Endolysins of Deep-Blue and Deep-Purple phages preying on Bacillus weihenstephanensis

Leprince, Audrey [UCL] Nuytten, Manon [UCL] Hock, Louise [UCL] Gillis, Annika [UCL] Mahillon, Jacques [UCL]

Introduction There is a renewed interest in phages and their derived proteins for various applications, including in the agro-food sector. Endolysins are peptidoglycan degrading enzymes synthetized at the end of the lytic cycle. In combination with holins, they are responsible for the lysis of the host cell allowing the release of newly synthetized virions. Endolysins from phages infecting Gram-positive bacteria are organized into two domains: a C-terminal Cell Wall Binding domain (CBD) and an N-terminal Enzymatically Active Domain (EAD) that cleaves the peptidoglycan. This study focuses on endolysins encoded by Deep-Blue [1] and Deep-Purple [2], two phages infecting Bacillus weihenstephanensis and belonging to the Myoviridae and Siphoviridae families, respectively. Materials and Methods Endolysin candidates were selected by bioinformatics and whole genes were cloned into the expression vector pET30a. Similarly, the corresponding CBD were cloned into pET30a with a N-terminal GFP fusion. The constructions were transformed into the strain BL21(DE3) and expression of each recombinant protein was induced by addition of IPTG. The proteins were purified on a Ni-NTA column. The lytic activity of the putative endolysins was assess by CFU reduction assay and reduction in Optical Density. As for the CBD, the interaction between the GFP fused domain and the bacteria was detected by fluorescent microscopy. Results and Discussion In silico analyses revealed that Deep-Blue gp221 and Deep-purple gp032 are good endolysin candidates. Gp221 has a Peptidase_M15_4 conserved domain from the VanY superfamily as EAD, which is also present in the endolysins of phages B4 [3] (LysB4) and Phrodo [4] (PlyP56) infecting members of the Bacillus cereus group. Gp032 possesses a GH25_PlyB-like domain belonging to the GH25_muramidase superfamily. PlyB is an endolysin from phage BcpI that displays activity against Bacillus anthracis [5]. As for the CBD, two SH3 domains were detected at the C terminus end of gp221 and one at that of gp032. Gp221 has a broader spectrum than its related phage and showed lytic activities not only against B. weihenstephanensis but also against other members of the B. cereus group that cannot be infected by Deep-Blue phage. Additionally, gp221 CBD is able to bind the surface of several strains of the B. cereus group. Characterization studies of both endolysins, including host spectrum, pH, temperature and salt concentration for optimal activity will be presented and discussed.