Molecular characterization of phage Deep-Purple infecting the Bacillus cereus group

As bacterial parasites, phages are generating considerable interest due to their potential applications in the development of antimicrobials and detection tools. The understanding of crucial steps of phage infection cycle is fundamental for phage research. This includes the adsorption stage which involves the interaction of bacterial receptors with phages proteins termed Receptor Binding proteins (RBPs) and the lysis process occurring at the end of the phage infection cycle upon bacterial peptidoglycan degradation performed by phage endolysins. Previous studies have mainly focused on Gram-negative targeting phages and much less information is currently available on phages preying on Gram-positive bacteria. This work aimed to shed light on the adsorption and lysis processes of Deep-Purple, a tailed phage belonging to the Siphoviridae family and infecting the Bacillus cereus group. Bioinformatic analyses of Deep-Purple tail proteins retained two putative RBP candidates namely gp28 and gp29. Gp28 was chosen for its putative evolved Dit function, reported to feature carbohydrate binding modules able to interact with the bacterial surface and sometimes even replacing the RBP in some phages including the Lactobacillus casei phage J-1. Gp29 was investigated for its putative tail fiber function and due to its C-terminal intramolecular chaperone domain (IMC), structurally similar to the IMC of Salmonella phage S16 RBP. Both candidates were tagged with GFP and recombinant proteins were successfully produced in Escherichia coli cells and purified. Their capacity to bind to Bacillus cereus was assessed in a cell wall decoration assay. This allowed to observe that both proteins are capable of interacting with B. cereus cells. Further investigations are required to determine if both are bona fide RBP and their precise roles in phage adsorption. Gp32 was investigated as sole potential endolysin candidate because of its sequence similarity with other endolysins. It displayed a putative N-terminal domain associated with 1,4-Nacetylmuramidase activity (EAD, Enzymatically Active Domain) and a C-terminal SH3 domain putatively involved in host recognition (CBD, Cell wall Binding Domain). This work managed to produce and purify gp32 protein and its lytic activity was confirmed by turbidity reduction assay on B. cereus cells and extracted peptidoglycan. The spectrum of activity of the protein was assessed and revealed that all tested members within the B. cereus group were sensitive to gp32 and some tested members outside of the B. cereus group also showed endolysin susceptibility. Then, the lytic activity of gp32 was assessed under varying conditions showing that gp32 was effective in conditions ranging from pH 4 to 8 and under 37◦C but was highly sensitive to NaCl concentrations. Full-length gp32 and its CBD were tagged with GFP in order to evaluate their binding capacity in a cell wall decoration assay. These experiments confirmed their capacity to tightly bind to bacteria and corroborated the activity spectrum assessed before. Finally, the spectrum of activity of gp32 was compared to Deep-Purple spectrum determined via top agar experiments and revealed that endolysin can act upon phage resistant bacteria. All in one, our data allowed the identification of two tail proteins interacting with B. cereus cells during adsorption and collected considerable information regarding the endolysin activity of Deep-Purple phage.