## 3 Results  
### PlyB221 and PlyP32 Display the Canonical Features Of Endolysins  
Analysis of phage Deep-Blue genome revealed that gene product (gp) 221 possesses the common organization of phage endolysins (Figure 1A). The gp of this 942-bp gene, renamed PlyB221, indeed displays a putative N-terminal L-Ala-D-Glu_peptidase_like EAD domain (accession number, cd14845, residues 12-135) belonging to the Peptides_M15 superfamily (cd14814). This suggests that the cleavage site occurs between the alanine and glutamine residues of the peptide chain. PlyB221 presents five potential active sites (Arg50, His80, Asp87, Asp129, His132) of which three (His80, Asp87, His132) are involved in Zn\({}^{2+}\) binding (Figure 2A). The C-terminal CBD of PlyB221 contains two putative SH3b domains (residues 171-225 and 251-305) from the SH3 superfamily (smart00287) which was confirmed by HHpred analysis that detected two regions (residues 173-230 and 241-304) of structural homologies with the SH3 domains of phage PBC5 endolysin targeting _B. cereus_ (Probability: 97%, Protein data bank (PDB) ID: 6 ILU_B) [33]. BLAST analysis also showed that PlyB221 is closely related to phage BigBertha endolysin (YP_008771084) as they share 80% identity with 100% coverage (Figure 2A). Besides, this protein is the only one that matches PlyB221 with a high coverage as the other endolysins only cover 48% of PlyB221 sequence, corresponding to its EAD. PlyB221 CBD thus seems to be highly variable and different from the other SH3b domains identified so far. The in silico analysis and protein sequence alignments also revealed that PlyB221 EAD is closely related to that of LysB4 (88% identity) and PlyP56 (85% identity), the endopeptidase of _B. cereus_ phages B4 and Phrodo, respectively (Figure 2A) [18,20]. This was confirmed through a comparison gathering all the characterized endolysins encoded by _B. cereus_ phages and showing that PlyB221 clusters with LysB4 and PlyP56 (Figure 3). Figure 1: In silico analysis of Deep-Blue PlyB221 and Deep-Purple PlyB32. (**A**) PlyB221 two domain organization. The EAD is a putative L-Ala-D-Glu peptidase domain (superfamily Peptidase_M15) and the CBD contains two potential SH3 domains (superfamily SH3). (**B**) PlyP32 displays a GH25_PlyB_like domain as EAD (GH25 superfamily) characteristic of a muramidase activity and a single SH3 domain as CBD. Figure 2: Multiple sequence alignment of PlyB221, PlyP32, and related endolysins. (**A**) Multiple sequence alignment of PlyB221 with endolysins from phages BigBertha, B4 (LysB4), and Phrodo (PlyP56). (**B**) Multiple sequence alignment of PlyP32 with endolysins from phages Bcpl1 (PlyB) and BcCS33 (PlyB33). Residues considered as participating to the active sites are highlighted by red frames, while red stars indicate the Zn\({}^{2+}\) binding sites in PlyB221. The putative EAD region is underlined in blue, the ’linker’ sequence with a dashed red line and the CBD in orange. Regarding phage Deep-Purple, gp32 (789 bp) encodes the endolysin PlyP32 (Figure 1B) with a putative N-terminal GH25_PlyB_like domain (cd06523, residues 3-173) as EAD, which is part of the GH25_muramidase superfamily (cd00599) (i.e., involved in glycosidic bonds cleavage). The 10 potential active sites are conserved in PlyP32 (Asp6, Arg28, Tyr57, Phe59, Asp89, Glu91, Tyr120, Trp140, Gln157, Asp171) and no metal binding site was detected (Figure 2B). One putative SH3b domain was detected using InterPro (G3 DSA:2.30.30.40, residues 200-262). Similarly, HHpred highlighted structural homologies with the SH3b domains of endolysins LysF1 from the _Staphylococcus_ phage K (Prob. 91%, PDB ID: 5 OIQ_A) and LysPBC5 of the _Bacillus_ phage PBC5 (Prob. 96%, PDB ID: 6 ILU_B) as well as other phage and bacteria-related SH3 domains [33,34]. BLAST analysis indicated that PlyP32 is related to PlyB (coverage 86%, identity 59%) and PlyBt33 (coverage 71%, identity 67%) from _Bacillus_ phages Bcp1 and BtCS33, respectively (Figure 3) [19,35].