the other hand, Blp family class II bacteriocin has been shown to mediate intraspecies competition among pneumococci [10].

This report highlights the potential of L. agilis C7 for the production of bacteriocin to control L. monocytogenes in dairy and meat products. In addition, this paper contributes to the scarce genetic information on L. agilis.


The genome sequence of L. agilis C7 is available in the DDBJ/ENA/GenBank databases under accession no. JAMGEC000000000. The BioSample accession no. is SAMN28229336, and the BioProject accession no. is PRJNA837731.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the High Value-added Food Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant no. 321035052HD040).


Not applicable.

Availability of data and material

Upon reasonable request, the datasets of this study can be available from the corresponding author.

Authors’ contributions

Conceptualization: Kang DK.

Formal analysis: Mendoza RM, Hwang IC.

Writing - original draft: Yoo JM, Mendoza RM.

Writing - review & editing: Yoo JM, Mendoza RM, Hwang IC, Kang DK.

Ethics approval and consent to participate

This article does not require IRB/IACUC approval because there are no human and animal participants.



Kajikawa A, Midorikawa E, Masuda K, Kondo K, Irisawa T, Igimi S, et al. Characterization of flagellins isolated from a highly motile strain of Lactobacillus agilis. BMC Microbiol. 2016; 16:49


Kajikawa A, Suzuki S, Igimi S. The impact of motility on the localization of Lactobacillus agilis in the murine gastrointestinal tract. BMC Microbiol. 2018; 18:68


Shi S, Cheng B, Gu B, Sheng T, Tu J, Shao Y, et al. Evaluation of the probiotic and functional potential of Lactobacillus agilis 32 isolated from pig manure. Lett Appl Microbiol. 2021; 73:9-19


van Heel AJ, de Jong A, Song C, Viel JH, Kok J, Kuipers OP. BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins. Nucleic Acids Res. 2018; 46:W278-81


Medema MH, Blin K, Cimermancic P, de Jager V, Zakrzewski P, Fischbach MA, et al. antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acid Res. 2011; 39:W339-46


Li J, Tai C, Deng Z, Zhong W, He Y, Ou HY. VRprofile: gene-cluster-detection-based profiling of virulence and antibiotic resistance traits encoded within genome sequences of pathogenic bacteria. Brief Bioinform. 2018; 19:566-74


Tymoszewska A, Diep DB, Wirtek P, Aleksandrzak-Piekarczyk T. The non-lantibiotic bacteriocin garvicin Q targets man-PTS in a broad spectrum of sensitive bacterial genera. Sci Rep. 2017; 7:8359


Wescombe PA, Upton M, Dierksen KP, Ragland NL, Sivabalan S, Wirawan RE, et al. Production of the lantibiotic salivaricin A and its variants by oral streptococci and use of a specific induction assay to detect their presence in human saliva. Appl Environ Microbiol. 2006; 72:1459-66


Barbour A, Philip K. Variable characteristics of bacteriocin-producing Streptococcus salivarius strains isolated from Malaysian subjects. PLOS ONE. 2014; 9e100541


Dawid S, Roche AM, Weiser JN. The blp bacteriocins of Streptococcus pneumoniae mediate intraspecies competition both in vitro and in vivo. Infect Immun. 2007; 75:443-51