*2.3. Bacillus Species Have the Lowest Number of Secondary Metabolite BGCs*

In order to identify P450s involved in the biosynthesis of secondary metabolites, the gDNA and plasmid DNA of each *Bacillus* species (Table S2) has been subjected to secondary metabolite BGCs analysis using anti-SMASH [30]. In total, 203 plasmids were identified in 60 *Bacillus* species (Table S2). Analysis of 128 *Bacillus* species genomes revealed the presence of 1098 and 26 secondary metabolite BGCs on gDNA and plasmid DNA, respectively (Figure 6 and Table S3).

**Figure 6.** Comparative analysis of secondary metabolite BGCs in 128 *Bacillus* species (gDNA and plasmid DNA). Numbers next to bars indicate the number of secondary metabolite BGCs. Detailed analysis of secondary metabolite BGCs in each species is presented in Table S3.

The number of secondary metabolite BGCs varied from a maximum of 14 to one in *Bacillus* species gDNA. Interestingly, among 203 plasmid DNAs from 60 *Bacillus* species (Table S3), only 21 plasmid DNAs from 18 *Bacillus* species were found to have secondary metabolite BGCs (Figure 6 and Table S3). The number of secondary metabolites BGCs on plasmid DNAs varied from a maximum of four to one (Figure 6 and Table S3).

Analysis of types of secondary metabolite BGCs revealed the presence of 33 and 10 types of BGCs on gDNA and plasmid DNAs in *Bacillus* species (Figure 7 and Supplementary Dataset 4). The types of BGCs in individual *Bacillus* species varied from a maximum of 10 types to one (Figure 7). Among types of BGCs, Nonribosomal peptides secondary metabolite (Nrps) BGCs were dominant in *Bacillus* species, both on gDNA and plasmid DNAs (Figure 7).

**Figure 7.** Comparative analysis of types of secondary metabolite BGCs in *Bacillus* species. (**A**) The number of types of secondary metabolite BGCs in *Bacillus* species. (**B**,**C**). Comparative analysis of types of secondary metabolite BGCs on gDNA and plasmid DNAs. Standard abbreviations representing secondary metabolite BGCs as indicated in anti-SMASH [30] were used in the figure.

Analysis of types of BGCs on gDNA and plasmid DNAs revealed the presence of seven types of common BGCs between gDNA and plasmid DNAs (Figure 7). This suggests that these plasmids might be involved in horizontal gene transfer of different BGCs among *Bacillus* species. It is important to note that horizontal gene transfer of BGCs is a common phenomenon among *Bacillus* species [21]. Interestingly, three distinct types of secondary metabolite gene clusters, namely Sactipeptide-Lantipeptide-T1pks-Nrps, Arylpolyene-Nrps, and Lantipeptide-T1pks-Nrps, were only identified on plasmid DNAs (Figure 7).
