Members of the genus Terribacillus were originally classified into the genus Bacillus (Claus and Berkeley, 1986); however, molecular and phenotypical analyses revealed a lineage distinct from that of Bacillus. At present (September, 2021), the genus Terribacillus comprises four species: T. saccharophilus and T. halophilus from field soils in Japan (An et al., 2007); T. goriensis from coastal water off of Korea (Kim et al., 2007; Krishnamurthi and Chakrabarti, 2008); and T. aidingensis from sediments of the saline Ayding Lake in China (Liu et al., 2010). Terribacillus aidingensis is a phenotypically aerobic rod-shaped endospore-forming bacterium that is motile by means of peritrichous flagella (Liu et al., 2010). In addition, T. aidingensis species include moderately halo-tolerant bacteria. However, information on T. aidingensis species, as well as other members of the genus Terribacillus, such as the normal habitats and genetic backgrounds, are limited. We isolated T. aidingensis DMT04 from the traditional Korean fermented vegetables, kimchi, and DMT04 exhibited a lipolytic activity on a tributyrin agar (Sigma-Aldrich) supplemented with 1% tributyrin (v/v) and 4% NaCl (w/v). Additionally, strain DMT04 grew on tryptic soy agar (BD) containing up to 10% NaCl (w/v) as a final concentration. The lipase activities contribute to flavor enhancement through lipolysis in food fermentation and salt-tolerant of bacteria might contribute to sustain in fermented food (Arnold et al., 1975; Jeong et al., 2020). We analyzed the complete genome of T. aidingensis DMT04 to shed light on its genetic background on lipase activity and salt-tolerant.

Strain DMT04 was grown in tryptic soy broth (BD) at 37°C for 24 h. Genomic DNA of strain DMT04 was purified using a Wizard Genomic DNA Purification Kit (Promega) and DNA contamination was determined by sequencing the 16S rRNA gene. Whole-genome sequencing of T. aidingensis DMT04 was analyzed using the PacBio Sequel 10K system (Pacific Bioscience) at ChunLab, Inc. Two contigs were obtained from generated sequencing reads (294,575 reads and 593.0× coverage) using the Flye assembler (ver. 2.8.3) in SMRT Link (PacBio). Two contigs DNA were used as templates for the PCR amplification to check the circular identification by DNA walking. The DMT04 genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (ver. 4.6) (Tatusova et al., 2016). Genes were predicted using the Clusters of Orthologous Groups database of the COGNITOR program (Tatusov et al., 2000) and CLgenomics^TM v. 1.55 software. Additional prediction of genes was performed with the RAST server in USA (https://rast.nmpdr.org/rast.cgi) and the SEED database.

The complete genome of T. aidingensis DMT04 consists of a circular 3,404,391-bp chromosome and a circular 115,498-bp plasmid pDMT04 (Table 1). The G + C content of the genome is 42.30%. The 16S rRNA sequence of T. aidingensis DMT04 showed 99.7% and 98.5% nucleotide similarity with T. aidingensis CGMCC 1.8913 and T. halophilus T-h1, respectively. DMT04 genome showed 91.7% similarity to the draft genome of the T. aidingensis CGMCC 1.8913 (11 scaffolds; incomplete genome) based on the average nucleotide identity (ANI) values and 99.8% similarity by tetranucleotide analysis (TNA) (https://www.ezbiocloud.net). The chromosome was predicted to contain 3,325 coding sequences (CDS), 76 tRNA genes, 25 rRNA genes and 4 ncRNA genes. The plasmid was predicted to contain 112 CDS, and most of the annotated CDS encoded hypothetical proteins. The 3,437 CDS in the DMT04 genome were functionally categorized using the Clusters of Orthologous Groups database. Major functional classifications, except function unknown, were related to amino acid transport and metabolism (268 genes, 8.4%), as well as transcription (204 genes, 6.4%) and carbohydrate transport and metabolism (197 genes, 6.2%).

Table 1

Genome features of Terribacillus aidingensis DMT04

Feature	Value
Genome size (bp)	3,519,889
Chromosome	3,404,391
Plasmid pDMT04	115,498
G + C content (%)	42.30
Total number of genes	3,594
Protein coding genes (CDS)	3,437
rRNA genes	25
tRNA genes	76
ncRNA genes	4

The T. aidingensis DMT04 genome possesses two putative lipase genes, alpha/beta hydrolase (KS242_RS15720) and alpha/beta fold hydrolase (KS242_RS12310). Those genes possess the conserved domain of acetyl esterase/lipase (COG0657) and triacylglycerol esterase/lipase (COG1075), respectively. We hypothesized that they confer lipolytic activity to strain DMT04. To determine the salt-tolerant of strain DMT04, we searched for genes related to osmoprotectants, such as glycine betaine, proline betaine, trehalose, glutamate and cardiolipin, using keywords in annotated gene list. Derived genes and functional roles were identified by COG and SEED databases. Terribacillus aidingensis DMT04 possesses four osmoprotectant uptake systems, OpuA, OpuB, OpuD and OpuE, for choline, glycine betaine and/or proline betaine (Fig. 1). Additionally, the DMT04 genome contains trehalose and glycerol uptake systems. Furthermore, the DMT04 genome has genes for synthesizing cardiolipin, glutamate and proline. Thus, we hypothesized that these genes conferred halo-tolerant properties to strain DMT04.

Fig. 1. Predected somoprotectant transport system and synthesis pathways in Terribacillus aidingensis DMT04. Osmoptectans are depicted letters in blue box. Genes are marked in green italics with locus number. Transporters are presented as protein names and their locus is blue. The black arrows correspond to the potential enzymatic reactions catalyzed by gene products encoded in Terribacillus aidingensis DMT04.

Nucleotide sequence accession numbers

The complete genome sequence of T. aidingensis DMT04 has been deposited in DDBJ/ENA/GenBank under accession numbers CP077639 and CP077640, and the strain has been deposited in the Korean Culture Center of Microorganisms under accession number KCCM 90491.

김치에서 분리한 지방분해활성과 내염성 특성을 보이는 Terribacillus aidingenesis DMT04 균주를 분리하여 유전체를 분석하였다. Terribacillus aidingenesis DMT04는 3,404,391-bp 크기의 chromosome과 115,498-bp 크기의 plasmid를 보유하고 있었다. DMT04 균주의 G + C 함량은 42.30%이고, 2개의 지질 분해 유전자와 고염에서 생존을 위한 위한 glycine betaine, proline betaine, trehalose, glycerol 유입을 위한 유전자 및 cardiolipin, glutamate, proline 합성 유전자를 보유하고 있었다.

This research was supported by the Dongduk Women’s University grant of 2020.

The authors have no conflict of interest to report.