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Complete genome sequence of Bacillus licheniformis strain 0DA23-1, a potential starter culture candidate for soybean fermentation
Korean J. Microbiol 2018;54(4):453-455
Published online December 31, 2018
© 2018 The Microbiological Society of Korea.

Do-Won Jeong1, Byunghoon Lee2, Sojeong Heo1, Mihyun Jang2, and Jong-Hoon Lee2,*

1Department of Food and Nutrition, Dongduk Women’s University, Seoul 02748, Republic of Korea
2Department of Food Science and Biotechnology, Kyonggi University, Suwon 16227, Republic of Korea
Correspondence to: E-mail: jhl@kgu.ac.kr; Tel.: +82-31-249-9656; Fax: +82-31-253-1165
Received September 12, 2018; Revised October 11, 2018; Accepted October 25, 2018.
Abstract

Bacillus licheniformis strain 0DA23-1, a potential fermentation starter candidate, was isolated from doenjang, a Korean high-salt-fermented soybean food. Strain 0DA23-1 contains a single circular 4,405,373-bp chromosome with a G + C content of 45.96%. The complete genome of strain 0DA23-1 does not include any of the virulence factors found in the well-known pathogens Bacillus cereus and Staphylococcus aureus. Additionally, no genes associated with resistance to eight antibiotics (chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin), hemolysis, or biofilm formation were identified.

Keywords : Bacillus licheniformis, doenjang, soybean fermentation, starter
Body
The genus Bacillus is the predominant bacterial group found in the fermented soybean foods of East Asia, including Korea (Kim et al., 2009; Lee et al., 2010). Bacillus licheniformis, a predominantly identified Bacillus species, exhibits growth on tryptic soy agar (TSA; Difco) supplemented with 14% (w/v) NaCl and enzyme, including amylase, protease, and lipase, activities (Jeong et al., 2014, 2017b). Because of these properties, B. licheniformis has the potential to be applied as a fermentation starter culture in soybean foods, including the Korean doenjang, a high-salt-fermented soybean paste ripened at NaCl concentrations of > 12% (w/w). Recently, we isolated B. licheniformis ‘0DA23-1’ from a commercial doenjang. This strain sustained growth on TSA containing 14% NaCl, and exhibited proteolytic and lipolytic activities on TSA containing 3% and 4% NaCl, respectively. When the safety of strain 0DA23-1 was evaluated using the European Food Safety Authority guidelines for the safe use of microorganisms as food/feed materials (EFSA, 2004; Jeong et al., 2017a), it was found to be susceptible to eight antibiotics (chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin) and did not exhibit either hemolysis or biofilm formation (data not shown). To confirm the genetic background of B. licheniformis 0DA23-1 as a safe fermentation starter candidate, its complete genome was sequenced.

Whole-genome sequencing was performed using the PacBio Single-Molecule Real-Time sequencing system by ChunLab, Inc. One contig was generated from a hybrid assembly of reads from the PacBio system (173.26 × coverage) for B. licheniformis 0DA23-1. The reads were assembled using CLC Genomics Workbench ver. 7.5.1 (CLC Bio) and CodonCode Aligner (CodonCode Co.). Gene predictions were performed using Glimmer 3 (Tatusov et al., 1997), followed by annotation through a search against the Clusters of Orthologous Groups (COG) database (Tatusov et al., 1997).

The complete genome of strain 0DA23-1 consists of a single circular 4,405,373-bp chromosome with a G + C content of 45.96% (Table 1). The genome is predicted to contain 4,518 protein coding sequences, 81 tRNA genes, and 24 rRNA genes. In total, 4,104 genes were functionally assigned to categories based on COG. The majority of the genes are related to transcription [331 genes (8.1%)], followed by carbohydrate transport and metabolism [327 genes (8.0%)] and amino acid transport and metabolism [312 genes (7.6%)]. The high proportion of genes in nutrient utilization indicates that strain 0DA23-1 has the ability to degrade a wide range of soybean carbohydrates and proteins. The genome of strain 0DA23-1 does not encode any of the virulence factors found in the food pathogens Bacillus cereus and Staphylococcus aureus. Additionally, no genes involved in the resistance to eight antibiotics (chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin), hemolysis, or biofilm formation were identified from the annotated genes based on COG. The genomic traits of strain 0DA23-1 satisfied the requirements of a functional and safe fermentation starter candidate that can be applied to produce fermented soybean foods. The complete genome sequence of strain 0DA23-1 provides information that will aid in the selection of functional and safe Bacillus starter culture candidates for soybean food fermentation.

Genome features of Bacillus licheniformis strain 0DA23-1

Features Value
Genome size (bp) 4,405,373
G + C content (%) 45.96
Open reading frames 4,518
CDS assigned by COG 4,104
rRNA genes 24
tRNA genes 81

CDS, coding DNA sequence; COG, Cluster of Orthologous Groups of proteins.


Nucleotide sequence accession numbers

B. licheniformis 0DA23-1 has been deposited in the Korean Collection for Type Cultures under accession number KCTC 43013, and its complete genome sequence has been deposited in DDBJ/ENA/GenBank under accession number CP031126.

적 요

된장으로부터 콩발효 종균후보 Bacillus licheniformis 0DA23-1가 분리되었다. 0DA23-1 균주는 GC 함량 45.96%, 약 4.4 Mb 크기의 단일 chromosome을 보유하고 있었고, 식중독균 Bacillus cereus 및 Staphylococcus aureus가 보유한 위해성 유전자는 유전체로부터 확인되지 않았다. 또한, 8종의 항생물질(chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, vancomycin) 저항성 및 혈청분해활성, 바이오필름 생성 관련 유전자도 확인되지 않았다.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D 1A1B01011421 and NRF2016R1D1A1B03930239). Mihyun Jang was supported by Kyonggi University’s Graduate Research Assistantship 2018. We thank Lesley Benyon, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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