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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2024; 52(2): 200-203

Received: April 23, 2024; Revised: May 7, 2024; Accepted: May 8, 2024

Complete Genome Sequence of Bacillus safensis DMB13 Exhibiting Non-Antibacterial Activity

Do-Won Jeong*

Department of Food and Nutrition, Dongduk Women’s University, Seoul 02748, Republic of Korea

Correspondence to :
Do-Won Jeong,

Strain Bacillus safensis DMB13 exhibiting protease and lipase activity was isolated from fermented kimchi in the previous study. Phenotypically, strain DMB13 showed no antibacterial activity. Thus, the complete genome sequence was analyzed to understand the phenotype of strain DMB13. The genome is 3,856,276-bp with a G+C content of 41.61 mol% and consists of a single circular 3,849,633-bp chromosome and one circular plasmids. Two genes related to bacteriocin production, skfF and skfG, were identified; however, six other genes in the skf operon were not detected. The genome includes 54 protease- and 5 lipase-encoding genes.

Keywords: Bacillus safensis, kimchi, bacteriocin, skf operon, protease, lipase

Bacillus safensis was initially discovered in the spacecraft-assembly facility at the Jet Propulsion Laboratory [1]. Since then, it has been isolated from various sources, including food products, such as honey, bread, and syrup. In addition to its presence in extreme environments, B. safensis shows high antimicrobial activity and is under investigation owing to its potential as a biopreservative in aquaculture [2, 3]. In a previous study, we isolated B. safensis from kimchi [4]. Antibiotic susceptibility testing and functional assays revealed four starter candidates out of 65 Bacillus strains, one of which was B. safensis DMB13 (initially designated as strain GN5_10) [4]. In this study, we evaluated the antimicrobial activity of strain DMB13. However, B. safensis DMB13 showed no antibacterial activity against nine species of foodborne pathogens, Bacillus cereus KCCM 11341, Enterococcus faecalis KCTC 2011, Listeria monocytogenes ATCC 13932, Staphylococcus aureus ATCC 12692, Alcaligenes xylosoxidans KCCM 40240, Escherichia coli O157:H7 EDL 933, Flavobacterium sp. KCCM 11374, Salmonella enterica KCCM 11862, and Vibrio parahaemolyticus KCTC 2729, contradicting previous findings indicating that B. safensis shows strong antibacterial activity [2, 3]. Therefore, to uncover genetic factors influencing the antibacterial properties the strain and explain variation among studies, we analyzed the genome of B. safensis DMB13. Additionally, through genomic analyses, we evaluated its susceptibility to antibiotics, lack of hemolysis, and protease and lipase activity.

Whole-genome sequencing of B. safensis DMB13 was conducted at CJ Bioscience, Inc. (South Korea) using the PacBio Sequel 10K system. Sequencing yielded 50,427 reads with a coverage of 522.37×, from which two contigs were generated using the FLYE assembler (version 2.8.3) in SMRT Link (PacBio). Annotation was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (version 4.6). Annotated genes were classified into functional categories using the Clusters of Orthologous Groups (COG) database [5], and metabolic pathways were examined using Rapid Annotation using Subsystem Technology [6].

The complete genome of B. safensis DMB13 consisted of a circular 3,849,633 bp chromosome and a 6,643 bp plasmid pDMB13. The G+C content was 41.61 mol%. The average nucleotide identity between the genome of B. safensis DMB13 and those of B. safensis subsp. safensis FO-36bT and B. safensis subsp. osmophilus CECT 9344T were 97.29% and 96.25%, respectively. The genome harbored 3,968 open reading frames, including 3,862 protein-coding genes, 81 tRNA genes, 24 rRNA genes, and 1 additional RNA gene. Using COG, functional annotations were obtained for 3,561 genes, with significant enrichment for amino acid transport and metabolism (304 genes, 8.5%), transcription (291 genes, 8.2%), and carbohydrate transport and metabolism (255 genes, 7.2%). In a SEED subsystem analysis, 1,577 genes were annotated, and the most abundant subsystem category was amino acids and derivatives (280 genes, 17.8%), followed by carbohydrates (236 genes, 15.0%).

Although B. safensis strain DMB13 lacked antimicrobial activity, it contained genes related to the bacteriocin gene cluster, skfF and skfG (Fig. 1). However, the genome of strain DMB13 did not possess the entire operon necessary for the production of sporulation killing factor (skf) (Fig. 1). In contrast, Bacillus subtilis 168, reported to exhibit antimicrobial activity, had the skf operon (Fig. 1) [7]. Additionally, B. safensis JRS3 harbored the skf operon. The exact influence of Skf on interspecific antimicrobial activity is not well understood, although it is known to lyse sibling cells [8]. Nevertheless, if the sporulation killing factor generated from the skf operon is responsible for antimicrobial activity, the lack of antimicrobial activity in strain DMB13 can be attributed to the lack of the full-length skf operon.

Figure 1.Comparison of the skf (sporulation killing factor) operon in Bacillus safensis and Bacillus subtilis. The homology of each gene is color-coded based on comparisons with B. subtilis 168.

B. safensis strain DMB13 exhibited sensitivity to eight antibiotics: ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin [4]. Specific antibiotic resistance genes for these eight antibiotics were not detected in the genome. Although hemolysis was not observed, a hemolysin- 3 like protein gene (WNC56_10320) was detected. However, our previous research indicated that this gene is ineffective [9]. Consistent with these previous findings, hemolysis was not observed in this study, supporting the ineffectiveness of this gene.

