*2.1. Bacillus Species Have the Lowest Number of P450s*

Genome data-mining and annotation of P450s in 128 *Bacillus* species revealed the presence of the lowest number of P450s in their genomes (Figures 1 and 2). In total, 507 P450s were found in 114 *Bacillus* species, where 14 species did not have P450s in their genomes (Figures 1 and 2). On average, four P450s were found in *Bacillus* species, with the highest number, 11, found in *Bacillus* subtilis subsp. *spizizenii* TU-B-10. The number of P450s found in *Bacillus* species is very low compared to mycobacterial species (60 species); the latter species have, on average, 35 P450s in their genomes [25]. The P450 count in *Bacillus* species, apart from *B. subtilis* subsp. *spizizenii* TU-B-10, which has 16, is as follows: 9 in 2 species, 8 in 4 species, 7 in 20 species, 6 in 21 species, 5 in 10 species, 4 in 9 species, 3 in 28 species, 2 in 7 species, and a single in 14 species (Figures 1 and 2). This indicates that most *Bacillus* species (28 species) have three P450s in their genomes. P450s identified in each *Bacillus* species and respective P450 sequences were presented in Supplementary datasets 1 and 2.

**Figure 1.** Phylogenetic analysis of *Bacillus* species P450s. Dominant P450 families were indicated in different colors. CYP51B1 from *Mycobacterium tuberculosis* H37Rv is used as an outgroup. A high-resolution phylogenetic tree is provided in the supplementary Figure S1.

**Figure 2.** Comparative analysis of P450s in *Bacillus* species. The numbers next to bars indicate the number of P450s in *Bacillus* species. *Bacillus* species' names with respect to their codes can be found in Supplementary Dataset 1.

#### *2.2. Bacillus Species Have the Lowest Number of P450 Families and Subfamilies'*

As per the International P450 Nomenclature Committee rules [27–29], all 507 P450s found in 114 *Bacillus* species can be grouped into 13 P450 families and 28 subfamilies (Figures 3 and 4). Phylogenetic analysis of *Bacillus* P450s revealed P450s belonging to the same family grouped together, suggesting that the annotation of P450s in this study is accurate (Figure 1). The number of P450 families and subfamilies found in *Bacillus* species is lower compared to mycobacterial species (60 species), which have 77 P450 families and 132 subfamilies [25]. Because of the presence of the lowest number of P450 families, the P450 diversity percentage in *Bacillus* species was found to be lowest (3.9%) compared to mycobacterial species (72%) [25]. Among 13 P450 families, the CYP107 P450 family has the highest number of P450s (165 P450s) contributing 31.5% of 507 P450s (Figure 3), followed by CYP102 (143 P450s), CYP109 (79 P450s), CYP106 (40 P450s), CYP152 (36 P450s), CYP113 (18 P450s), CYP134 (13 P450s), CYP1756 (4 P450s), CYP1221 (3 P450s), CYP1179 and CYP223 (2 P450s), CYP1341 and CYP197 (single P450s) (Figure 3).

**Figure 3.** Comparative analysis of P450 families in *Bacillus* species. The numbers next to the family bar indicate the total number of P450s and percentage contribution (parenthesis) by a respective family to the total number of P450s. The data on the number of P450s in each P450 family in *Bacillus* species is presented in Table S1.

**Figure 4.** Comparative analysis of P450 subfamilies in *Bacillus* species. The numbers next to bars indicate the total number of members in a particular subfamily. Data on the number of P450s in each P450 subfamily in *Bacillus* species is presented in Table S1.

P450 subfamily analysis revealed that most P450 families have a single subfamily (Figure 4). Among P450 families, CYP107 has the highest number of P450 subfamilies (eight subfamilies) followed by CYP109 (six subfamilies), CYP152 (three subfamilies), and CYP106 (two subfamilies) (Figure 4). The remaining nine P450 families, CYP102, CYP113, CYP1179, CYP1221, CYP1341, CYP134, CYP1756, CYP197, and CYP223, all have a single subfamily (Figure 4). It is interesting to note that the CYP102 P450 family, despite having the second largest number of P450s, has a single subfamily "A". Analysis of P450 subfamily profiles revealed that specific subfamilies are dominant in a particular family (Figure 4). Subfamily "J" is dominant in the CYP107 family, subfamily "B" is dominant in the CYP109 family and subfamily "A" is dominant in the CYP152 family (Figure 4). Analysis of P450 family profiles revealed that no P450 family is conserved across *Bacillus* species (Figure 5). Most *Bacillus* species have CYP102, CYP107, CYP109, CYP106, and CYP152 P450 families (Figure 5). CYP197, CYP223, and CYP1341 are present in a single *Bacillus* species (Supplementary Dataset 1).

**Figure 5.** Heatmap of presence or absence of cytochrome P450 families in 128 species of *Bacillus*. The data have been represented as −3 for family presence (green) and 3 for family absence (red). A hundred and twenty-eight *Bacillus* species form the horizontal axis and CYP family numbers form the vertical axis. The respective data used in the generation of Figure 5 is presented in Supplementary Dataset 3.
