## 3 Results
### PlyB221 and PlyP32 Show a Bacteriolytic Spectrum Broader than That of Their Parental Phages  
The full _plyB221_ and _plyP32_ genes were cloned in the expression vector pET30, expressed in _E. coli_ BL21(DE3) and purified on a Ni-NTA affinity column to characterize the bacteriolytic activity of the corresponding enzymes. Figure 4 shows the purified recombinant proteins displaying the expected MW of 39 (lane 2) and 34 kDa (lane 3) for PlyB221 and PlyP32, respectively. Figure 3: Dendrogram of relationships between the characterized endolysins of _B. cereus_ phages. The tree was built using the maximum likelihood method and JTT matrix-based model with 100 bootstrap iterations. Endolysins are indicated by the name of the phage they derived from. Endolysins with the same EAD are grouped in the same cluster. Muramidases, amidases, and endopeptidase are highlighted as yellow, blue, and gray rectangles, respectively. Deep-Blue and Deep-Purple endolysins are indicated by red asterisks. The bar indicates 0.5 substitutions per amino acid site. The numbers refer to the percentage of trees in which the associated endolysins clustered together. The lytic activity was assessed in a turbidity reduction assay using _B. cereus_ ATCC 10,987 as model strain. The endolysins were serially diluted (from 100 to 3 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro g}\)/mL) and incubated with the bacteria resuspended in a Tris-HCl buffer (pH 8 for PlyB221 and pH 7 for PlyP32, see below). The test was performed at 30 \(\mathrm{\SIUnitSymbolCelsius}\) and the OD was monitored during 30 min. Figure 5A shows that at the highest concentration (i.e., 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), PlyB221 induced a 50% reduction in OD within 4 min of incubation. The same reduction was observed after 15 min at an endolysin concentration of 6 \(\mathrm{\SIUnitSymbolMicro g}\)/mL. The same dose response behavior was observed for PlyP32 and 50% of inhibition was reached within ca. 5 min at a 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL protein concentration (Figure 5B). Figure 4: SDS-page gel of purified endolysins PlyB221 and PlyP32. The recombinant proteins were expressed in _E. coli_ BL21(DE3) and purified thanks to a six-histidine tag on a Ni-NTA column. Lane 1: Protein ladder in kDa; Lane 2: PlyB221; Lane 3: PlyP32; Lane 4: GFP::PlyB221_CBD; Lane 5: GFP::PlyP32_CBD. Figure 5: Bacterolytic activity of PlyB221 (**A**) and PlyP32 (**B**) assessed by OD monitoring. The activity test was performed on exponentially growing _B. cereus_ ATCC 10,987 at 30 \(\mathrm{\SIUnitSymbolCelsius}\) in the optimal buffer for each endolysin and using enzyme concentrations ranging from 3 to 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL. The activity ranges of PlyB221 and PlyP32 were then established by subjecting strains from the _B. cereus_ group (_n_ = 14) and other Gram-positive bacteria (_n_ = 8) to 100 ug/mL of protein in a turbidity reduction assay. In parallel, the susceptibility of these strains to the parental phages, Deep-Blue and Deep-Purple, was assessed via spot-on-plate. Three types of bacterial responses were recorded (Table 3): sensitive strains (i.e., formation of phage plaques), insensitive strains and those affected by the process of 'lysis from without'--i.e., no individual plaques during spot assay--but nonetheless cell lysis when phages were applied at a high multiplicity of infection (MOI) [36]. As shown in Table 3, all the tested strains inside the _B. cereus_ group were sensitive to both endolysins, although to different extents, ranging from 47% to 76% of inhibition for PlyB221 and from 15% to 79% for PlyP32. Interestingly, phages Deep-Blue and Deep-Purple themselves displayed narrower host spectra as they infected three and four strains, respectively, out of the 14 tested strains of the _B. cereus_ group. Thus, the PlyB221 and PlyP32 endolysin activity spectra are much broader than those of their parental phages. Outside the _B.