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Complete genome sequence of Lactiplantibacillus plantarum KM2 from low-temperature aging beef
Korean J. Microbiol. 2021;57(4):303-306
Published online December 31, 2021
© 2021 The Microbiological Society of Korea.

Sojeong Heo1, Jong-Hoon Kim2, Mi-Sun Kwak2, Do-Won Jeong1*, and Moon-Hee Sung2,3*

1Department of Food and Nutrition, Dongduk Women’s University, Seoul 02748, Republic of Korea
2Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 02707, Republic of Korea
3KookminBio Corporation, Seoul 02826, Republic of Korea
Correspondence to: (M.H. Sung) E-mail: smoonhee@kookmin.ac.kr;
smoonhee@kmbio.co.kr; Tel.: +82-2-910-4808; Fax: +82-910-5739 /
(D.W. Jeong) E-mail: jeongdw@dongduk.ac.kr;
Tel.: +82-2-940-4463; Fax: +82-2-940-4610
Received November 17, 2021; Revised December 28, 2021; Accepted December 28, 2021.
Abstract
We previously isolated Lactiplantibacillus plantarum strain KM2 from low temperature aging beef. Its complete genome sequence was determined in the current study to understand KM2’s genetic background. The genome contains a single circular 3,233,034 bp chromosome and five plasmids. The genome G + C content is 44.24%. Significantly, strain KM2 contains a plantaricin A gene cluster, an antifungal determinant, and exopolysaccharide biosynthetic genes. The complete genome sequence of strain KM2 will contribute to understanding the genetic background of antimicrobial activities.
Keywords : Lactiplantibacillus plantarum, genome, low temperature aging beef, probiotics
Body

Lactiplantibacillus plantarum was recently reclassified from Lactobacillus plantarum on the basis of whole genome sequencing (Zheng et al., 2020). Lactiplantibacillus plantarum predominantly occurs in fermented foods but is also found in human and other animal intestines. Generally, it is described as a food-grade bacteria due to a long history of use in fermented foods. Lactiplantibacillus plantarum produces lactate from glucose, which lactate contributes to the taste and smell of fermented foods and inhibits pathogen growth. Also, it is a known probiotic species that affects regulation of the immune system and can be used in the treatment of gastrointestinal diseases (Molin, 2001). Some Lp. plantarum strains produce bacteriocins (Todorov, 2009), and some also show antifungal activity (Yang and Chang, 2010). This has led to investigation of this species for use as probiotics, and some strains have been commercially used as probiotics. Probiotic strains with antimicrobial and antifungal activities can prevent the growth of bacterial pathogens and fungi; therefore, these strains have unique, natural preservative qualities that make them useful as starter cultures in fermented foods. We previously isolated Lp. plantarum strain KM2 from low-temperature aging beef. In the current study, we checked the antimicrobial activities of strain KM2 against food-borne pathogens (Table 1). Furthermore, to provide additional insight into the genes involved in this antimicrobial activity, the complete genome sequence of Lp. plantarum strain KM2 was determined.

Antibacterial activities of Lactiplantibacillus plantarum KM2 against food-borne pathogens

Indicator strain Activity
Gram-positive bacteria
Enterococcus faecalis KCTC 2011 +
Listeria monocytogenes ATCC 19111 -
Staphylococcus aureus ATCC12692 ++
Gram-negative bacteria
Escherichia coli O157:H7 (EDL 933) +++
Salmonella enterica KCCM11862 +++
Vibrio parahaemolyticus ATCC17802 ++

Diameter of growth-inhibition zone: -, 0 mm; +, ~20 mm; ++, 20~40 mm; +++, > 40 mm.



Whole-genome sequencing of strain KM2 was performed using a combination of the Illumina MiSeq system (Illumina) and the PacBio RSII platform (Pacific Bioscience) at ChunLab, Inc. The generated PacBio sequencing reads (83,183 reads at 170-fold coverage) were assembled using the MaSuRCA algorithm (version 3.3.9). Six contigs were generated from the reads, of which one was assumed to be a chromosome, and the others to be five plasmids. All contigs were confirmed to be circular structures using gene walking techniques. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (version 4.6; Tatusova et al., 2016). Open reading frames (ORFs) were predicted using Glimmer 3, followed by manual annotation via a search against the Clusters of Orthologous Groups (COG) database.