B. safensis strain DMB13 exhibited lipase and protease activities and its genome possessed five lipase genes and 54 protease genes (Table 1). These genes may influence the lipase and protease activities of strain DMB13. This study provides a basis for further studies aimed at the identification of genes associated with the antimicrobial activity of B. safensis and provides insight into its functionality.

Table 1 . Putative lipase and protease genes in the genome of Bacillus safensis DMB13.

Gene locusE.C. no.ProductCOGGene
WNC56_02285-Spore germination lipase LipCE-
WNC56_027303.1.1.-Hormone-sensitive lipaseIaes
WNC56_138303.1.1.3Triacylglycerol lipaseSlip
WNC56_00285-Sporulation-specific protease YabGS-
WNC56_013903.4.21.-Cell wall-associated proteaseOwprA
WNC56_014503.4.21.-Minor extracellular protease EprOepr
WNC56_01635-Cysteine protease StiPS-
WNC56_02530-Putative rhomboid protease YdcAS-
WNC56_02980-Putative membrane peptidase YdiLS-
WNC56_044703.4.21.89Signal peptidase IUlepB
WNC56_047553.4.-.-Probable peptidoglycan endopeptidase LytEM|MlytE
WNC56_051203.4.24.84Ste24 endopeptidaseO-
WNC56_055153.4.21.89Signal peptidase IUlepB
WNC56_059753.4.24.-Oligoendopeptidase F like proteinEpepF
WNC56_065003.4.21.107Peptidase DoOdegP
WNC56_065453.4.14.13Gamma-D-glutamyl-L-lysine dipeptidyl-peptidaseMykfC
WNC56_066603.4.21.-Major intracellular serine proteaseOisp
WNC56_067553.4.24.-Protease HtpX like proteinOhtpX
WNC56_06860-ATP-dependent Clp protease ATP-binding subunit ClpEO-
WNC56_07095-Putative L,D-transpeptidase YkuDS-
WNC56_072703.4.21.89Signal peptidase IUlepB
WNC56_077253.4.23.-Sporulation sigma-E factor-processing peptidaseSspoIIGA
WNC56_078203.4.23.36Signal peptidase IIMUlspA
WNC56_081703.4.25.2HslU--HslV peptidaseOhslV
WNC56_08175-ATP-dependent protease ATPase subunit ClpYO-
WNC56_083803.4.24.-Probable protease eepMrseP
WNC56_085353.4.24.-Uncharacterized zinc protease YmxGOpqqL
WNC56_085803.4.21.92Endopeptidase ClpOclpP
WNC56_087503.4.21.-Serine protease AprXOaprX
WNC56_090203.4.21.19Glutamyl endopeptidaseOsspA
WNC56_100853.4.-.-D-gamma-glutamyl-meso-diaminopimelic acid endopeptidase CwlSM|McwlS
WNC56_101553.4.21.102C-terminal processing peptidaseMprc
WNC56_10900-Protease PrsWS-
WNC56_115253.4.21.116SpoIVB peptidaseMspoIVB
WNC56_117303.4.21.89Signal peptidase IUsipW
WNC56_118253.4.19.11Gamma-D-glutamyl-meso-diaminopimelate peptidaseEyqgT
WNC56_118453.4.21.105Rhomboid proteaseSgluP
WNC56_121903.4.24.78GPR endopeptidaseOgpr
WNC56_125953.4.-.-Uncharacterized protease YrrOO-
WNC56_126003.4.-.-Uncharacterized protease YrrNO-
WNC56_130603.4.21.53Endopeptidase LaOlon
WNC56_130653.4.21.53Endopeptidase LaOlonB
WNC56_13070-ATP-dependent Clp protease ATP-binding subunit ClpXO-
WNC56_136853.4.21.-Putative signal peptide peptidase SppAOUsppA
WNC56_142053.4.14.5Dipeptidyl-peptidase IVE-
WNC56_15220-Putative membrane protease YugPS-
WNC56_15640-Uncharacterized peptidaseE-
WNC56_157003.4.-.-L-Ala--D-Glu endopeptidaseMlytH
WNC56_159503.4.21.107Peptidase DoOdegP
WNC56_166753.4.21.92Endopeptidase ClpOclpP
WNC56_167453.4.-.-Peptidoglycan DL-endopeptidase CwlOM|ScwlO
WNC56_169653.4.21.102C-terminal processing peptidaseMprc
WNC56_169753.4.-.-Peptidoglycan DL-endopeptidase CwlOS|McwlO
WNC56_183753.4.21.-Minor extracellular protease vprOvpr
WNC56_194403.4.21.107Peptidase DoOdegP
WNC56_195203.4.21.26Prolyl oligopeptidaseS-

The Enzyme Commission (EC) number is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. The EC numbers are based on the genes of strain DMB13 and gene are assigned by BlastKoALA. The Clusters of Orthologous Group (COG) categorization was generated by annotated gene functions.

The complete genome sequence of Bacillus safensis DMB13 has been deposited in DDBJ/ENA/GenBank under accession number CP150833 and CP150834.

This study was conducted in support of the sabbatical program of Dongduk Women's University in 2023.

The authors have no financial conflicts of interest to declare.

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