Genomic Analysis of Bacillus licheniformis CBA7126 Isolated from a Human Fecal Sample

Bacillus licheniformis is a Gram-positive, endospore-forming, saprophytic organism that occurs in plant and soil (Veith et al., 2004). A taxonomical approach shows that it is closely related to Bacillus subtilis (Lapidus et al., 2002; Xu and Côte, 2003; Rey et al., 2004). Generally, most bacilli are predominantly aerobic; however, B. licheniformis is a facultative anaerobe compared to other bacilli in ecological niches (Alexander, 1977). The commercial utility of the extracellular products of B. licheniformis makes this microorganism an economically interesting species (Kovács et al., 2009). For example, B. licheniformis is used industrially for manufacturing biochemicals, enzymes, antibiotics, and aminopeptidase. Several proteases such as α-amylase, penicillinase, pentosanase, cycloglucosyltransferase, β-mannanase, and certain pectinolytic enzymes are synthesized industrially using B. licheniformis (Rodríguez-Absi and Prescott, 1978; Rey et al., 2004). The proteases are used in the detergent industry and the amylases are utilized for starch hydrolysis, desizing of textiles, and sizing of paper (Erickson, 1976). In addition, certain strains are utilized to produce peptide antibiotics, specialty chemicals, and poly-γ-glutamic acid (Nierman and Maglott, 1989; Rey et al., 2004). The annotated genome sequence of B. licheniformis has been previously analyzed to assess the biotechnological importance of the organism (Veith et al., 2004). Since the first sequencing, the genomes of specific B. licheniformis strains have been sequenced to completely realize its industrial potential. In this study, genome sequencing of B. licheniformis CBA7126 isolated from a human fecal sample was performed to understand bacterial specificity. The genome sequence of CBA7126 revealed features such as stress response genes, antibiotic-resistance genes, and genes for resistance to toxic compounds, which are of considerable biotechnological value.


INTRODUCTION
Bacillus licheniformis is a Gram-positive, endospore-forming, saprophytic organism that occurs in plant and soil (Veith et al., 2004). A taxonomical approach shows that it is closely related to Bacillus subtilis (Lapidus et al., 2002;Xu and Côte, 2003;Rey et al., 2004). Generally, most bacilli are predominantly aerobic; however, B. licheniformis is a facultative anaerobe compared to other bacilli in ecological niches (Alexander, 1977). The commercial utility of the extracellular products of B. licheniformis makes this microorganism an economically interesting species (Kovács et al., 2009). For example, B. licheniformis is used industrially for manufacturing biochemicals, enzymes, antibiotics, and aminopeptidase. Several proteases such as α-amylase, penicillinase, pentosanase, cycloglucosyltransferase, β-mannanase, and certain pectinolytic enzymes are synthesized industrially using B. licheniformis (Rodríguez-Absi and Prescott, 1978;Rey et al., 2004). The proteases are used in the detergent industry and the amylases are utilized for starch hydrolysis, desizing of textiles, and sizing of paper (Erickson, 1976). In addition, certain strains are utilized to produce peptide antibiotics, specialty chemicals, and poly-γ-glutamic acid (Nierman and Maglott, 1989;Rey et al., 2004).
The annotated genome sequence of B. licheniformis has been previously analyzed to assess the biotechnological importance of the organism (Veith et al., 2004). Since the first sequencing, the genomes of specific B. licheniformis strains have been sequenced to completely realize its industrial potential. In this study, genome sequencing of B. licheniformis CBA7126 isolated from a human fecal sample was performed to understand bacterial specificity. The genome sequence of CBA7126 revealed features such as stress response genes, antibiotic-resistance genes, and genes for resistance to toxic compounds, which are of considerable biotechnological value.

MATERIALS AND METHODS
Bacterial Isolation, Culture Conditions, and DNA Extraction B. licheniformis CBA7126 was isolated from the feces of a 74-year-old man in Geochang-gun, South Korea and was cultured under anaerobic conditions in Gifu Anaerobic Medium (GAM) (containing per liter of deionized distilled water: 10 g peptone, 3 g soytone, 10 g proteose peptone, 13.5 g bovine serum albumin, 5 g yeast extract, 2.2 g beef extract, 2.5 g monopotassium phosphate, 1.2 g liver extract, 3 g sodium chloride, 0.3 g L-cystein, 0.3 g sodium thioglychollate, 3 g dextrose, 5 g soluble starch) at 37 • C for 48 h. Genomic DNA of strain CBA7126 was extracted using the QIAamp DNA extraction kit (Qiagen, USA) and QuickGene DNA tissue kit S (Kurabo, Japan), and purified using the MG genomic DNA purification kit (Doctor Protein, Korea) according to the manufacturer's instructions. The purity and concentration of the extracted genomic DNA were measured using the Nanodrop spectrophotometer (NanoDrop Technologies, UK).

Ethics Approval
The study protocol was approved by the institutional review board of the Theragen ETEX Bio Institute (700062-20160804-JR-005-02).

Comparative Genomic Data
Analysis of the orthoANI values among Bacillus genome sequences with symmetric identity of >97% revealed that B. licheniformis CBA7126 has higher than 99% genome sequence similarity with other species. The genome of strain CBA7126 was closest to that of B. licheniformis VTM3R78 (99.99% orthoANI), followed by B. licheniformis B4164 (99.98%), Bacillus sp. H15-1 (99.85%), B. licheniformis B4124 (99.81%), and B. licheniformis V30 (99.80%) (Supplementary Figure 2). Similar results were also obtained using ANI. Based on the results of Lee et al. (2016), similarity values >95-96% indicated that two strains belong to the same species. Therefore, strain CBA7126 was confirmed to be a species of B. licheniformis. The genome of strain CBA7126 was aligned with more than 97% symmetric identity with those of strains B. licheniformis B4164, B. licheniformis VTM3R78, B. licheniformis V30, Bacillus sp. H15-1, and B. licheniformis B4124 FIGURE 1 | Graphic circular map of the Bacillus licheniformis CBA7126 genome. The outer circle shows RNA genes (red, tRNA; blue, rRNA) and genes on the sense and antisense strands (colored according to COG categories), shown from the outside of the circle to the center. The inner circle shows the GC skew, with yellow and blue indicating positive and negative values, respectively; the GC content is indicated in red and green. This genome map was visualized using CLgenomics 1.55 (Chun Lab Inc.).
using MAUVE. The genomic representations of the other strains were rearranged based on the structure of strain CBA7126. Gene order comparison was established for seven regions with Local Collinear Blocks (LCBs). The structure of strain CBA7126 was similar to that of B. licheniformis B4124 and B. licheniformis V30 (Supplementary Figure 3). Comparison of strain CBA7126 genomic structure with that of Bacillus sp. H15-1 showed that two major regions were in opposite direction. Analysis based on the POG of strain CBA7126 and the closely related strains identified 4,108 shared genes and 137 unique genes (Supplementary Figure  4). Strain CBA7126 possessed 19 genes among the unique genes: 1 poly (glycerol-phosphate) alpha-glucosyltransferase, 1 thymidylate synthase (FAD), 2 prophage-derived protein, and 15 hypothetical proteins. The three genes among the 19 unique genes of strain CBA7126 were classified to one carbon pool by folate, pyrimidine metabolism, and metabolic pathways, based on KEGG analysis. In addition, CRISPR analysis indicated that strain CBA7126 did not harbor any known CRISPRs.

Phage and Pathogenesis-Related Genes
PHAST analysis was performed for identifying prophage contamination in the genome sequence of strain CBA7126. Contig 1 contained three intact and two incomplete prophages, whereas contig 2 contained only one incomplete prophage (Supplementary Figure 5). Intact regions of prophages were located between positions 1,596,547-1, 623,555, 1,775,723-1,820,161, and 3,429,284-3,483,201 bp, respectively. Strain CBA7126 was identified to be a human pathogen with 0.81 probability in PathogenFinder 1.1. Analysis of pathogenesisrelated genes showed that all the 238 analyzed genes encoded pathogenesis-associated proteins.

Stress Response Genes and Resistance to Toxic Compounds
Comparison with NCBI PGAP 4.1 showed that the genome of strain CBA7126 harbors several stress response genes and various genes required for resistance to antibiotics and toxic compounds (Tatusova et al., 2016). The identified

DATA ACCESS
The genome sequence of B. licheniformis CBA7126 has been deposited in DDBJ/ENA/GenBank under the accession numbers BDJJ01000001-BDJJ01000002.

AUTHOR CONTRIBUTIONS
SR and YN designed and coordinated all the experiments. HS performed cultivation, DNA extraction, and purification. CL, JK, HS, YK, YC, and CY performed the sequencing, genome assembly, gene prediction, gene annotation, and comparative genomic analysis. CL, YN, and SR wrote manuscript. All authors have read and approved the manuscript.