Bacillus licheniformis is one of the predominant bacterial species identified in fermented soybean foods of Korea (Kim et al., 2009; Lee et al., 2010). We considered the species as a potential starter candidate of doenjang, a Korean high-salt-fermented soybean food ripened at NaCl concentrations of > 12% (w/w), because of its salt tolerance on tryptic soy agar (TSA; Sigma- Aldrich) supplemented with 14% (w/v) NaCl and activities of amylase, protease, and lipase which can contribute to degrade macromolecules in soybeans (Jeong et al., 2014, 2017). In the process of selecting B. licheniformis starter candidates with biosafety from our stock cultures, we identified strain 14ADL4 resistant to clindamycin (Jeong et al., 2017). The minimum inhibitory concentration of clindamycin for strain 14ADL4 determined by the broth micro-dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (2007) was 64 mg/L and the strain exhibited growth on TSA containing 30 mg/L clindamycin. The present study performed a complete genome analysis of strain 14ADL4 to shed light on the genetic background behind the phenotypic resistance to clindamycin.

Whole-genome sequencing was performed using the PacBio Single-Molecule Real-Time (SMRT) sequencing system by ChunLab, Inc. One contig was generated from a hybrid assembly of reads from the PacBio system (105,988 reads and 345.13 coverage) for B. licheniformis 14ADL4. The reads were assembled using CLC Genomics Workbench ver. 7.5.1 (CLC Bio) and CodonCode Aligner (CodonCode Co.). Gene prediction was performed using Glimmer 3 (Delcher et al., 2007), followed by annotation through a search against the Clusters of Orthologous Groups (COG) database (Tatusov et al., 1997).

The complete genome of strain 14ADL4 consists of a single circular 4,332,232 bp chromosome with a G + C content of 45.86% (Table 1). The genome is predicted to contain 4,419 protein coding sequences, 81 tRNA genes, and 24 rRNA genes. A total of 3,900 genes were functionally assigned to categories based on COG assignments. Gene category analysis shows that the majority of the genes are related to carbohydrate transport and metabolism (329 genes [8.4%]), followed by transcription (319 genes [8.2%]) and amino acid transport and metabolism (312 genes [8.0%]).

Table 1

Genome features of Bacillus licheniformis 14ADL4

Features	Value
Genome size (bp)	4,332,232
GC content (%)	45.86
Open reading frames	4,419
CDS assigned by COG	3,900
rRNA genes	24
tRNA genes	81

Strain 14ADL4 has two potential lincomycin resistance genes, lmrA (designated BL14DL4_03907, putative HTH-type transcriptional regulator gene) and lmrB (BL14DL4_03548, lincomycin resistance protein gene). The lmrAB genes, which encode a transcriptional repressor and a multidrug efflux protein, have been reported to be involved in the resistance of Bacillus subtilis to puromycin and lincomycin (Murata et al., 2003). Erythromycin ribosome methylase gene (erm) has been reported to confer resistance to clindamycin as well as erythromycin (Leclercq, 2002), while any homolog of erm was not found in the genome of strain 14ADL4. In this context, the lmrAB operon found in strain 14ADL4 may confer resistance to lincosamide antibiotics including clindamycin. Comparative genomic analysis of B. licheniformis 14ADL4 with clindamycin sensitive B. licheniformis strains will provide further genetic insight into the strain-specific clindamycin resistance of Bacillus species. Illumination of the clindamycin resistance ortholog for B. licheniformis will contribute to introduce a safety-approved Bacillus starter culture in food fermentations.

Nucleotide sequence accession number

B. licheniformis 14ADL4 has been deposited in the Korean Collection for Type Cultures under accession number KCTC 33983 and its complete genome sequence has been deposited in DDBJ/ENA/GenBank under accession number CP026673.

Clindamycin 내성 Bacillus licheniformis 14ADL4 균주는 된장으로부터 분리되었다. 14ADL4 균주의 유전체분석 결과, GC 함량 45.86%, 약 4.3 Mb 크기의 염색체가 확인되었고, 유전체 상의 lmrA와 lmrB 추정 유전자가 clindamycin 내성에 관여할 것으로 예상된다.

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-2016R1D1 A1B01011421 and NRF2016R1D1A1B03930239). This work was also supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Technology Commercialization Support Program funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, 815003-3). Byunghoon Lee was supported by Kyonggi University’s Graduate Research Assistantship 2018.