The complete Lp. plantarum KM2 genome consists of a circular 3,233,034 bp chromosome and five circular plasmids, pKM2-1 (38,668 bp), pKM2-2 (7,301 bp), pKM2-3 (75,285 bp), pKM2-4 (13,623 bp), and pKM2-5 (50,246 bp). The G + C content of the chromosome is 44.56%, while that of the plasmids are 37.35%, 34.46%, 39.01%, 35.62%, and 40.89%, respectively. The average nucleotide identity (ANI) of the KM2 genomic sequence showed 99.26% similarity with Lp. plantarum WCFS1. Genome analysis predicts 3,219 ORFs, 70 tRNA genes, and 16 rRNA genes. Of the 3,219 ORFs, 2,890 genes are assigned to functional categories based on COG analysis. Gene category analysis shows that the majority of these genes are related to carbohydrate transport and metabolism (n = 296; 10.24%), followed by transcription (n = 248; 8.58%), and amino acid transport and metabolism (n = 214; 7.40%) (Fig. 1).

Fig. 1. Functional categories of Lactiplantibacillus plantarum KM2 genome based on COG. Relative abundance > 2% are shown as Figures.

Strain KM2 possesses the plantaricin A gene, plnA (JQC82_RS01800). Plantaricin A is a class II bacteriocin, which are small, heat-stable, non-lanthionine peptides (Diep et al., 1994; Todorov, 2009). The KM2 plnA gene encodes a 48-amino acid precursor peptide with membrane permeabilizing antimicrobial activity (Kristiansen et al., 2005). Two-component system, annotated plnB (sensor kinase; JQC82_RS01805), and plnD (response regulator; JQC82_RS01815), were also detected to regulate the transcription of plnA gene. Additionally, two bacteriocin operons (plnEFI and plnJKLR; JQC82_RS01765–JQC82_RS01775 and JQC82_RS01820–JQC82_RS01830, respectively), which show fungicidal activity against Candida albicans (Sharma and Srivastava, 2014), occur in the KM2 genome. Other antifungal substances associated with Lactiplantibacillus spp. include phenylactic acid, phenolic amines, and fatty acid methyl esters (Yang and Chang, 2010; Yu et al., 2014). The KM2 genome encodes the phosphoethanolamine N-methyltransferase gene (JQC82_RS13240) and 12 lactate dehydrogenase (ldh) genes to produce these compounds. Exopolysaccharide (EPS) is a major component of extracellular biofilms. EPS contributes to surface adhesion including that within human intestinal mucosa and it is resistance to environment stress (Schmid et al., 2015). These properties are important for probiotic strains to reside in the gut and also provide protection against antibiotics and enzymes, as well as against stomach acid. The KM2 genome has several EPS biosynthetic genes (JQC82_RS04835–JQC82_RS04860 and JQC82_RS09170–JQC82_RS09210). Therefore, we assume that these genes contribute to the enhanced probiotic properties of KM2.

Further genome analysis revealed that Lp. plantarum KM2 does not encode any enterotoxin genes including hemolysin genes through keyword search for annotated genes based on COG database. In addition, the KM2 genome does not encode acquired antibiotic resistance genes against chloramphenicol, clindamycin, erythromycin, gentamicin, penicillin, streptomycin, or tetracycline. The absence of these virulence related genes, along with the presence of a bacteriocin cluster, antifungal determinants, and EPS biosynthetic operons satisfies the requirements of an excellent potential probiotic bacterial strain. The complete genome sequence of Lp. plantarum KM2 provides the genetic basis for further comparative and functional genomic analyses of antimicrobial activities and will help with the development of this strain for use in human food, animal feed, and medicinal industries.

Strain deposition and nucleotide sequence accession numbers

Lactiplantibacillus plantarum KM2 has been deposited in the Korean Collection for Type Cultures under the number KCTC 14637BP. The complete genome sequence of KM2 has been deposited in GenBank under accession numbers CP06 9282–CP069287.

적 요

Lactiplantibacillus plantarum KM2 균주를 저온숙성 한우로부터 분리하여 유전체를 분석하였다. 균주 KM2의 유전체는 약 3.4-Mb 크기로 GC 함량은 44.24%였다. 유전체 분석 결과 박테리오신 오페론, 항진균 유전자 및 exopolysaccharide 생합성 유전자들을 다양하게 보유하고 있었다. 이는 KM2 균주의 항균 및 항진균 활성과 프로바이오틱 특성에 대한 유전적 배경을 이해하는데 활용될 수 있음을 제공한다.

Acknowledgments

This work was supported by the Innopolis Korea innovation Foundation 2020 grant funded by the Korea government (MEST) (No. 2020-JB-RD-0218), Korea Environmental Industry and Technology Institute (KEITI) grant funded by the Ministry of Environment of Korea, and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A2C100 3639 for DWJ and NRF-2020R1A6A3A13077341 for SH).

Conflict of Interest

The authors have no conflict of interest to report.

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