## 4 Discussion  
Endolysins are phage-encoded enzymes that mediate the bacterial lysis during the last step of the viral lytic cycle. Thanks to their EAD enzymatic properties and their CBD allowing recognition and binding to the host cell, endolysins have gained much attention as alternative antimicrobials and detection tools for pathogenic bacteria [46]. In this study, we focused on PlyB221 and PlyP32 endolysins, encoded by two phages preying on the _B. cereus_ group. Deep-Blue is a lytic phage belonging to the _Herelleviridae_ whose potential for biocontrol purposes was previously investigated [23]. It was able to efficiently reduce the contamination by emetic _B. weihenstephanensis_ in food matrices and biofilms, but its main drawback remained its narrow host range, therefore indicating that it should be included in a phage cocktail. Similarly, the siphovirus Deep-Purple was only able to infect a limited number of strains belonging to the _B. cereus_ group but in addition, it was hypothesized that it might be a lytic  
Figure 8: Binding ability of PlyB221 and PlyP32 CBD. Exponentially growing cultures were used in a cell wall decoration assay based on the observation of the GFP fused CBD adsorbed to bacteria by fluorescent microscopy. Images on the left show bright field microscopy while images on the right display the corresponding fluorescent images. (**A,B**) _B. cereus_ ATCC 10,987 - PlyB221_CBD; (**C,D**). _B. weihenstephanensis_ Si0239 - PlyB221_CBD; (**E,F**) _B. megaterium_ Si0003 - PlyP32_CBD; (**G,H**) _B. weihenstephanensis_ LH002 - PlyP32_CBD. Scale bar = 25 \(\upmu\)m for all panels. variant of a lysogenic phage, making it unsuitable for biocontrol purposes [24]. To get around these drawbacks, their respective endolysins were investigated in this study. As for other endolysins encoded by phages infecting Gram-positive bacteria, PlyB221 and PlyP32 have an EAD located at their N-terminal end. BLAST and phylogenetic analysis of PlyB221 indicated that it is close to LysB4 and PlyP56 encoded by _B. cereus_ phages B4 and Phrodo, respectively. Similarly, its enzymatic function is fulfilled by an L-Ala-D-Glu_peptidase domain that is predicted to have three metal-binding sites. It was previously shown for LysB4 and PlyP56, that the activity is lost in the presence of EDTA and then restored with the addition of metal ions--such as zinc, calcium, and manganese--thus reinforcing the fact that PlyB221 also relies on divalent cations for its activity [18; 20]. PlyP32 possesses a GH25_muramidase EAD that is similar to that of PlyB, the endolysin encoded by the _Bacillus_ phage Bcp1, and PlyBt33 from phage BtCS33 [47; 48]. Both PlyB221 and PlyP32 displayed high lytic activity within the first 10 min of incubation with the most sensitive bacteria when 100 \(\upmu\)g/mL of protein was tested, which corresponds to 2.6 and 2.9 \(\upmu\)M, respectively. This antimicrobial activity is similar to what has been previously described for endolysins targeting the _B. cereus_ group. Endolysins encoded by phages infecting the _B. cereus_ group show differences regarding the targeted bacteria, ranging from a narrow to a broad activity spectrum. For instance, LysPBC5 is only able to lyse members of the _B. cereus_ group whereas LysPBC2 is also active against other _Bacillus_ spp., and even _L. monocytogenes_ and _Clostridium perfringens_[11; 33]. PlyB221 and PlyP32 both have intermediate activity spectra, as they are active against strains of the _B. cereus_ group but also _B. megaterium_, _B. pumilus_, and _B. licheniformis_. Noteworthy, PlyB221 and PlyP32 were not tested against _B. anthracis_, but based on their close relationships to PlyP56 and PlyB whose activity against _B. anthracis_ was demonstrated [20; 47], their activity spectrum is expected to include this pathogenic bacterium. Also, PlyB221 and PlyP32 are active on a much wider range of bacteria than that of their parental phages. These broad activity spectra could target most, if not all, of the pathogenic strains of the _B. cereus_ group.