Molecular characterization of LysPBC2, a novel endolysin harboring a Bacillus cereus spore binding domain.

To control a spore-forming human pathogen Bacillus cereus , we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus , Listeria , and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapped with its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and binding assay indicate that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure. Importance Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive infecting phages have modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. Single point mutation on the spore binding domain greatly enhance the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide an insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus .