*2.2. KOF112 Is a Strain of Bacillus velezensis*

The 16S rDNA nucleotide sequence of KOF112 had high homologies to the corresponding sequences of *B. velezensis* BIM B-1312D (100%), *B. velezensis* FJAT-52631 (100%), and *B. velezensis* CR-502 (99.9%). Phylogenetic analysis of the nucleotide sequences of KOF112 and *Bacillus* isolates showed that KOF112 formed a cluster with *B. velezensis* FZB42, which has a capacity to produce antimicrobial secondary metabolites (Figure 2) [21].

**Figure 2.** Phylogenetic analysis of 16S rDNA nucleotide sequence of KOF112 compared with those of *Bacillus* isolates. A phylogenetic tree was designed by means of the neighbor-joining (NJ) method using MEGA X program (bootstrap value with 1000 replicates). Bar indicates 1% band dissimilarity. Distance corresponds to the number of nucleotide substitutions per site. *B. velezensis* FZB42 (accession no. CP000560). *B. amyloliquefaciens* DSM7 (accession no. FN597644). *B. amyloliquefaciens* Mk4 (accession no. MT131178). *B. nakamuraii* NRRL B-41091 (accession no. KU836854). *B. subtilis* NRRL B-4219 (accession no. NR\_116183). *B. tequilensis* VrN5 (accession no. LT986216). *B. subtilis* subsp. *spizizenjii* TU-B-10 (accession no. CP602905). *B. paralicheniformis* KJ-16 (accession no. KY694465). *B. pumilus* ATCC 7061 (accession no. AY876289). *B. megaterium* BF245 (accession no. MK491077). *B. circulans* ATCC 4513 (accession no. AY724690). *B. coagulans* NBRC 12583 (accession no. KX261624). *B. mycoides* DSM 11821 (accession no. AB021199). *B. wiedmannii* FSL W8-0169 11821 (accession no. KU198626). *B. toyonensis* BCT-7112 (accession no. CP006863).

The draft genome of KOF112 includes a circular genome (3,916,789 bp) and a plasmid (13,003 bp). The annotation predicted 3746 coding sequences, 27 rRNA genes, and 86 tRNA genes. Comparison of KOF112 genome sequence with the genome sequences of antagonistic *Bacillus* isolates, *B. velezensis* FZB42 and *B. amyloliquefaciens* DSM7, which formed a cluster by 16S rDNA comparison (Figure 2), revealed significant differences among the genome sequences (Figure 3). For example, the gene clusters responsible for the nonribosomal synthesized antimicrobial peptides and polyketides in KOF112 genome were different from those in FZB42 and DSM7 genomes. KOF112 genome has gene clusters for the biosynthesis of surfactin, bacillibactin, bacilysin, and bacillaene, but not gene clusters for the biosynthesis of iturin, bacillomycin, fengycin, difficidin, and macrolactin.

**Figure 3.** Genome comparison of KOF112, *B. velezensis* FZB42, and *B. amyloliquefaciens* DSM7. The whole genomes of KOF112 (outermost circle), FZB42 (middle circle), and DSM7 (innermost circle) were aligned using the CGView Server. The server used BLAST to compare the KOF112 genome sequence with FZB42 and DSM7 genome sequences. BLAST results and feature information for coding sequences were converted into a graphical map with black, blue, and green colors. Gene annotation of KOF112 coding sequences is partially shown in the figure.

Surfactin synthase subunit 1 (*srfAA*), surfactin synthase subunit 2 (*srfAB*), surfactin synthase subunit 3 (*srfAC*), and surfactin synthase thioesterase subunit (*srfAD*) were present in KOF112 genome (Figure 3). Surfactin biosynthetic gene cluster was selected as a nonribosomal peptide synthase cluster and compared among KOF112, *B. velezensis* FZB42, and *B. amyloliquefaciens* DSM7 (Figure 4). The organization of *srfAA*, *srfAB*, *srfAC*, and *srfAD* showed a perfect match among the isolates (Figure 4A). Phylogenetic analysis of each gene indicated that KOF112 surfactin biosynthetic genes formed a cluster with *B. velezensis* FZB42 surfactin biosynthetic genes (Figure 4B).

Taken together, the results suggest that KOF112 is a strain of *B. velezensis*.

**Figure 4.** Comparison of surfactin biosynthetic gene clusters among KOF112, *B. velezensis* FZB42, and *B. amyloliquefaciens* DSM7. (**A**) Organization of surfactin biosynthesis genes. (**B**) Phylogenetic analysis of *srfAA*, *srfAB*, *srfAC*, and *srfAD* of KOF112 compared with those of FZB42 and DSM7. A phylogenetic tree was designed by means of the NJ method using MEGA X program (bootstrap value with 1000 replicates). Bar indicates 1% band dissimilarity. Distance corresponds to the number of nucleotide substitutions per site.
